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UA / Biology / BSC 300 / What is the multi-adhesive matrix proteins?

What is the multi-adhesive matrix proteins?

What is the multi-adhesive matrix proteins?


School: University of Alabama - Tuscaloosa
Department: Biology
Course: Cell Biology
Professor: John yoder
Term: Fall 2018
Tags: Biology
Cost: 50
Name: BSC 300-001 Final Studyguide
Description: This final will not be comprehensive - only on material in class
Uploaded: 12/07/2018
65 Pages 41 Views 10 Unlocks

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BSC 300-001 Week 12 Notes

What is the multi-adhesive matrix proteins?

I. Chapter 20 – Integrating Cells into Tissues

a. Cell-Cell and Cell-Extracellular Matrix Adhesion: An Overview

i. General Concepts about Cell-Cell and Cell-Extracellular Matrix Adhesion 1. Cell-cell and cell-extracellular matrix interactions are critical for  

assembling cells into tissues, controlling cell shape and function,  

and determining developmental fate of cells and tissues

a. Dynamic – nothing is really permanent

2. Cell-adhesion molecules mediate direct cell-cell adhesions, and  

adhesion receptor proteins mediate cell-matrix adhesions

3. The extracellular matrix is a dynamic, complex meshwork of  

proteins and polysaccharides that contributes to the structure and  

What is the cell adhesion and morphogenesis?

If you want to learn more check out What does the mulvery describes?

function of tissues

a. As cells interact with it, they change and modify it and vice  


ii. Major cell-cell and cell-matrix adhesive interactions

1. In multicellular animals cell-cell and cell-matrix adhesions  

aggregate cells into distinct tissues in order to cooperatively  

perform common functions

2. Humans have hundreds of unique cell types while simpler  

metazoans, like the round worm C. elegans have only 12

3. Despite these differences all animal cells can be classified into 5  If you want to learn more check out What is the capital lease?

major tissue types:

a. Muscle  

b. Neural

c. Blood

d. Epithelial – sheets of tightly connected cells composed of  

What is the mechanotransduction?

one of more layers that cover or line the major body and  

organ surfaces

e. Connective – found in between other tissues throughout  

the body. They support and protect other tissues and  

collagenous fibers, autonomous, non-connected cells and  

ground substance: the gel-like material secreted by cells in  

which they and the fibers are embedded. Collectively, the  

ground substance and fibers are referred to as the  

extracellular matrix

4. Cells adhere to one another and associate with the extracellular  

environment through dynamic spatial and temporal expression and  

functional regulation of diverse adhesion molecules

a. Cell-adhesion molecules (CAMs) generate cell to cell  

interactions. Cytosolic domains bind diverse intracellular  

adapter proteins that link them to the cytoskeleton.  Don't forget about the age old question of What type of diabetes is more common?
Don't forget about the age old question of Where did psychology originate from?

Extracellular domains associate with other CAM proteins

b. Adhesion receptors link cells to the extracellular matrix.  

Similarly are cytoplasmically linked to the cytoskeleton

i. Can transduce info to the interior of the cell

BSC 300-001 Week 12 Notes

c. Both types of adhesion link to the cytoskeleton and  

participate in intracellular signaling pathways

i. In doing so, through these associations cells can  

transduce signals and respond to environmental  

cues and be influenced to change their shape and  


ii. Likewise, cells can communicate to the  

extracellular environment and alter activity and  If you want to learn more check out What does the concept of empiricism promote?

function of molecules there

iii. These processes are referred to, generally as  

outside-in and inside-out signaling

iii. Cell-adhesion molecules (CAMs)

1. We don’t necessarily have to define these terms on an exam, but  they are referenced a lot in this lecture:

a. Some bind homophilic – only members of the same family  can interact

i. CAM molecules themselves

b. Others bind heterophilic – associate with members of  

another CAM family

c. May mediate homotypic interactions – association of cells  of the same type or

d. Heterotypically – association of cells of different types Don't forget about the age old question of How does air circulation affect climate?

2. Four major families of CAMs – each family with functionally  diverse members

a. Cadherins

b. Immunoglobin (Ig) superfamily

i. Antibodies are a member of this family

c. Integrins – can function as both CAMs, as well as adhesion  receptors

d. Selectins

e. Members of each family share conserved domains (often  found as repeated units within the cell) that are present in  

multiple copies and therefore define the distance between  

membranes of joined cells

i. Each family represents families as a result of  

evolution – many members of each family

3. Two types of CAM interactions promote cell-cell adhesion: a. Trans interactions occur between CAMs on adjacent cells,  while

b. Lateral interactions occur between CAMs on the same cell i. Cluster CAMs and promote more efficient trans  

interactions between cells

c. Trans and lateral interactions are mutually reinforcing

4. General features of cell-cell adhesions

BSC 300-001 Week 12 Notes

a. Cell-cell interactions are dynamic – changing according to  

the needs of the cell, the type of cell or extracellular matrix,  

signals received and forces applied to the cells

b. Some CAMs require calcium for interactions, so regulated  

release of Ca2+ plays important roles in their regulation

c. Association of intracellular molecules can alter CAM  

binding, as well as promote clustering that enhances cell

cell associations

d. Other variables that affect CAM dynamics include:

i. Binding affinity of CAMs (thermodynamic  


ii. Overall rate of association vs dissociation (kinetic  


iii. Spatial distribution and abundance of CAMs

1. How many are produced

iv. Regulated active vs inactive state of CAMs

v. External forces like mechanical stress  


vi. Turbulence of surrounding fluids

b. The Extracellular Matrix I: The Basal Lamina

i. The Extracellular Matrix (ECM) (Table 20-2 might be a good reference  point)

1. A complex combo of proteins and polysaccharides secreted and  assembled by cells into a cross-linked network

a. 4 major types of polysaccharides and proteins that re  

secreted by the cells with which they are associated

2. Holds cells in place within tissues (example – autonomous cells of  connective tissue)

3. Anchors epithelial cells to their substrate

4. Also provides positional and signaling info to cells, serving as a  reservoir and conduit for the diffusion of signaling molecules

5. Serves as a lattice that can either prevent or promote (through  guidance cues) the migration of cells – especially important during  embryogenesis and a process hijacked by cancer during metastasis

6. Because individual cells of a tissue all associate with the ECM, it  also serves as an indirect physical link between cells in tissue

7. ECM is connected to cell through transmembrane adhesion  


a. Which through both mechano-sensation (AKA pulling on  

our skin) and altered confirmation transduce info into the  


8. The ECM is not a static structure, but in a constant state of  

dynamic remodeling through diverse cell regulated enzymatic  

modification of its constituent components

9. Such remodeling results from enzymatically regulated:

a. Phosphorylation state

BSC 300-001 Week 12 Notes

b. Sulfation state

c. Cross-linking

d. Cleavage by proteases and glycosidases

e. Oxidation

f. Addition of glucose (glycation)

10. Principle ECM components:

a. Proteoglycans – core protein attached to sugar chains  (GAGs – glycosaminoglycans)

b. Collagens – structural protein fibers that provide tensile  strength

i. Absorb water and generate a cushion around the cell  – important in receiving signals (Wnt and  


c. Multi-adhesive matrix proteins – principally Fibronectin and laminin – important organizers of the ECM

i. Long, flexible molecules that contain multiple  


ii. Bind various collagens, other matrix proteins,  

polysaccharides, and extracellular signaling  

molecules and link the ECM to adhesion receptors

iii. Often serve as guidance cues for migrating cells  

during embryogenesis

11. Relative density of cells and ECM varies

a. Relative volumes occupied by cells and ECM vary greatly  among different animal tissues

b. Dense connective tissue contains mostly tightly packed  ECM fibers interspersed with rows of relatively sparse  fibroblasts (cells that synthesized ECM of connective  tissue)

c. Sparse ECM is typical of epithelial cells – here squamous  epithelial cells are tightly packed into a quilt-like pattern  with little ECM between the cells

12. Molecular and Species Specificity

a. While CAMs and adhesion receptors can be divided into  clear homolog families, their binding is so specific that  even between closely related species cells with the same  identity will preferentially associate with same-species cells  following mechanical disruption (a classic developmental  biology experiment by HV Wilson)

i. These specific interactions are driven by CAMs,  

adhesion receptors and ECM molecules – as shown  

in C & D. Beads coated with a collagen ECM  

protein from two species of sponge preferentially  

associate only with beads coated with the collagen  

from the same species

BSC 300-001 Week 12 Notes

ii. Separated two sponges and the mixed them together  – assumed orange would only interact with orange  

and yellow only with yellow – not the case

iii. Evidence that cell interactions are driven by  

different interactions with CAM proteins

13. Cell adhesion and morphogenesis

a. Cell-cell and cell-ECM associations play critical roles  during embryo morphogenesis – when cell movements and  rearrangements create the 3D structure of tissues and  organs

i. Branching is a common theme in many tissues  

(blood vessels, lung alveoli, etc)

ii. Some antibodies that bind and inhibit function of  

fibronectin impede branching

b. Adhesion molecule diversity

i. The evolution of adhesion molecules was a critical  step in the evolution of multicellular animals  


ii. Many of these molecules are shared (have  

homologs) in organisms as distantly related as  

humans and sponges – others evolved as  

components of unique types of tissue

iii. A common feature of adhesive proteins is repeating,  nearly identical domains – referred to as repeats –

that generate the larger protein. Each domain, or  

repeat, often encoded by a single exon

iv. The number and combination of these repeats  

determines the overall size and binding specificity  

as well and number and type of interactions

v. The evolution of gene families arose through gene  duplication and divergence as well as accidental  

“exon shuffling” between genes with similar  


vi. Protein isoform diversity (the number of similar  

proteins expressed in a species) is largely the result  


1. Families of multiple genes that arose by  

duplication and divergence

2. Alternative splicing of exons that generates  

multiple distinct isoforms from the same  

gene (primary transcript)  

c. Cell-adhesion molecules mediate signal transduction i. CAMs and adhesion receptors do not simply hold  

cells in place, but also respond to changes in the  

ECM, an interacting cell’s CAM expression or  

mechanical force to transduce signals

BSC 300-001 Week 12 Notes

ii. Binding of ECM ligands to an integrin adhesion  

receptor can stimulate many of the same  

transduction pathways we have previously  

described, and in doing so regulate:

1. Migration

2. Proliferation

3. Apoptosis

4. Enzymatic activities

d. Cell-adhesion molecules mediate mechanotransduction

i. Mechanotransduction – the reciprocal  

interconversion of mechanical force and  

biochemical processing (intracellular signaling) that  

can result in altered gene. Expression, enzymatic  

activity and/or mechanical action

1. In other words, CAMs and adhesion  

receptors can serve as activators of signal  

transduction into the cell (outside n  

signaling), while signaling pathways that  

converge on these molecules can alter CAM  

and adhesion receptor  

function/conformation and lead to inside out  

signaling (communication with the ECM or  

another cell)

2. Such mechanotransduction results from  

physical stress on tissue that stretches  

components of the ECM – like fibronectin  

and talin

a. This alters the proteins’  

conformation and exposes  

interaction domains

b. This can, in the case of fibronectin,  

allow the proteins to polymerize and  

form fibers that strengthen the ECM,  


c. In the case of talin (an intracellular  

adapter for integrins) mechanical  

stress exposes binding sites for an  

actin binding protein called vinculin  

which links integrins to the actin  

cytoskeleton forming focal adhesions  

in migrating cells or cells undergo  

physical stress

c. Cell-Cell and Cell-Extracellular Matrix Junctions and Their Adhesion Molecules i. Principal types of epithelia

1. Epithelia – sheets of tightly connected sessile (non-motile) cells  polarized into apical, lateral, and basal surfaces. Critical roles in

BSC 300-001 Week 12 Notes

animal development as migrating sheets that contribute the  reorganization of germ layers during gastrulation and organ  sculpting

a. All surfaces have distinct characteristics and function

b. General epithelial arrangements:

i. Simple columnar for absorbing/secreting (like  

digestive tract)

ii. Simple squamous – line blood vessels (rapid  

diffusion through the cells)

iii. Transitional line cavities subject to expansion

iv. Stratified protective layers (like our skin or  


c. All epithelial cells are:

i. Anchored to a specialized ECM at their basal  

surfaces via cell junctions called hemidesmosomes  

and focal, fibrillary and 3D adhesions. The  

specialized ECM is a dense framework that  

contributes to organ/tissue morphology called the  

basal lamina

ii. Tightly associated to one another through a number  

of types of cell junctions called:

1. Tight junctions

2. Adherens junctions

3. Desmosomes

4. Gap junctions

iii. All of these structures link the extracellular  

environment to the cytoskeleton and diverse signal  

transduction pathways

ii. Principal types of cell junctions connecting the columnar epithelial cells  lining the small intestine (we need to be familiar with the definition and  function of each type of cell adhesion and the intracellular adapters that  function with them) (Table 20-3 is a good reference for the subsequent  



1. Anchoring Junctions

a. Adherens junctions

i. Adhesion type – cell-cell

ii. Principal CAMs or Adhesion Receptors – Cadherins

1. Cadherins mediate cell-cell adhesions in  

adherens junctions and desmosomes

a. Transmembrane glycoproteins that  

mediate Ca2+ dependent cell-cell  


b. Mediate cell-cell adhesions through  

homophilic interactions

BSC 300-001 Week 12 Notes

i. Can only interact with  

cadherins of the same  


c. Homophilic interactions that are  

Ca2+ dependent:

i. Intestinal endocrine cells do  

not express cadherins and do  

not aggregate into sheets

ii. Same cells expressing  

cadherin transgene:

iii. Ca2+ treatment, cells adhere  

into a sheet

iv. Without Ca2+ cells do not  


2. More than 100 protein types – but four  

general families

a. E-cadherin (epithelial)

i. E-cadherin mediates adhesive  

connections in cultured  

MDCK epithelial cells

ii. E-cadherin clusters mediate  

initial attachment of cells into  


iii. Experiment:

iv. Time course study of  

transgenic fluorescent E

cadherin in cultured kidney  


v. Fluorescent E-cadherin  

distribution changes over  

time to localize only the cell  


b. N-cadherin (neural)

c. P-cadherin (placental)

d. Desmosome cadherins

iii. Cytoskeletal Attachment – Actin filaments

iv. Intracellular adapters – catenins, vinculin

v. Function – shape, tension, signaling, force  


vi. Protein constituents of typical adherens junctions 1. Adherens junctions – a cell junction basal  

to tight junctions, whose cytoplasmic face is  

linked to the actin cytoskeleton. They  

contribute to epithelial cell shape, changes  

in cell shape and establish tensile strength in  

the epithelia

BSC 300-001 Week 12 Notes

a. E-cadherin: principle CAM of  

adherens junctions

i. Exoplasmic domains  

clustered by cis and trans  

interactions at adherens  

junctions on adjacent cells

b. Cytosolic domains

i. Bind directly/indirectly  

through adapter proteins  

(principally catenins) to actin  

filaments in the cytoskeleton

ii. Such adapter proteins can  

transduce signals to cell  

interior – Beta-catenin, for  

example can function as  

transcriptional activator

b. Desmosomes

i. Adhesion type – cell-cell

ii. Principal CAMs or Adhesion Receptors –

Desmosomal Cadherins

iii. Cytoskeletal Attachment – Intermediate filaments iv. Intracellular adapters – Plakoglobin, plakophilins,  desmoplakins

v. Function – Strength, durability, signaling

vi. Desmosomes utilize two specialized cadherins – desmoglein and desmocollin

1. Strong cell-to-cell adhesion types found in  

tissue that experience lots of mechanical  


2. Randomly scattered throughout lateral  

surfaces of epithelial cells

3. Cytosolic domains bind intracellular adapter  proteins (plakoglobin and plakophilins)  

which bind a third adapter, desmoplakin

a. Collectively these adapters create the  

thick cytoplasmic plaques  

characteristic of desmosomes – do  

not need to memorize plakoglobin  

and plakophilins

b. Desmoplakins directly bind  

intermediate filaments which radiate  

into the cytoplasm and provide  

strength to these cellular rivets

c. Hemidesmosomes

i. Adhesion type – cell-matrix

BSC 300-001 Week 12 Notes

ii. Principal CAMs or Adhesion Receptors – Integrin  (alpha 6 beta 4)

1. Integrins mediate cell-ECM adhesions

a. Epithelial cells are anchored to the  

basal lamina via integrin proteins,  

both within and outside of anchoring  

complex called hemidesmosomes

b. Integrins function both as adhesion  

receptors as well as CAMs

c. 24 known alpha/beta heterodimers  

promote broad ligand binding  


d. 3 principle binding domains:

i. RDG domain – binds  

proteins containing Arg-Gly

Asp sequence

ii. Laminin binding domain

iii. The I-domain – collagen  


e. Relatively high Kd (low binding  

affinity), but the hundreds of  

thousands of interactions in a single  

cell promotes robust attachment

f. Diversity of integrin dimers and their  

associations allow them to  

participate in a variety of cellular  

processes ranging from  

inflammatory response to cell  


g. Like cadherins, integrin dimers of  

focal, fibrillary and 3D adhesions are  

linked via adapter to the actin  

cytoskeleton – where they maintain  

or modify cell shape, participate in  

cell signaling and movement

h. Integrins of hemidesmosomes  

interact with intermediate filaments –

where they maintain cell shape,  

rigidity, and participate in signal  


i. In addition to binding the ECM,  

integrins are also bound by a diverse  

ligand pool that can mediate signal  

transduction via altered  

conformation of the dimer and  

signaling through various adapter

BSC 300-001 Week 12 Notes

proteins. Also, intracellular protein  

interactions can alter integrin  

conformations in such a way as to  

promote inside-out signaling and the  

interaction with other cells or the  


iii. Cytoskeletal Attachment – intermediate filaments iv. Intracellular adapters – plectin, dystonin/BPAG1

v. Function – shape, rigidity, signaling

d. Focal, fibrillar, and 3D adhesions

i. Adhesion type – cell-matrix

ii. Principal CAMs or Adhesion Receptors – Integrins iii. Cytoskeletal Attachment – Actin filaments

iv. Intracellular adapters – talin, kindlin, paxillin,  

vinculin kinase

v. Function – shape, signaling, force transmission, cell  movement

2. Tight Junctions

a. Adhesion type – cell-cell

b. Principal CAMs or Adhesion Receptors – Occulin,  claudins, JAMs

c. Cytoskeletal Attachment – Actin filaments

d. Intracellular adapters – ZO – 1,2,3, PAR3, cingulin e. Function – Controlling solute flow, signaling

f. Tight junctions seal off body cavities and restrict diffusion  on membrane components

i. Tight junctions – specialized contacts at the apical most regions of the lateral surface of epithelial cells

1. Seals off body cavities

2. Located at apical end of the junctional  

complex between adjacent cells

3. Serve as barrier to free diffusion of water  

and solutes from the extracellular  

compartment between cells

4. Some are permeable to specific ions or  


g. Protein components of tight junctions

i. Primarily composed of the two TM proteins  

occludins and claudins

ii. Link tight junctions to the cytoskeleton via various  adapter proteins possessing PDZ domains

iii. Also contain Junction Associated Molecules  

(JAMs) that promote homophilic adhesion via Ig  


iv. Transport across epithelia requires either  

transcellular pathway requiring membrane

BSC 300-001 Week 12 Notes

transporters (example glucose absorption in the  

intestine) or paracellular transport – small  

molecules and ions that are selectively permeable to  

cell-type specific tight junctions

3. Gap Junctions

a. Adhesion type – cell-cell

b. Principal CAMs or Adhesion Receptors – Connexins,  innexins, pannexins

c. Cytoskeletal Attachment – Via adapters to other junctions d. Intracellular adapters – ZO- 1,2,3

e. Function – Communication, small-molecule transport  between cells

f. Gap Junctions: Mediating Intracellular Communication i. Gap Junctions – regulated channels between animal  cells for intracellular communication

1. Composed entirely of the membrane protein  

connexin – 21 different human connexin  

genes – different sets of connexins are  

expressed in different cell types

a. Two connexons join together to  

create a gap junction

2. Connexins are organized into a barrel  

shaped complex called connexon – 6  

individual connexin proteins

3. Allows small molecules to pass directly  

between cytosols of adjacent cells

a. Remain open most of the time but  

some can be gated

ii. Compatibility differences between connexins either  promote or prevent communication between  

different cells

iii. Gap-junction intercellular communication (GJIC)  allows the passage of low-weight molecules like  

ions, hormones, and second messengers

iv. Many types of neurons are connected by GAP  

junctions to aid in increasing the speed of neural  


v. Gap junctions can allow integration of activities of  individual cells of a tissue into a functional unit

vi. Also promote metabolic coupling, in which a cell  transfers nutrients or intermediate metabolites to a  

neighboring cell that is incapable of producing them  


1. Sharing

vii. Open-closed state can be regulated by a number of  factors

BSC 300-001 Week 12 Notes

1. Eg post-translational modification  


2. As well as pH, Ca2+ concentration,  

membrane potential, and intercellular  

potential between adjacent interconnected  

cells (“voltage gating”)

4. Plasmodesmata

a. Adhesion type – cell-cell

b. Principal CAMs or Adhesion Receptors – Undefined

c. Cytoskeletal Attachment – Actin filaments

d. Intracellular adapters – NET1A

e. Function – Communication, molecule transport between  


f. Plasmodesmata are cytoplasmic channels passing through  

cell walls of adjacent plant cells

i. Lined by plasma membrane

ii. Contain a central structure, the desmotubule, a  

membranous extension of the ER

iii. Serve as sites of cell-cell communication

iv. Many types of molecules pass cell to cell through  

plasmodesmata – including some transcription  

factors, nucleic acid/protein complexes, metabolic  

products, and plant viruses

g. Only plant-specific structure we need to know

II. Lecture 21 – Continuation of Chapter 20

a. The extracellular Matrix I: The Basal Lamina

i. The Extracellular Space

1. Extracellular matrix (ECM) – an organized network beyond the  

plasma membrane that plays key regulatory roles in determining  

cell shape and activities. Extensively involved in cell signaling as  

it regulates diffusion of ligands and their interactions with  


a. Unique composition of fibrous proteins and proteoglycans  

are secreted by and surround all cell types and this ECM is  

involved in regulating diverse cellular physiology including  

cell-cell contact, proliferation, migration, cell signaling,  

gene expression, and differentiation

b. Organizes cells into tissues and coordinates cellular  

functions by activating transduction pathways that control  

cell growth, division, and gene expression

c. Controls rates of signaling molecule diffusion and serves as  

a repository of inactive or inaccessible signals that are  

released when the ECM is disassembled or digested by  


BSC 300-001 Week 12 Notes

d. The collective proteins that compose and modify the ECM  is known as the matrisome – over 1000 human genes  

encode the matrisome

e. Adhesion receptors bind to three types of molecules  

abundant in the ECM of all tissues:

i. Proteoglycans – a group of glycoproteins that  

cushion cells and bind diverse extracellular  


ii. Collagen – fibers which provide structural integrity  

and mechanical strength and resilience

iii. Multi-adhesive matrix proteins – soluble proteins  

that bind and cross-link adhesion receptors and  

other ECM components

ii. Basal lamina – specialized ECM under the basal surface of epithelia 1. Maintains cell attachment

2. Provide signals for cell survival (survival of most epithelial cells is  anchorage dependent)

3. Separate distinct tissues within an organ

4. Serves as substratum for cell migration (critical during  


5. Forms a barrier to macromolecules (for example the blood-brain  barrier)

6. Major protein components of the basal lamina

a. Four ubiquitous protein types, synthesized by the cells  

anchored to the basal lamina. Basal lamina of each tissue  

composed of different combinations of these four principle  building blocks

i. Each protein is composed of multiple distinct  


1. Proteoglycans are interspersed between all 4  


ii. Type IV collagen – trimeric molecules with rod

like and globular domains that form a 2D network  

onto which the other proteins are attached

1. All collagens, regardless of their type, are  

homo- or heterotrimers, composed of a  

combination of the collagen alpha chains  

encoded b 43 human genes

2. These three subunits coil together into a  

collagenous triple helix

3. 28 types of human collagen participate in  

the formation of distinct ECMs in various  


a. Do not memorize the 28 types –

recognize the 5 different divisions  

(fibrillar collagens, fibril-associated

BSC 300-001 Week 12 Notes

collagens, sheet-forming and  

anchoring collagens, transmembrane  

collagens, and host defense  


4. The collagen triple helix

a. Fibrillar collagens

i. Trimeric protein composed of  

three alpha chain  

polypeptides – can be  

homotrimer or heterotrimer

b. Collagen structure – longitudinal  


i. Repeating Gly-X-Y amino  

acids of collagen alpha chains  

(X-Y can be any protein, but  

most often proline and  


ii. Each chain is twisted into a  

left-handed helix – three  

chains wrap around one  

another in a right-handed  

triple helix held together by  


iii. Triple-helical structure with  

left-handed twist of the  

individual collagen alpha  


c. Collagen structure – cross-sectional


i. Glycine proteon side chains  

project into center of the  

triple helix

d. The triple helix forms because of the  

abundance of three amino acids:  

glycine, proline and the modified  

proline hydroxyproline

i. Proline is unique because it  

folds onto itself

ii. Glycine is unique because it  

only has H as its side chain

e. T-alpha chains are composed of  

many repeated triple residues with  

the sequence Gly-X-Y: where:

i. G = glycine

ii. X and Y can be any amino  

acid, but predominantly

BSC 300-001 Week 12 Notes

X=proline and  

Y=hydroxyproline (OH  

attached to it) whose unique  

cyclical structures promote  

the curvature of the strands

iii. The small glycine (remember  

it does not have a side chain,  

but instead an H) allows the  

crowded arrangement of the  

triple helix

iv. The three strands are held  

together by H-bonds

f. These triple helical structures are  

considered a collagen monomer, but  

assemble into the larger, more  

complex 28 types of collagen

i. Diverse adhesion receptors  

and signal transduction  

receptors bind these distinct  

types of collagen

g. Unique properties and affinities of  

each type of collagen are due mainly  

to differences in:

i. The number and length of the  

triple-helical monomers

ii. Segments that flank or  

interrupt the helical segments  

and fold into other 3D  


iii. Covalent modifications to the  

alpha chains, principally:  

glycosylation, hydroxylation,  

oxidation, and cross-linking

5. Structure and assembly of type IV collagen

a. For example, type IV collagen forms  

the structural sheets of the basal  


b. The principle units of mammalian  

type IV collagen are called IV alpha  

chains – there are 6 homologous  

proteins that assemble into 3  

different heterotrimers – each with  

distinct properties

c. All type IV collagen forms a 400-

nm-long triple helix that is  

interrupted by about 24 non-helical

BSC 300-001 Week 12 Notes

segments, that provide flexibility to  

the helix

d. Globular domains at the termini  

interact to form the branching,  

irregular 2D lattice of the basal  


iii. Laminin – multi-adhesive cross-shaped proteins  that underlies the collagen network and binds  

integrins and other adhesion receptors

1. Heterotrimeric multi-adhesive matrix  

protein that cross-links basal lamina proteins

2. 16 vertebrate laminin isoforms from 5 alpha,  3 beta, 3 y chains – each with tissue and  

developmental stage expression specificity

3. Coiled-coil region – these peptides  

covalently linked by disulfide bonds

4. Globular domains at N-termini of each  

subunit bind to one another mediating self


5. Five C-terminal globular LG domains of the  alpha subunit mediate Ca2+ dependent  

binding to cell0surface laminin receptors,  

including integrins, sulfated glycolipids,  

syndecan, and dystroglycan (do not worry  

about these names that they bind to – just  

know that it is the most diverse)

iv. Nidogen and perlecan molecules crosslink basal  lamina networks

1. The proteoglycan perlecan cross-links  

components of the basal lamina and cell

surface receptors

a. Perlecan is the major secreted  

proteoglycan in basal lamina

b. Like all proteoglycans, it contains a  

large filament-like core protein to  

which many oligosaccharides are  


c. The protein core has multiple copies  

of three protein interaction domains  

(LG, EGF, and IG domains)

d. And is covalently linked to three  

types of oligosaccharides: N-linked,  

O-linked, and the  

glycosaminoglycan (GAGs) called  

heparan sulfate

BSC 300-001 Week 12 Notes

e. It is the presence of GAGs that  

define proteoglycans as a specific  

category of glycoproteins

f. The multiple protein interaction  

domains and three types of  

oligosaccharides give perlecan a  

wide-range of potential interacting  

partners including cell surface  

receptors, ECM components and  

growth factors

i. It therefore serves as the  

principle cross-linking agent  

of basal laminae as well as a  

repository and conduit for  

cell-cell signaling

b. The Extracellular Matrix II: Connective Tissue

i. Diversity of collagens

1. Most collagen of the body is fibrillary and located primarily in  connective tissue (90%, Types I, II, and III)

2. These proteins are produced by fibroblasts of connective tissue and  secreted into the ECM

3. Less abundant collagen includes:

a. Fibril-associated collagens that cross-link other collagens

b. Sheet forming and anchoring collagens that generate a 2D  

network in basal laminae

c. Transmembrane collagen which functions as adhesion  


d. Host defense collagens which help the immune system  

identify and eliminate pathogens

ii. Synthesis and secretion of collagen

1. Because of their large size when mature, ECM collagens cannot  assemble as fibrils intracellularly. Rather, after translation into the  ER they contain N- and C- termini propeptides that inhibit  


2. Such immature collagen proteins are called pro-alpha chains 3. In the ER:

a. The pro-alpha chains receive N-linked glycosylation on  

their C-terminal propeptide

b. Propeptides associate to form trimers and are covalently  

linked by di-sulfide bonds

c. Prolines and hydroxyprolines of G-X-Y are hydroxylated  

and some prolines undergo cis to trans isomerization

d. Some hydroxylysines are covalently attached to galactose

e. These modifications facilitate formation of the triple helix,  

which is stabilized by a chaperone protein to prevent  

premature aggregation

BSC 300-001 Week 12 Notes

4. In the Golgi

a. Lateral association via H-bonding occurs and the chains are  secreted from the cell

b. Procollagen peptidases remove N and C termini

i. Exposes interaction domains to allow different fiber  

aggregates to assemble with each other

c. Trimers assemble into staggered arrays forming fibrils and  are covalently cross-linked via their exposed termini

d. Within these termini lysine and hydroxylysine residues are  oxidized to aldehydes that form the covalent cross-links  

that hold the trimers together

i. Forms very long and stable structures

5. Human health relevance – the post-translational modifications to  the alpha chains are essential for forming the mature collagen  fibers

6. For example, ascorbic acid (vitamin C) is a necessary cofactor for  the hydroxylase that add hydroxyl groups to prolines and lysines a. In the absence of vitamin C hydroxylation of the monomers  does not take place and collagen fibers disassemble

b. The ancient mariner disease Scurvy resulted from lack of  vitamin C in sailor’s diets causing degeneration of  

connective tissue

i. No vitamin C = no collagen formation = lots of  

nasty stuff (loss of teeth, etc)

ii. This is why British soldiers are called Limeys

iii. Type I and II collagen

1. Type I collagen form long fibers with great tensile strength a. For example, tendons are primarily composed of type I  

aligned in long parallel fibers capable of stretching and not  


b. Other collagens (type VI) and proteoglycans associate with  these fibers and non-covalently link them together into  

strong collagen fibers

c. Type I is also used in as reinforcing rods in bones

2. Type II does not form fibers, but instead generates more mesh-like  networks, like that found in cartilage

a. Here, other accessory collagens (type IX) cross-link type II  collagen to matrix proteoglycans

3. Diverse human health implications:

a. Osteogenesis imperfecta: aka brittle bone disease. Can  result from a single point mutation in the alpha 1 genes that  encode osteogenic collagen. A single mutation to almost  

any of the encoded glycines promotes poorly formed and  

unstable triple helices. Autosomal dominant recessive –

meaning only copy (allele) of the gene must be mutated for  

the disease to manifest

BSC 300-001 Week 12 Notes

b. Dominant or recessive muscular dystrophy: muscle  

weakness, respiratory insufficiency, muscle wasting and  

joint abnormalities

c. A variety of skin and vascular diseases

iv. Proteoglycans and their constituent GAGs play diverse roles in the ECM 1. As we saw with perlecan, proteoglycans are secreted glycoproteins  with a core protein covalently cross-linked to specialized  

polysaccharide chains called glycosaminoglycans (GAGs)

a. GAGs are long, linear polymers of specific repeated  

disaccharides: one sugar is usually a uronic acid or  

galactose, while the other is N-acetylglucosamine or N


i. One or both sugars have at least one anionic group  

(carboxylate or sulfate), so each GAG bears many  

negative charges

v. Gels of polysaccharides resist compression

1. Four major GAGs:

a. Hyaluronan

b. Chondroitin sulfate

c. Heparin/Heparan sulfate

d. Keratan sulfate

2. All but hyaluronan are sulfated, and all but hyaluronan are the  major GAGs that occur naturally as a part of proteoglycans

3. The arrangement of sugars in GAG chains and their various  covalent modifications determine their function and that of the  proteoglycan in which they are found

a. For example, certain GAGs of heparin sulfate  

proteoglycans control the binding of specific growth factors  or morphogens to their receptor or the activities of protein  

involved in blood clotting

4. Typically, many GAG chains are attached to a core protein, which  may then be linked end to end to form a huge molecule

5. GAGs can assemble as complex chains

6. The negatively charged molecules attract cations like Na+ which  draws water into the matrix forming a gel-like solution that  

provides cushioning to the embedded cells

vi. The repeating disaccharides of glycosaminoglycans (GAGs) 1. Syndecans are cell-surface proteoglycans expressed in epithelial  and non-epithelial cells that bind collagen and multi-adhesive  matrix proteins

a. Link ECM to cytoskeleton

2. As with adhesion receptors, the cytosolic portion of syndecan  interacts with the cytoskeleton and intracellular regulatory proteins 3. For example, in the hypothalamic region of the brain that controls  eating behavior in response to food depravation and regulates  binding of anti-satiety peptides to their receptors

BSC 300-001 Week 12 Notes

a. Anti-satiety peptides tell our brain that we are no longer  


b. People who cannot seem to control their appetite may have  an issue with anti-satiety peptides

c. Mice engineered to over-express syndecan1 never  

experience fullness after eating and continue to overeat,  

becoming obese

vii. Hyaluronan, aka hyaluronic acid (found in many lotions because it is very  large sized)

1. The only non-sulfated GAG is a major component of the ECM  surrounding migrating and proliferating cells, especially during  embryogenesis

2. Also forms the backbone on large complex proteoglycan  aggregates found in many ECMs, particularly cartilage

3. It imparts stiffness and resilience as well as lubricating properties  to many types of connective tissues and joints

4. Typical hyaluronan molecule has 10,000 repeats that bend and  twist into many configurations

5. Because of the large number of anionic residues on hyaluronan it  attracts a large amount of water and swells into a hydrated sphere  that cushions cells and maintains osmotic pressure – preventing  cell swelling in hypotonic solutions

6. The predominant proteoglycan in cartilage, called aggrecan,  assembles. With hyaluronan into a large complex aggregate that gives cartilage its gel-like properties and resistance to deformation

7. A core hyaluronan molecule is bound by hundreds of aggrecan  proteoglycans to generate one of the largest macromolecules  known – about the size of a bacterial cell

viii. Organization of fibronectin and its binding to integrin

1. Fibronectin (Fn) is an abundant multi-adhesive domain matrix  protein found in all vertebrates

2. About 20 isoforms derived from alternative splicing of a single  transcript

3. Essential for cell migration and differentiation during  

embryogenesis, as well as wound healing through promotion of  blood clotting

4. Dimers of two polypeptides linked by disulfide bridges at their C termini

5. Each protein composed of several functional regions each with  distinct binding affinities

6. Each region contains multiple copies of three types of domains: Fn  I, II, and III repeats

a. Only be familiar with Type III

7. Type III repeats contain the tri-peptide RGD motif (Arg-Gly-Asp)  which is recognized by integrins – other integrin binding proteins  likewise possess RGD motifs

BSC 300-001 Week 12 Notes

ix. Integrins mediate linkage between fibronectin in the ECM and the  cytoskeleton

1. Inside out signaling: to promote robust adhesion to the cell’s  substrate, binding of cell-surface integrins to fibronectin in the  

ECM induces cytoskeletal dependent movement of integrin in the  plasma membrane

a. This generates force on fibronectin proteins, causing them  

to unfold

b. Activated fibronectin molecules aggregate into fibrils that  

mature into stable matrix components via covalent cross


2. Matrix metalloproteases remodel and degrade the ECM

a. Processes like cell migration, wound healing, immune  

response and proliferation require not only an ECM but  

also its remodeling or degradation

b. This is driven by cell synthesized zinc-dependent proteases  

– the principle class being matrix metalloproteases – 23  

different ones in humans

i. Do not memorize the names of any of them

ii. Cancer hijacks the expression of MMPs, which  

promotes metastasis

c. Adhesive Interactions in Motile and Nonmotile Cells

i. Interactions of Cells with Extracellular Materials

1. Focal Adhesions (all we really need to know about integrins at this  point is that their conformation can promote inside-out and  

outside-in signaling)

a. Focal adhesions – docking sites where cells adhere to their  

substratum and send signals to the cell interior

i. Cytoplasmic domains of integrins contain binding  

sites for a variety of cytoplasmic proteins

ii. These proteins relay both mechanical and chemical  

information to the cell interior altering cytoskeleton  

dynamic and gene expression

iii. Can be rapidly assembled/disassembled in order to  

promote cellular migration

ii. Regulation of integrin-mediated adhesion and signaling controls cell  movement

1. Integrin binding: integrins exist in low affinity, inactive  

conformations and active high affinity states

2. In the inactive state the dimer subunits are closely positioned and  bent, while in the active state, the dimer is extended and its ligand  binding domains are exposed with higher affinity and its  

cytoplasmic tails are separated allowing binding to adapter proteins 3. This dual conformational change explains the ability of integrins to  participate in both outside-in and inside-out signaling

BSC 300-001 Week 12 Notes

4. Binding of extracellular ligands or ECM components alters  cytoplasmic conformation and accessibility to adapter proteins like  talin

5. Similarly, binding by adapter proteins like talin, causes the  extracellular domains to extend and allow interaction with the  ECM

BSC 300-001 Week 13 Notes

I. Lecture 22 – Stem Cells, Cell Asymmetry, and Cell Death (Ch 21) a. Early Mammalian Development

i. Overview of the birth, lineage, and death of cells

1. Not all cell divisions are symmetric – meaning both daughter cells  receive the same amount of cytoplasm and the various proteins in  

the parental cytoplasm

2. Examples of symmetric division include yeast, fungi, and most  

protists. Similarly, mature liver hepatocytes divide symmetrically,  

producing both genetically as well as functionally identical  

daughter cells

a. Don’t have a lot of differentiation

3. But, while every cell in a multicellular organism has the exact  

same genotype (with a couple of exceptions like immune cells as  

we’ll see in Ch 23) there are many unique cell types, each  

expressing only a portion of the genes within their genome

a. Cells in our body have all the same genes but don’t express  

the same ones

4. How does cell diversity arise in multicellular organisms?

a. One mechanism that contributes to cell diversity is  

asymmetric cell division, in which daughter cells receive  

different amounts or components of the cytoplasmic  


i. Such daughter cells may differ in size, shape,  

protein composition – all of which may cause them  

to function/behave differently and importantly  

transcribe different sets of genes, which lead them,  

and their descendants, among very different fates

ii. Asymmetric division during animal development is  

one of the fundamental mechanisms used to  

establish cell lineages – a line of descent from a  

founder cell that acquired a difference in gene  

expression compared to a sibling cell

iii. Separate cell lineages become more and more  

restricted in developmental potential as they  

proliferate and acquire unique combinations of gene  


1. Ultimately such cells exit the cell cycle as  

fully differentiated cells

5. Metazoan development begins with the union of two gametes: ova and sperm (haploid) which combine their haploid genomes to  

generate the first cell of a metazoan organism – the zygote


a. For some organisms, the first cell divisio of the zygote is  

asymmetric – producing daughter cells with unique protein  

complements that direct differential gene exression, and  

those whose developmental fate is therefore restructured  

very early

BSC 300-001 Week 13 Notes

b. In other organisms, like humans, early cell divisions  

produce daughter cells that are totipotent – capable of  

giving rise to any cell lineage

i. In humans, however, at the 16 cell-stage – known as  

blastula state, some cells become committed to  

generating the extraembryonic tissues population  

(trophoblasts), while a smaller cell population –

the inner cell mass – in the interior of the embryo  

remains pluripotent – capable of generating all of  

the lineages of the embryo body

6. Major Stages During Mammalian Embryo Development

a. Blastula have a fluid-filled interior called the blastocoel and  

the ICM

i. Both ICM and TE cells are stem cells -they can  

proliferate indefinitely in cell culture and can be  

induced to adopt any of the cell fates that these cell  

types would generate during development, provided  

they are cultured with the appropriate growth and  

differential factors to push them down that ppath

b. Embryonic Stem Cells and Induced Pluripotent Stem Cells

i. Genomic equivalence and animal cloning

1. In 1996 Ian Wilmut at the University of Edinburgh proved that  mammalian adult cells possess genomic equivalence – all somatic  cells of an adult possess the same genetic material

a. They are difference because each cell type expresses a  

different subset of gees

b. Wilmut showed that the nucleus of a differentiated adult ell  

could be transplanted into an enucleated ovum and be  

induced to begin development and give rise to a genetic  

clone of the animal that donated the nucleus. This  

technique is called somatic cell nuclear transfer

i. Showed that all cells in the adult body have the  

exact same genetic material in them

c. The first mammal that was successfully cloned was a sheep  

named Dolly. During SCNT the donor genome undergoes  

reprogramming and most of the epigenetic code from the  

donor genome is erased – wiping the slate clean of the  

epigenetic programming of the differentiated donor cell

i. Does not erase all programming, so they get  

different diseases that manifested in their life much  

faster than usual

d. Dolly and subsequent clones generated by SCNT  

experienced diverse medical problem that stemmed from  

the fact that not all epigenetic codes were cleared

i. Epigenetic codes are the various histone and DNA  

covalent modifications like acetylation and  

methylation that accompany genomic differentiation

ii. Genomes display genetic totipotency

BSC 300-001 Week 13 Notes

1. Somatic cell nuclear transfer for a while held the promise of  incredible scientific investigation and therapeutic advancement a. Ex: this technique can be used to generate an early embryo  clone of any individual, and from the blastula stage embryo  harvest embryonic stem cells for research or therapy

b. However, this technique still had great ethical challenges.  Embryos must be destroyed to harvest the embryonic stem  


iii. Transcriptional network regulating pluripotency of ES cells 1. Shinya Yamanaka shared the 2012 Nobel Prize I nMedicine of  Physiology for his discovery that a small number of transcription  factors are exressed in ICM cells and maintain them as stem cells 2. Three master transcription factors – Oct4, Sox2, and Nanog a. Oct4 and Nanog – expressed only in ES cells

i. Sox2 is also expressed in multipotent neural stem  

cells that give arise exclusively to neuronal and glial  

cell types

b. These transcription factors bind one anothers’ promoters,  establishing positive autoregulatory loops

c. They also regulate expression of many other genes, which: i. Encode proteins important for the proliferation and  

self-renewal of ES cells

ii. Repress genes and micro-RNAs that are essential  

for cell differentiation

3. Amazingly, Yamanaka found that forced expression of these  transcription factors in differentiated cells reprograms them into  cells virtually identical to embryonic stem cells. Such  

reprogrammed cells are now known as induced pluripotent stem  cells (iPSCS), and obviate the need to create an embryo

a. Cultured differentiated cells from any individual can be  

reprogrammed as iPSCs

b. Work subsequently to Yamanaka showed that one of the  critical jobs of Oct4 is to remove epigenetic silencing of  

genes to establish stem-cell fate

i. Doing this, they worked out mechanisms for erasing  

the genetic code and created embryonic stem cell

4. Two potential medical applications of iPSCs

a. iPSCs can be induced to develop and differentiate as a  

mature cell type

b. Mature cells from a patient can be harvested and  

transformed into patient-specific iPSCs

c. Ways that medicine is advancing new techniques to cure  diseases using embryonic cells

i. New technology (like CRISPR-Cas9) could be used  

to repair genetic mutations

1. Repaired iPSCs would then undergo in vitro  

differentiation before transplant back into  

the patient. Importantly, because this

BSC 300-001 Week 13 Notes

technique uses patient-specific cells there is  

no fear of host-rejection  

ii. Alternatively, differentiated patient-specific cell  

types affected by a specific genetic disorder could  

be used to screen for drugs that reverse or treat the  

specific disease

d. Such thereapeutic approaches are close for diseases like  

ALS, Cystic fibrosis, diabetes, etc

5. Production of normal insulin-secreting Beta islet cells from human  iPS or ES cells

a. What is required for such approaches, however, is knowing  

the appropriate combination of growth factors to induce  

iPSCs to specific developmental fates

i. Doug Melton, at Harvard, for example has  

researched the discovered the correct sequence and  

timing of growth factors required to transform  

iPSCs into pancreatic insulin secreting Beta cells

ii. Mice pancreas transplanted with human iPSC  

derived B cells repond to increase blood sugar  

levels and appropriately promote its uptake and  


6. Multipotent stem cells generate a continuous supply of terminally  differentiated cells

a. Terminally differentiated cells do not divide, so where do  

replacement cells come from?

i. Most tissue types contain populations of  

underdifferentiated self-renewing cells known as  

multipotent stem cells (aka somatic or adult stem  


1. These are distinct from embryonic  

pluripotent ste cells in that they are not  

pluripotent. Their developmental potential is  

restricted by expression of tissue-specific  

transcriptional regulators

c. Stem Cells and Niches in Multicellular Organisms

i. The pathway from stem cells to lineage-restricted progenitors to  differentiated cells

1. Multipotent somatic stem cells have three key properties:

a. They can give rise to multiple types of differentiated cells

b. They are undifferentiated  

c. They increase in number during embryogenesis and then  

remain relatively constant over an individual’s life. In this  

sense they are immortal

i. Give rise to a set number that will reside for the rest  

of our lives

2. MP stem cells often divide asymmetrically – producing daughters  that remain as undifferentiated stem cells and non-stem cell

BSC 300-001 Week 13 Notes

daughters that undergo multiple rounds of division as transit  amplifying cells before differentiating as mature cells

ii. Patterns of stem-cell differentiation

1. However, some stem cell populations responod to hormonal  signals that can alter this pattern of division and produce identical  stem cell daughters in order to increase stem cell number, or  produce identical transit amplifying daughter cells to increase the  pool of proliferating cells that will differentiate

iii. The stem cell niche

1. Stem cells require intrinsic regulatory signals, like the expression  of specific transcription factors to maintain their fate

2. Additionally, stem cells require a specialized microenvironment  known as the stem cell niche, that possesses extrinsic regulatory  signals like hormones and growth factors

a. Stem cell niche provides the appropriate source of signals  to maintain stem cells in their proliferative and self

renewing state

b. As well as providing the physical context for signals that  will be received by proliferating precursor cells to receive  

the signals that will promote their differentiation

3. Intestinal stem cells and their niche

a. For example, intestinal epithelial cells turn over every 3-5  days

b. The intestinal epithelium generates microvilli – long  

protrusions into the lumen of the gut in order to increase  

surface area

i. At the base of each microvillus lies a specialized  

compartment known as the intestinal crypt: the  

stem cell niche for intestinal epithelial cells

ii. Within the crypt lie the intestinal stem cells which  

express the GPCR LGR5

iii. And a second population of cells called Paneth  


1. Paneth cells secrete many anti-microbial  

peptides, and therefore play an important  

role in immune function

2. They also express a Wnt ligand which  

stimulates neighboring stem cells to  

maintain their stem cell fate

3. Paneth cells secrete the ligand R-spondin  

which binds the LGR receptor and  

transduces a signal the potentiates Wnt  


4. Therefore, Paneth cells – which are progeny  

of intestinal stem cells themselves –

constitute much – if not all – of the niche for  

intestinal stem cell maintenance

BSC 300-001 Week 13 Notes

c. As transit amplifying cells move up and away from the  

stem cell population they are no longer exposed to the Wnt  

and R-spondin signals

i. They proliferate as transit amplifying cells and  

terminally differentiate as secretory and absorptive  

cells within the villus

ii. Other precursor cells migrate downward and adopt  

the Paneth cell fate

d. Mechanisms of Cell Polarity and Asymmetric Cell Division

i. General features of cell polarity and asymmetric cell division

1. Three core principles of cell polarity:

a. Cells have intrinsic cell polarity program that allows them  

to polarize in the absence of external cues

b. The intrinsic program can be directed by internal and  

external cues

c. Polarity of individual cells is often maintained by  

intracellular mutually antagonistic complexes

2. Although the mechanism of location varies across the Eukarya, the  small G protein Cdc42 plays a critical role in establishing cell  


a. Once localized to discrete cell locations Cdc42 polarizes  

the actin cytoskeleton and provides tracks for the  

asymmetric distribution of secretory granules and  

cytoplasmic proteins that promote asymmetric localization  

and distribution of cytoplasm and protein within cells as  

well as between daughter cells during division

3. Cell polarity determinants – mRNAs, proteins, and lipids:

a. Asymmetrically localized in mother cell

b. Mitotic spindle – positioned so that polarity determinants  

are segregated differentially into daughter cells during cell  


4. Cdc42 – master regulator of cell polarity

a. Intrinsic polarity program – establishes asymmetry in  

cells before asymmetric cell division that yields cells with  

different fates

b. Cdc42-GTP is required to establish cell polarity

i. Cdc42-GTP locally activates formins

ii. Recall, formins nucleate and elongate actin  

filaments with (+) ends oriented toward Cdc42-GTP  

at membrane

iii. Myosin V motor transports secretory vesicles  

toward actin filament (+) ends

5. Establishing polarity in vertebrate epithelial cells

a. Vertebrate polarized epithelial cells – cues from adjacent  

cells and the extracellular matrix orient polarization axis by  

regulating location of three protein complexes

b. Interactions between adherens and tight junctions signal  

epithelial cells to recruit the Par complex to these sites

BSC 300-001 Week 13 Notes

(Par stands for partitioning defective). The Par complex  

includes regulatory proteins and the Cdc42 G protein

c. Apical to the Par complex another complex called Crumbs  

is assembled, while basally the Scribble complex is  


d. Though not completely understood, this arrangement of  

protein complexes reorganizes the cytoskeleton:

i. Actin filaments – distinct arrangements associated  

with the apical and basolateral membranes

ii. Microtubues – not all associated with a centrosomes

iii. Intermediate filaments – associated with basolateral  

cell-cell adhesion junctions (desmosomes

hemidesmosomes) and ends of microvilli actin  


e. This arrangement also polarizes membrane traffic to direct  

transport vesicles to their appropriate membranes

e. Cell Death and Its Regulation

i. Apoptosis (Programmed Cell Death)

1. Apoptosis is an ordered and molecularly activated process  

involving killing: molecules that activate the process, Destruction: proteins that destroy the cell from the inside, promoting cell  

shrinkage, loss of adhesion to other cells, degradation of  

chromatin, and Engulfement: phagocytosis by scavenger white  

blood cells

a. Plays a key role during animal development as well as to  

remove damaged cells from tissue

b. Apoptotic changes are activated by proteolytic enzymes  

called caspases, which target:

i. Protein kinases, some of which cause detachment of  


ii. Lamins, which line the nuclear envelope

iii. Proteins of the cytoskeleton

iv. Caspace activated DNase (CAD) which degrade  


c. Very deeply conserved process with genetic pathways  

shared by all Metazoa

2. Death by apoptosis

a. Cells undergoing necrosis (uncontrolled cell death) exhibit  

very different morphological changes:

i. Swell and burst

ii. Release intracellular contents that can damage  

surrounding cells and cause inflammation

3. Apoptosis during development

a. Apoptosis may be activated due to a lack of an external  

signal (trophic factor) that is necessary for survival. In its  

absence, cells essentially commit suicide

i. Alternatively, other signals may signal the cell to  

initiate apoptosis – essentially cellular murder

BSC 300-001 Week 13 Notes

b. During embryogenesis apoptosis sculpts tissue and organs  by the controlled removal of specific cells

i. For example, vertebrate limb buds begin  

development a paddle shaped appendages.  

Apoptosis removes interdigital tissue to sculpt  

hands, toes, paws, etc

c. Apoptosis is also active in the adult, where about 1010 – 1011 cells die everyday

d. Reduced or elevated apoptosis is linked to several human  diseases:

i. Cancer – murder pathway can’t be activated

ii. Parkinson’s, Alzheimer’s, and Huntington’s  

diseases – increased death

iii. Diabetes type I – increased death

4. Evolutionarily conserved proteins participate in apoptosis a. Comparison between major components of the apoptotic  pathway in C. elegans and humans – color coded to  

identify. Conserved homologous proteins

b. Pathways in both organisms converge on the activation of  caspases – proteases bearing a conserved cysteine in their  active site

c. In vertebrates and initiator caspase (Caspase 9) is activated  by dimerization induced by upstream activatory proteins  (principally Apaf-1 following inernal or external stimuli)

d. Activated initiator caspases cleave and activate effector  caspases (principally Caspase 3 and 7, though there are  many in vertebrates)

e. Prior to cleavage and activation, caspases and referred to as  procaspases – inactivated form

5. Mitochondrial Mediated Apoptosis

a. Key regulatory proteins lie upstream of caspaces in the  apoptotic pathway and fall into one of two categories: pro apoptosis or anti-apoptosis (pro-survival)

b. All of these proteins are always expressed and the pro survival proteins bind and inhibit the activities of the pro apoptotic proteins

c. All are members of a large family of proteins called the  Bcl-2 family, which share up to 4 characteristic domains  called the BH 1-4 domaisn (Bcl2 homology domains)

i. Through these domains the proteins oligomerize  

and regulate one another’s activities and ability to  

bind other proteins

6. Vertebrate Apoptosis and Mitochondria: Intrinsic Apoptosis a. In healthy vertebrate cells the anti-apoptotic protein Bcl-1  binds to Bak and Bax, which span the outer mitochondrial  membrane – preventing them from oligomerizing

BSC 300-001 Week 13 Notes

b. In response to intrinsic signals like excessive DNA damage  or loss of trophic factors or pro-apoptotic signals, other  members of the Bcl-2 family are released

i. Puma is released from the nucleus, following  

excessive DNA damage, and Bad being released  

upon loss of trophic factors

ii. These pro-apoptotic factors bind either Bcl-2 or  

Bak/BAx disrupting the complex

iii. Bak and Bax are able to oligomerize

1. Bak and Bax oligomerize to form a pore in  

the outer mitochondrial membrane

2. This pore is specific to the Cytochrome C  

peripheral membrane protein (of the ETC),  

which is released into the cytoplasm

3. In the cytoplasm, Cyt C binds Apaf-1,  

causing it to dissociate from Caspase-9

a. Caspase-9 cleaves the effector  

procaspase-3 to an active form and  

caspase-9 promotes proteolytic  

cleavage of its many targets

7. Apoptosis: The Extrinsic pathway – Death receptor-regulated a. Initiated by external stimuli

i. Tumor necrosis factor (TNF) is detected by a TNF  cell surface receptor

ii. The activated receptor recruits Receptor  

Associated Death Domain proteins (TRADD and  


iii. These proteins bind and dimerize a different  

initiator caspase (Caspase 8)

iv. Which cleaves other effector procaspases to active  forms

v. Caspase 8 also cleaves a Bcl-2 family member  

called Bid into an active form, which similar to the  

intrinsic pathway promotes Bax and Bak activity  

and the release of Cytochrome C peripheral  

membrane protein – leading to activation of the  

effector caspase Caspase-9

8. Apoptosis Clearance

a. Apoptotic cell death occurs without spilling cellular  contents to prevent inflammation, compared to necrosis b. During apoptosis, a phospholipid “scramblase” moves  phosphatidylserine molecules to the outer leaflet of the  plasma membrane where they are recognized as an “eat  me” signal by specialized macrophages

c. Apoptotic cells are cleared by phagocytosis

BSC 300-001 Week 14 Notes

I. Lecture 23 – Immunology Part I (Ch 21 Part I)

a. Overview of Host Defenses

i. Overview of Immune Defenses

1. Pathogenic attack has spawned an evolutionary arms race. Metazoa  have evolved diverse mechanisms to destroy infectious agents, and  

pathogens likewise continue to evolve means of evading or  

disarming these systems

2. Many organisms possess an innate immune system that serves as  

a rapid, somewhat specific, system for recognizing and destroying  

invading pathogens

3. Vertebrates have evolved a complex adaptive immune system.  

This system is highly specific and changes in response to new or  

increased numbers of pathogens

ii. The three layers of Vertebrate Immune Defenses

1. Mechanical and chemical defenses provide protection against most  pathogens – such protection is immediate and continuous, but has  

little specificity

2. Mechanical defenses consist of epithelia and skin that serve as  

barriers to infection, with associated cell derived molecules that  

weaken potential pathogens

3. The innate immune system includes phagocytic cells, complement  

proteins and glycoproteins called interleukins secreted by certain  

white blood cells. Innate defenses are active within minutes to  

hours of infection and have a small degree of specificity

4. Pathogens not cleared by the innate immune system are dealt with  

by the cells and highly specific molecules of the adaptive immune  


5. The adaptive immune response of vertebrates is activated within  

days of the primary infection and includes four key features  

lacking from the innate response shared by many Metazoa:

a. Specificity – it can distinguish between closely related  


b. Diversity. -it can recognize millions, if not billions, of  

unique molecules, or molecular structures

c. Memory – the host’s immune system can remember  

previous infection and mount a faster and more specific  

response upon re-exposure

d. Tolerance – it can avoid mounting an immune response  

against the host’s own cells

6. These features arise from the production of diverse proteins  

including antibodies and cell-surface receptors, each with unique  

affinity for target molecules

iii. A few key terms:

1. Antigen – any material that can evoke an immune response

2. Antibody – specialized members of the Immunoglobin  

superfamily that bind to specific molecular features of antigens

3. B-cells – White blood cells (lymphocytes) that mature in the bond  marrow and express antigen-specific receptors. Once encountering

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an antigen, B-cells mature to become antibody-expressing plasma  cells

4. T-cells – a lymphocyte that matures in the thymus and expresses  antigen-specific receptors

a. Two major classes:

i. Cytotoxic T-cells that kill virus infected and tumor  


ii. Helper T-cells that are required for activation of B


iv. Overview of Host Defenses

1. Our skin, GI and genital tracts collectively possess a surface area  of approximately 4000 square feet, constantly being exposed to  bacteria and viruses

2. Many bacteria are commensal – they live on and in us without  causing harm, and in many cases are beneficial (they represent our  microbiota)

3. Rupture of epithelia is the most effective and common route for  viral and bacterial infection

4. Viral replication is restricted to host cell cytoplasm or nuclei, and  can then spread to other cells following release as free viral  particles or direct transfer from adjacent cells

5. Most bacteria replicate extracellularly, though some are  intracellular obligates – either in intracellular vesicles or free in the  cytoplasm

6. Therefore an effective host immune system must be capable of  eliminating both extracellular pathogens as well as host cells  harboring pathogens

v. Vertebrate immune defense: leukocytes

1. Leukocytes (white blood cells) include a variety of cell types  including B and T cells, monocytes (precursors to scavenger cells  called macrophages), dendritic cells, neutrophils and Natural Killer  cells – each with distinct functions in the immune response

2. Lymphocytes (B and T cells) are born in the bone marrow and are  mobilized in the circulatory system to sites where they mature and  become activated (thymus gland, lymph nodes, tonsils and spleen)

3. The circulatory system also moves mature lymphocytes throughout  the body where they exit to sites of infection

vi. Vertebrate Immune Defense: Lymph nodes

1. The pressure of the circulatory system forces cell-free fluid across  the blood vessels (interstitial fluid) that bathes all cells in the body 2. A portion of this fluid is absorbed by lymphatic vessels and is  conducted to enlarged capsules of the lymph system called lymph nodes (vis afferent vessels)

3. The arriving lymph carries cells that have encountered antigens, as  well as soluble antigens themselves

4. Within lymph nodes the cells and molecules of the adaptive  response encounter these antigens and count an appropriate

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response. They are activated, leave the lymph node (via efferent  vessels) in lymph that is returned to the circulatory system

vii. The innate immune response following infection

1. Innate immune response is activated once the mechanical/chemical  barrier is breached. It includes:

a. Phagocytes – a diverse group of cells (including  

leukocytes: the phagocytic white blood cells macrophages,  

neutrophils and dendritic cells) capable of ingesting foreign  

cells and destroying them by phagocytosis. They are  

widespread through our circulatory system, tissues and  


b. Several soluble proteins that are constitutively present –

known as the complement system

c. The phagocytic white blood cells are known as  

professional phagocytes because they are much more  

effective than non-professional cells at identifying and  

removing foreign cells

d. Their professional status is a result of expressing specific  cell surface receptors known as pattern recognition  

receptors (PRRs) that recognize and bind broad patterns of  

pathogen-specific markers like bacterial cell wall  

molecules, nucleic acids lacking methylated guanines, and  

double-stranded RNA

e. The most important of these PRRs is the Toll-like family  of receptors (TLRs). Cells activated by their TLRs secrete  

numerous effector molecules, including antimicrobial  

peptides that destabilize biological membranes, causing  

targeted bacteria to rupture

f. Macrophages and dendritic cells, once activated by their  TLRs, become antigen presenting cells (APCs), by  

processing and displaying foreign material on their cell  

surfaces. These presented antigens are bound by and  

activate antigen-specific T cells. Therefore the TLR system  

of macrophages and dendritic cells serves as a bridge  

between the innate and adaptive immune system

2. The innate response: Inflammasome and non-TLR nucleic acid  sensors

a. A mammalian family of leucine rich repeat receptors

(LRRs) recognizes a wide array of foreign molecules  

(bacteria cell wall components, uric acid crystals, bacterial  

DNA etc)

b. In response to these cytosolic insults these proteins  

stimulate assembly of a large protein complex called the  

inflammasome, which activates a caspase called caspase-1

that stimulates secretion of interleukin cytokines

i. Cytokines – small secreted proteins that promote  

inflammation and attract white blood cells of the  

immune response

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3. The innate response: The Complement System

a. The complement system is a system of serum proteins that  can bind directly to bacterial cell walls and fungal surfaces

b. Distinct pathways in response to different insults lead to the  assembly of a bacterial membrane attach complex. For  

example, in the classic pathway, pathogens bound by  

antibodies recruit the complement system to destroy the  


c. All three pathways converge on the function of the protein  C3 which promotes assembly of the membrane attack  


i. This promotes cell lysis as well as attracts  

macrophages which ingest and destroy the  


ii. Additionally, this labeling of pathogenic bacteria  

attracts and activates T-Cells of the adaptive  

immune system- hence its name the complement  


4. The innate response: Natural killer Cells

a. The innate immune system also functions to eliminate cells  that have been infected by viruses

b. Cells infected by viruses secrete cytokines called type 1  

interferons, which activate Natural Killer Cells

i. NK Cells release perforin, which oligomerizes to  

form pores in the target cell membrane

1. The pores allow for the passive diffusion of  

a family of pro-apoptotic proteases, known  

as the granzymes, into the target cell

c. NK Cells not only kill viral infected cells. They also secrete  type II interferon y, which promotes diverse anti-viral  

responses, and can target stressed or cancerous cells that  

have been bound by antibodies 

viii. The Inflammatory Response

1. Inflammation is characterized by redness, swelling, heat and pain  caused by:

a. Increased leakiness of blood vessels due to increased blood  flow

b. Attraction of immune system cells to the site of infection  due to localized release of cytokines

c. And production of additional soluble mediator of  


2. The response is initiated by tissue resident dendritic cells that  identify pathogens via their Toll-like Receptors, and respond by  releasing cytokines and chemokines – which attract other immune  cells – critically neutrophils

a. Neutrophils are phagocytic, and directly engulf invading  bacteria and fungi

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b. Neutrophils also release lysozymes and proteases that  

digest bacterial cell walls and small peptides with  

microbicidal activity called defensins that puncture  

bacterial cell walls

3. Additional inflammation responses are mediated by local mast  cells which secrete histamine, a small peptide that binds specific  GPCRs promoting increased vascular permeability and therefore  the migration of immune cells into the infection

4. If the bacterial burden in the infected tissue is high dendritic cells  also migrate to lymph nodes where they present bacterial antigens  to activate T-Cells that will mount an acquired immune response  by activating B-Cells that secrete antibodies against the pathogens  ix. Adaptive immunity – a vertebrate specific process

1. Acquired immunity imparted by lymphocytes bearing antigen  specific receptors in their membranes

2. In 1905 von Behring (won the first Novel in physiology) and  Kitasato showed that an antibiotic factor appeared in the serum of  infected rodents and that this factor conferred resistance to other  naïve rodents upon inoculation

3. This work also showed that serum contains a heat inactivated  substance that “complements” the serum from exposed rodents.  

Today, we know this as the complement system described earlier b. Immunoglobins: Structure and Function

i. Immunoglobins have a conserved structure consisting of heavy and light  chains

1. Several classes (isotypes) of immunoglobins involved in adaptive  response (IgM, IgA, IgE, IgD, and IgG)

2. Most abundant class (IgG) possesses two identical heavy chains covalently attached by disulfide bridges to two identical light  

chains and one another: the H2L2 configuration

3. Each isotype is distinguished by possessing distinct heavy chains  resulting from genetic recombination events in the gene encoding  the heavy chain, a process called class switching

4. Each monomer can be divided into two functionally distinct  regions, Fab – the antigen binding portion and Fc, the constant  

portion that is identical for all antibodies of the same isotype

5. Each B-cell lineage produces a slightly different version of heavy  and light chains in the Fab portion through another recombination  process called V(D)J recombination

6. As a result of this recombination an almost limitless amount of  antigen specificity is generated by the adaptive immune system

7. While there is a single heavy chain locus (gene) there are two  distinct light chain loci called κ and λ

8. Each B-Cell transcribes only one of the two light chain genes,  therefore the light chains are always identical in any given Ig

ii. Immunoglobin diversity

1. IgM is the principle B cell receptor, expressed in the membrane of  naïve B cells

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a. IgM is also secreted as a pentamer of H2L2 chains joined  by another protein, the J fragment

b. This arrangement greatly increases the affinity of  interaction, known specifically as avidity: the combination  of affinity and the number of binding interactions

c. The IgM conformation is highly conductive to activating  the complement cascade

2. IgA is also bound by J fragment, forming an H2L2 dimer a. IgA binds an IgA receptor on the basolateral side of  epithelial cells, where binding triggers receptor mediated  endocytosis

b. Following this, the receptor is cleaved and the dimer is  secreted to the apical side of the epithelium: a process  called transcytosis

c. This plays an important part of the immune response to  bacterial infection following tears and abrasions in epithelia 3. IgG is the major secreted antibody of B-Cells

a. It is important for neutralizing virus particles and for  preparing antigens from viruses or bacteria for cells  

equipped with receptors for the Fc portion of the antibody.  Such receptors are called FcRs

b. Phagocytic cells (dendritic cells and macrophages) can  recognize bacteria and virus coated with IgGs and  

endocytose them. Such coating of bacteria by antibodies is  called opsinization

c. Natural Killer cells can recognize and attack virally  infected cells that express viral proteins in their plasma  membraned bound to antibodies

4. Each naïve B cells produces a unique immunoglobin a. The clonal selection theory stipulates each naïve  lymphocyte (having not bound its cognate antigen)  

expresses an antigen binding receptor with unique binding  specificity

b. Once it binds an antigen there is rapid cell division to  generate a genetically identical lineage – a clone

c. Most antigens are complex 3D structures with a number of  potential binding sites for antibodies.

i. Such binding sites are referred to as epitopes

d. Thus, when exposed to an antigen many naïve lymphocytes  may be activated, each expressing a distinct antibody  specific for a different region (epitope) of the antigen

e. Therefore, antibody production against an antigen is  usually polyclonal – several, rather than just one, lineages  are established

5. Specificity determined by the complementarity-determining region  of each Ig

a. Over 1 million unique antibodies can be generated by naïve  B-cells in vertebrates

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b. Each heavy chain is composed of four globular domains,  

each light chain 2

c. The N-terminal domains of each chain is referred to as  

variable regions VL and VH

i. Within each variable region reside three  

hypervariable regions – HV1, HV2, and HV3 –

loops sandwiched between two B-sheets

d. Within the globular conformation of the variable domain,  

these loops are in close proximity to one another, and  

collectively the HV regions of the light and heavy chains  

constitute the complementarity determining region

(CRD) of each antibody

c. Generation of Antibody Diversity and B-Cell Development

i. Generating antibody diversity

1. Naïve B and T cells both express cell surface receptors with high  specificity for an epitope they have never encountered

2. If there are only 3 genes that encode the chains of antibodies,  where does all of this diversity come from? Somatic gene  

arrangement. The principles of which are similar for T-Cell  

receptors as well as B Cell receptors and their soluble isotypes

3. Because of the almost infinity number of epitopes that can be  recognized, there exists the potential for auto-immunity, so  

organisms must have a way of distinguishing self from non-self

ii. The Cellular and Molecular Basis of Immunity:

1. DNA Rearrangement of Light Chain loci: V(D)J Recombination a. The single heavy chain and two light chain loci possess a  

number of clustered gene segments that will encode the  

distinct domains of the peptide produced

b. These segments are akin to exons, in that they will be  

transcribed and spliced together after recombination occurs

c. The domains are the:

i. Variable gene segments

ii. Joiner gene segments

iii. Constant gene segments

iv. Diversity gene segments (Heavy chain only)

2. Focus on the kappa light chain

a. The C gene (constant) encodes the constant portion of the  

protein and is located at the 3’ end of the locus. A single C  

segment in the light chain loci, multiple segments in heavy  


b. The V locus (variable) contains multiple similar V genes –

40 in humans

c. The J locus (joining) contains a smaller number of  

segments – 5 in humans

d. As B Cells are generated in the marrow the heavy and light  

chain loci undergo recombination to produce one antibody  

species with unique epitope specificity

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e. A protein complex composed of the Rag1 and Rag2  recombinases is activated and excises a random number of  V and J segments. This is mediated by the complex  

recognizing Recombination Signal Sequences at the 3’  end of each V segment and 5’ end of each J segment

f. The complex then joins a single V gene and J segment into  one continuous exon

g. The RAG enzymes make single-stranded cuts, and the free  -OH groups attack the complimentary strands, generating  hairpin loops

i. RAGs open the hairpins, either symmetrically or  

asymmetrically, leading to single-stranded  

overhangs from asymmetric cleavage. A DNA  

polymerase added random nucleotides to  

symmetrically cleaved strands

h. As a result, random nucleotides are added to each junction  – increasing the amino acid diversity in the encoded CDRs i. DNA ligase IV then joins the two strands generating a new  light or heavy chain locus

j. Transcription initiates at the 3’ most V segment and  terminates at the end of the single κ C gene

k. Non-joined J fragments are removed by splicing (as if they  were an intron) to generate the mature mRNA

l. Such mature light chain mRNAs therefore contain coding  sequence for one C and one VJ domain

3. The lambda light chain and heavy chain

a. The same V(D)J recombinase complex similarly performs  recombination at these loci, with the added complexity of Diversity genes in the heavy chains – hence V(D)J  


b. A single B Cell expresses only kappa or lambda light  chains, not both

c. V(D)J recombination occurs only at one of the two alleles  for these three loci. The second allele is transcriptionally  silenced

4. Focus on the kappa light chain: V-J recombination a. Collectively these arrangements can produce approximately  2000 kappa chains (5 x 40) and an equivalent number of  lambda chains

b. Heavy chain recombination is estimated to produce  approximately 100,000 unique peptides

c. Therefore, over 200,000,000 unique antibody combinations  are possible from heavy and kappa light chains alone

d. Junctional imprecision:

i. Of the three hypervariable loops in the peptides two  are encoded within the V gene. The third falls at the  

joint between the V gene segment and the J gene

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ii. Recombination at this site is imprecise, such that  

the same V and J genes joined together in different  

cells will have different amino acid sequences in  

this loop due to random addition or loss of  


5. Somatic Hypermutation

a. When a B cell recognizes an antigen, it is stimulated to  


b. During proliferation the V region undergoes somatic  

mutation at a rate that is at least 105 to 106 greater than the  

rest of the genome. In other words, somatic  

hypermutation – increased single base pair substitution

c. Mechanisms:

i. Activation-induced cytosine deaminase: converts

C to U, leading to U:G mismatches and removal of  

the uracil

ii. Translesion DNA polymerases: error-prone DNA  

polymerases that fill in the spot where uracils were,  

creating mutations that are propagated when the cell  


d. As a result of somatic hypermutation, there is increased  

diversity in the hypervariable region of B cell antibodies

e. This can produce antibodies with even greater specificity  

for the offending antigen. And these cells will be  

preferentially selected following antigen re-introduction

6. Class Switching

a. Class switching – once a B cell is committed to producing  

a specific antibody it can be induced to change the class of  

antibody it produces

b. In response to T cell cytokines, during B cell maturation  

other enzymes rearrange the C genes encoding the different  

heavy chains

c. The VDJ region remains the same, but genes for class  

specific heavy chains are moved proximal to the VDJ gene

II. Lecture 24 – Immunology Part II (Ch 21 continued)

a. The MHC and Antigen Presentation

i. The MHC (Major Histocompatibility Complex) encodes many proteins  involved in immune response

ii. Class I and II MHC proteins are highly polymorphic and responsible for  graft acceptance/rejection

iii. Class I and II MHC proteins display antigens on the cell surface and  present them to antigen specific receptors on T cells – activating the T cell 1. Cytoxic T cells then produce cytokines and/or kill viral infected  


2. Helper T cells activate cognate B cells, stimulating them to mature

iv. Helper T Cells aide B Cell maturation

v. Both naïve B cells and T cells have receptors that allow them to interact  with specific epitopes – a similar mechanism to V(D)J recombination

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generates epitope specific receptors on the surface o cytoxic and helper T  cells

vi. While naïve B cells can directly interact with antigens via their IgM  receptors, the antigen-specific receptors of T cells can only recognize  epitopes that are associated with specific glycoprotein complexes on the  surface of antigen presenting cells

vii. The various antigen presenting cells that digest foreign pathogens present  portions of proteins from the pathogens associated with transmembrane  MHC proteins (class I and II)

viii. The genes encoding these glycoprotein complex proteins are contained  within a large genomic region called the Major Histocompatibility  Complex (MHC)

ix. The MHC locus determines the ability of members of the same species to  accept tissue grafts

1. Success of graft transplants is determined by genes encoded in the  MHC

2. The human MHC is called the HLA complex (human leukocyte  antigen) – which contains dozens of genes, most encoding proteins  with immunological relevance

3. Most cells in vertebrates express proteins from the MHC and thus  have the potential for presenting antigen peptides to the immune  system

4. Class I MHC proteins are single pass transmembrane  

glycoproteins, non-covalently associated with a non-MHC encoded  protein called B2-macroglobin  

5. Class II MHC proteins are heterodimeric, each a single pass  protein

x. The killing activity of Cytoxic T cells is Antigen-specific and MHC  restricted

1. MHC proteins play a critical role in recognition of viral infected  cells by cytoxic T cells – aka Cytolytic L lymphocytes (CTLs), or  Killer T Cells, which following identification of infected cells  promote lysis of the infected cell

2. The experimental assay depicted here simply shows that CTLs  from influenza infected mice are able to recognize and promote  lysis of target cells infected with the same strain of influenza (right  tubes), but not genetically identical cells that are not virally  


3. Importantly, killing activity is both restricted to cell infected with  the exact same strain of flu. Even a single amino acid change in flu  proteins (virus X vs virus Y) prevents CTLs from the host rat from  recognizing and destroying virally infected cells

a. Additionally, this killing activity is restricted to rate of the  same genotype – virally infected cells from rats that  

genotypically differ from the host rate cannot be recognized  

and destroyed by its CTLs

b. This phenomenon is referred to as MHC restriction and  the MHC molecule involved is called restriction elements

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xi. T cells with different functional properties are guided by two distinct  classes of MHC molecules

1. The MHC encodes two types of glycoproteins essential for  immune recognition: MHC class I and class II molecules – there  are multiple genes for each class

2. In addition the MHC encodes proteins involved in antigen  processing, presentation, and proteolysis

3. Also, the vertebrate MHC encodes components of the complement  cascade

4. Class I and class II MHC molecules are both involved in  presenting antigens to T cells, but serve two distinct functions: a. Class I: Present antigens to CTLs (Cytotoxic T Cells)  

triggering them to destroy virally infected cells. CTLs  

express a cell surface glycoprotein called CD8 which  

determines the ability of a T cell to interact with MHC  

Class I proteins. Most nucleated cells can support viral  

replication, and therefore all express MHC class I proteins

b. Class II: Are found exclusively on profession antigen  

presenting cells (B cells dendritic cells and  

macrophages), which present antigens to Helper T cells  

and initiate the cascade on interactions that will stimulate  

naïve B cells to differentiate as active plasma cells and give  

rise to Memory B cells. Helper T cells express the  

glycoprotein CD4 which is required for interaction with  

Class II MHC proteins

c. Like B Cell receptors and their isotypes, T Cell receptors,  CD4 and CD8 belong to the immunoglobin superfamily

5. Important Concept – B cell activation involves rapid proliferation  in the lymph system. A subset of B cells undergo class switching  to soluble antibody secreting cells. Such activated B cells are  referred to as plasma cells. In addition, a subset of these cells  undergo somatic hypermutation and remain in the lymph nodes  and spleen as memory B cells, which can be reactivated more  rapidly if the body is exposed to the same antigen in the future

xii. MHC molecules bind peptide antigens and interact with the T-Cell  receptor

1. The source of graft rejection is that both Class I and Class II MHC  molecules are highly polymorphic: there are many alleles of all  members of each class throughout the human population – more  

than 2000 alleles combined for all of the Class I and II genes a. As a result, except for closest relatives, the chances that any  two individuals share the same MHJC variants is very  


b. Regardless of this variation, all members of both Class I  and II MHC protein share a highly similar conformation as  

well as interaction mechanism with the T Cell receptors

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i. This is why transplant surgeons must MHC match  potential donors with patients in need of a  

tissue/organ transplant

2. Class I MHC Molecules

a. There are 3 MHC Class I genes (HLA-A, HLA-B, and  HLA-C), and the encoded single pass Ig glycoproteins non covalently interact with the non-MHC encoded B2-

macroglobulin, which has a conformation highly similar to  an Ig domain

b. Two Ig domains form the peptide binding cleft

c. Antigen peptides bound by Class I molecules are 8-10  residues long, and there are binding pockets in the MHC  protein for the charged N and C termini of the peptide

d. Affinity for the peptides is enhanced by a small number of  binding pockets that accommodate amino acid side chains  of each peptide

e. In this manner, a given MHC protein can accommodate a  large number of peptides of diverse sequence

f. Most allelic diversity among MHC protein lies in amino  acids in and around the peptide binding cleft, which  

therefore alters specificity of peptide binding between  variants

i. As well as generates distinct conformations of the  MCH protein itself. A T Cell which can interact  

with one Class I MHC allele will therefore not be  

able to physically interact with another – this is the  

basis of MHC restriction

g. The CD8 protein on cytoxic T Cells (CTLs) serves as a co receptor binding to conserved portions of Class I MHC  proteins

i. The CD8 protein, thus sets the Class I preference of  all mature T cells

3. Class II MHC Molecules

a. Six human genes

b. Encoded protein similar structure to Class I: two Ig  domains near the membrane. Both proteins of the  

heterodimer (alpha and beta) contribute to the peptide  binding cleft, which is therefore larger and can  

accommodate larger peptides than Class I

c. Similar to Class I polymorphisms affect architecture of  binding cleft and therefore peptide affinity and ability to  interact with T-Cell receptors (MHC restriction) and the co receptor CD4

i. CD4 is the protein through which HIV virus gains  entry into T cells to initiate immunodeficiency  

associated with AIDs

d. Binding of peptides to MHC Class II takes place in  endosomes and lysosomes

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i. MHC Class II proteins are targeted to these  

organelles by a chaperon called the invariant chain

xiii. Antigen processing and presentation

1. Class I is involved in presenting antigens produced by the cell  itself, including pathogen-associated proteins

2. Class II is involved in presenting antigens from endocytosed  pathogens

3. General steps to processing and presentation:

a. Acquisition and targeting of antigens

i. Class I: relies on error-prone translation that results  

in peptides destined for ubiquitin-mediated  

proteasomal degradation to produce peptide  

fragments for presentation

1. Such peptides are delivered to the ER by the  

TAP peptide transporter (an ABC  


a. The TAP complex has affinity for  

peptides terminating in Leu, Val, Ile  

and Met), which matches the binding  

preference of the MHC proteins

ii. Class II: relies on phagocytosis, pinocytosis,  

receptor mediate endocytosis followed by  

lysosomal degradation, which mature into late  


1. In naïve B cells the IgM receptors initiate  

receptor mediated endocytosis

2. Professional phagocytes (antigen presenting  

cells) possess Fc receptors (FcRs) that  

recognize opsonized bacteria initiating  

phagocytosis, degradation, and presentation  

at the cell surface

3. It is not only peptides that can be presented  

at the cell surface – a Class I like MHC  

protein called CD2 presents lipid antigens

b. Delivery and binding of peptides to MHC proteins:

i. Class I: In the ER newly synthesized MHC proteins  

are part of a complex called the peptide-loading  

complex, that includes the chaperones calnexin and  

calreticulin, which promote peptide association.  

Conformational change after loading releases the  

MHC and bound peptide from the loading complex

ii. Class II: MHC proteins are produced in the ER and  

assemble with the invariant chain, which provides  

the targeting signal for delivery to late endosomes.  

In the lysosomes peptides are trimmed in length by  

proteases which also remove the invariant chain  

from the MHC proteins. A distinct chaperone called  

DM helps load the peptides onto Class II proteins

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c. Presentation of MHC/peptide complexes

i. Class I: complexes are transported from ER via  

vesicles through Golgi and to the plasma membrane

ii. Class II: Complexes are mainly found within late  

endosomal multivesicular bodies. These are  

recruited to the endosomal membrane and bud off  

as tubular endosomes that are transported to the  

plasma membrane on microtubules

4. Naïve B Cells bind soluble antigen by their IgM B Cell receptors 5. Complex is endocytosed, degraded, and peptide fragments are  presented on cell surface in association with MHC Class II proteins a. This step, as we will see, is necessary for the ultimate  

activation of B cells

b. T-Cells, T-Cell Receptors, and T-Cell Development

i. T Cell receptors are structurally related to the F(ab) portion of B Cell Ig  isotypes

ii. Genes encoding these receptors undergo almost identical genomic  rearrangements to V(D)J somatic recombination of the Ig Heavy and light  chains – the details of which we will not review

iii. Unlike B Cell Ig’s T Cell receptors do not undergo somatic hypermutation  during differentiation

iv. In the course of their development T Cells must learn to recognize self derived from non-self-antigens to avoid an autoimmune response

v. T Cell Receptor Structure

1. Like B Cells, T Cells that have been activated through antigen  binding on their R Cell Receptors also undergo clonal selection  

and proliferation

2. They then acquire the ability to kill antigen-bearing target cells (as  cytoxic T cells, CTLs) or they secrete cytokines that will assist B  Cells in their differentiation (Helper T Cells)

3. A T Cell Receptor is a heterodimer compose of two glycoproteins,  each of which have undergone somatic rearrangement. Each  

heterodimer is composed of either an alpha or beta or y subunit  

with the N termini of the two subunits forming the antigen binding  site – as with B cell Ig’s, this is the region where somatic  

recombination generates diversity

4. Most T cell receptors contain one subunit that was the result of  VDJ recombination and another the result of VJ recombination – in  this respect they are highly similar to B Cell Ig’s

vi. T Cell and B Cell Proliferation and Differentiation

1. Antigen binding to TCR’s and BCR’s to naïve T and B Cells  

triggers their immune response

2. The antigens for TCRs are the peptide bound MHC proteins on the  surface of APCs

3. Antigens for BCRs do not need to be associated with antigen  presenting cells

4. Several auxiliary proteins function in collaboration with the TCR’s  and BCR’s to initiate a signal transduction event

BSC 300-001 Week 14 Notes

5. The cytoplasmic portion of the receptors themselves are too short  to interact with cytosolic proteins and initiate transduction

6. Instead auxiliary proteins with cytosolic ITAM domains (immunoreceptor tyrosine-based activation motif) associate with  TCR’s and BCR’s

7. Antigen binding by the receptors alters their conformation and  recruits members of the Src family of tyrosine kinases, which  phosphorylate the ITAMs on specific tyrosines

8. Phosphorylated ITAMs recruit additional non-Src kinases via SH2  domains, which in turn recruit and phosphorylate adapter proteins  that serve as scaffolds to recruit proteins and initiate signal  


9. Signal transduction includes activation of phospholipase C-y and  PI3-Kinase – which generate IP3 and Protein Kinase B, the major  effectors of signal transduction in B and T cell activation

10. T Cells depend on production of the cytokine Interleukin 2 (IL-2) for clonal expansion. It is one of the first genes to be transcribed  following antigen binding to TCRs. I:-2 stimulates the T Cell to  produce more IL-2 (autocrine signaling) in order to sustain  


11. The transcription factor that transcribes IL-2 is named NF-AT (nuclear factor of activated T Cells), and is always expressed but  sequestered in the cytoplasm in a phosphorylated state

12. The phosphatase that removes this inhibitory phosphorylation is  calcineurin, a Ca2+-dependent enzyme

13. IP3 release by PLCy stimulates Ca2+ efflux from the ER, thus  activating calcineurin, and as a result promoting NF-AT entry into  the nucleus

14. Human Health relevance – how can organs from unrelated  donors be successfully transplanted? Immunosuppressants, like  cyclosporin, bind and complex with calcineurin – inhibiting its  ability to dephosphorylate NF-AT. As a result T cells cannot  undergo clonal expansion, and therefore the tissue is not seen as  being foreign

vii. Comparison of T and B Cell Development

1. Both are born in the bone marrow. Pre-T cells migrate to the  thymus, while pre-B cells remain in the marrow

2. Before becoming mature both experience life as a pre-lymphocyte 3. The membrane bound receptors are not yet capable of binding  ligands, but instead the B cell IgM heavy chain and T cell Beta or  y subunit associate with different proteins and form pre BCRs and  TCRs – the function of which is to transduce signals via distinct  ITAM proteins that shut down RAG mediated recombination,  promote proliferation and promote transcription of the missing  subunit (K: κ or λ light chain or T:α or δ).

4. The receptor and lymphocyte are now considered to be mature, but  still naïve

viii. T Cell Differentiation

BSC 300-001 Week 14 Notes

1. In the thymus naïve T Cells are presented with antigens both  foreign and derived from self

2. T Cells that cannot interact with antigens, and T cells that react  with self-derived antigens and killed by extrinsic apoptosis

3. A protein called AIRE (autoimmune regulator) allows the  expression of tissue specific proteins (like those of the pancrease)  in the thymus and the screening of T cells with auto-affinity

4. Defects in AIRE lead to a wide array of autoimmune disorders 5. Pre-T Cells express both CD4 and CD8, while differentiated T  Cells express only one of the cofactors

6. The mechanism appears to be stochastic and determined by a battle  between two transcription factors, both of which are expressed in  pre-T Cells, but only one remains active in mature T cells

7. ThPOK promotes the CD4 lineage

8. Runx3 promotes the CD8 lineage

9. Both are regulated by pre-TCRs and appear to fight for supremacy,  suppressing one another’s expression until there is only one

ix. T Cells Require Two Signals for Activation

1. All T Cells require antigen binding

2. The second co-stimulatory signal is mediated via the receptor  CD28, expressed on all T cells, which is bound by other CD  protein on the surface of professional APCs

3. Upon activation, T Cells promote attenuation of activation by  expressing an additional protein, CLTA 24 that competes with  CD28 for these APC originating signals

4. In this manner, the T cell is able to fully commit to proliferation  and activation

x. Cytoxic T Cells

1. Once bound to virally infected cells through their TCRs and co receptors CD8 cytoxic T Cells (like Natural Killer Cells of the  Innate Immune System) release into the narrow clefts between the  cells the combination of perforin and granzymes

a. Perforin punctures the membrane of the infected cell while  granzymes activate caspases that promote apoptosis

xi. Interleukins

1. Lymphocytes and other cells in the lymph produce an array of  cytokines that control lymphocyte activity – primarily to  

proliferate or differentiate

2. Cytokines that act on leukocytes are called interleukins – of which  there are approximately 35 encoded by the human genome. Each  bound by distinct receptors in the array of leukocyte cell types.  Interleukins may act to alter the fate of cells, or as chemokines – attracting leukocytes to a site of infection

3. These signals act through cytokine receptor activating, principally  the JAK/STAT pathway

a. Ex: most B Cells bound by an antigen require direct contact  with a helper T cell bound by the same antigen in order to  

be activated and to initiate proliferation and class

BSC 300-001 Week 14 Notes

switching. This signal is mediate via Interleukin 4 (IL-4)

secreted by the helper T cells

4. Additionally, when tissue damage occurs resident fibroblasts  secrete IL-8, which attracts neutrophils to the site of infection

c. Collaboration of Immune-System Cells in the Adaptive Response i. A functional immune system requires constant interplay between the  various cells of the immune system. Resident APCs must be able to  activate T Cells as either cytoxic T Cell or helper T cells, and helper T  cells must be able to activate B Cell in order for antibodies to be produces ii. Langerhans cells represent one of the first line of defenses of the innate  immune system – they constitute a network of dendritic cells within the  skin that makes it virtually impossible for invading bacteria to not trip the  alarm

iii. These and other professional APCs primarily conduct their screening of  the environment via the action of Toll-like Receptors, discussed briefly in  the previous lecture

iv. Production of High Affinity Antibodies Requires Collaboration Between  B and T cells

1. Activation of naïve B cells requires both a local source of antigen  as well as a helper T Cell already activated by the same antigen

2. Soluble antigen reaches the lymph nodes where B and T Cells  reside, either as a result of bacterial lysis through the various  

mechanisms of the innate immune system, or through presentation  by professional APCs that have migrated into the lymph system  

after engulfing and digesting a pathogen

3. Dendritic cells present the antigen to CD4+ T Cells which mature  into helper T cells

4. Soluble antigens are bound by the BCRs of naïve B Cells

a. These B Cells then internalize the receptor and antigen,  

process the antigen via proteolytic degradation and present  

it in their own surface coupled to Class II MHC proteins

b. Activated helper T Cells interact with antigen presenting B  

Cells via interaction between the TCR and peptide bound  

MHC proteins on the B cell

c. The B cell also displays receptors for a number of cytokine  

and membrane bound signals from the helper T cell

d. And a second required interaction for maturation mediated  

by the B cell receptor CD40 and the T Cell membrane  

bound ligand CD40L

e. Both IL-4 and CD40 binding are required for B Cell  


f. Only then will B Cells proliferate

5. A subpopulation of these cells undergo class switching and  

become soluble antibody secreting plasma cells – these cells are  short lived – a few days – and are responsible for the immediate  immune response of the adaptive system

6. A second subpopulation of B-cells do not undergo class, but  instead experience somatic hypermutation and exit the cell cycle.

BSC 300-001 Week 14 Notes

These quiescent cells remain in the lymph nodes and spleen as  memory B cells that can mound a faster and, often more time  efficient, attack against future invasion by the same pathogen

7. Similarly, helper T cells proliferate and differentiate as either  activated T cells that aide the antigen exposed B cells, and  

additionally a subpop remains quiescent in the lymph system as  Memory T Cells (no somatic hypermutation

v. Vaccines elicit protective immunity against a variety of pathogens 1. Inoculation with compromised virus activates the immune system  and raises antibodies against viral protein

2. Viral inactivation can result from heating and denaturing the virus,  but this is not as effective as attenuation, in which the virus is  passaged through successive rounds of cell culture. Along the way  it becomes less potent (reason is unsure, but likely related to  mutation)

3. Inoculation with live attenuated strains results in activation of the  immune system and mild reaction caused by inflammation and  weak proliferation of the virus

4. Additional methods of vaccination include subunit vaccination, in  which only a single protein, rather than the entire virus, is  


vi. The immune system also defends against cancer

1. The elevated mutation rate of cancer – next lecture – not only  produces driver mutations that cause cancer, but also passenger  mutations that do not contribute to cancer itself

2. Rather passenger mutations may slightly alter the amino acid  sequence of membrane proteins – generating neo-antigens – and  allow the immune system to recognize the wayward cells and  eliminate them

3. Additionally, genetic mutation associated with cancer may lead to  the expression of fetal-specific genes whose protein products were  expressed prior to the development of the immune system. These,  likewise, can be recognized as abnormal, and their carrier cells  eliminated by the immune system

BSC 300-001 Week 15 Notes

I. Lecture 25 – Cancer

a. Intro

i. Cancer cells proliferate, invade, and metastasize

1. One in 5 people will die from cancer: an aggressive,  

hyperproliferative, malignancy of rogue cells that, through  

uncontrolled growth and hypermutability, wage an evolutionary  

arms race against one another and the healthy tissue they infest

a. Metastasis and secondary tumors are what are killers, not  

primary tumors

2. Inherited and acquired mutations allow cancerous cells to:

a. Sustain proliferative signaling

i. Either through uncontrolled production of mitogens  

or uncontrolled activation of their pathways

b. Evade growth suppressors

i. Often by disabling mutations in the suppressor’s  

receptors or the pathways they regulate

c. Activate invasiveness and metastasis

i. Allows cancer cells to leave a primary tumor and  

establish secondary tumors

d. Enable replicative immortality

i. Continue to proliferate past the number of  

generations when normal cells senescence and die

e. Induce angiogenesis

i. Stimulate growth of blood vessels to supply  

nutrients and oxygen to the tumor

f. Resist cell death

i. Inactivate the apoptotic pathway and evade the  

immune system

3. Cancer, like many diseases, results from an interplay between an  

individual’s genotype and environmental influences, often referred  

to as gene-by-environment interactions; i.e., some people are more  

susceptible to cancer promoting influences due to differences in  

our genetic makeup

a. Most cancer arises from combined genetic mutations that  

result from exposure to carcinogens, chemicals, and/or  

other environmental factors (UV light and radiation) that  

perturb the DNA replication process, as well as the inherent  

random error of DNA replication

b. Some people also inherit genetic mutations that increase  

their chances of developing cancer

c. Importantly, cancer causing mutations work through the  

combinatorial action of mutated genes. No single mutation  

is cancer-causing

d. This usually takes many years, therefore most cancer is a  

disease associated with advanced age. However, some  

mutations are more oncogenic than others and can lead to  

development much earlier in life

BSC 300-001 Week 15 Notes

i. 90% of all cancer is from epithelial cells because  

they are in a constant state of division

e. Because most cancer requires multiple mutations, and  mutation only takes place in proliferating populations of  cells, those cells with higher rates of proliferation (epithelia  like skin, lung, esophagus, colon) are the most common  sites of cancer formation

f. Cancer is usually classified based on the embryonic tissue  of origin:

i. Carcinomas – derived from epithelia (endodermal and ectodermal) and are the most common type of  

malignant tumors (greater than 90%)

ii. Sarcomas – derived from mesoderm (muscle, bone,  connective tissue, and blood)

iii. Both carcinomas and most sarcomas are solid mass  tumors

iv. Leukemias – cancers of individual types of white  blood cells

4. Mutations in three broad classes of genes are implicated in the  onset of cancer:

a. Protooncogenes

i. Their encoded proteins normally control the rate of  cell proliferation. Gain of function mutations 

convert protooncogenes into oncogenes

1. Because of this, mutation in only one copy  

of a protooncogene may convert into an  

oncogene (just a gamma mutation in mom or  

dad to convert)

b. Tumor Suppressor Genes

i. Their encoded proteins normally restrain growth

and function as regulators of the cell cycle. Loss of  

function mutations release these regulatory controls  

and allow cells to proliferate even following  

without appropriate checks on cell cycle  

progression or DNA damage

1. Mutations in both copies of a tumor  

suppressor gene are necessary to exert an  

oncogenic effect (both mom and dad)

c. Genome maintenance genes

i. Maintain stability and integrity of the genome and  prevent excessive mutation from occurring

1. As with tumor suppressor genes, loss of  

function mutation in both copies of such  

genes are required for full oncogenic effect

2. Hypermutation – increased mutation rate  

when affected

5. Cancer is a clonal process. A single cell acquires mutations that  allow it to begin proliferating outside of the controls of normal

BSC 300-001 Week 15 Notes

cells. If it evades the immune system, it can establish a tumor, a  benign pool of precancerous cells. Such cells acquire additional  mutations and establish subclonal populations – some better, some  worse – and growth and evasion

a. Eventually, a subpop will acquire mutations that allow  

metastasis: cells exiting the primary tumor and invading  

other tissues and organs

b. Most cancer deaths are due to metastasis and the formation of secondary tumors that spread throughout the body

ii. Genetic makeup of most cancer cells is dramatically altered 1. Point mutations are not the only, and usually not the most,  important drivers of cancer progression – tumors harbor all types  of genetic alterations, including:

a. Point mutations

b. Small and large deletions

c. Amplifications

d. Insertions

e. Translocations and inversions – movement of portion of  one chromo into another chromo or flipping a chromo left  

or right

f. Aberrant number of chromosomes, usually too many  


g. Mistakes in DNA replication, through loss of tumor  

suppressors activity, increases replication error and  

mutation number

2. Other mechanisms of increased mutation rate:

a. Kataegis (thunderstorm) – the broad-scale activation and  deployment of the enzymes that promote somatic  

hypermutation in B cell Ig domains, like activation induced  deaminase

b. Chromothripis (chromosome shattering) – in some  

aberrant cells following mitosis, some chromo are not re

integrated into nucleus, but form their own micronuclei,  

where replication and segregation is much less efficient.  

Rampant chromo breakage and random stitching together  

or the pieces produces dozens to hundreds of chromo  

fragments in odd combos

iii. Cancers have highly abnormal karyotypes

1. Chromo of most cancer cells are dramatically altered. In addition  to point mutations in individual genes:

a. Individual chromo number is altered (aneuploidy)

b. Chromo rearrangement (Composite chromo)

2. Normal karyotype vs SW403 colorectal adenocarcinoma cell line  chromo: analysis of the number, type and shape of the entire  complement of mitotic chromo of a eukaryotic cell

iv. Housekeeping functions are fundamentally flawed

1. Cancer cells are usually less differentiated than and visually  distinct from normal cells – high nucleus to cytoplasm ratio,

BSC 300-001 Week 15 Notes

prominent nucleoli, increased percentage of mitotic cells and little  differentiated structures

2. The increase in gene copy number, due to chromo and regional  duplications, leads to higher protein production that alters cell  physiology. This activates the stress response pathway – proteins  of which are principle targets of many cancer drugs: i.e., if you  block the stress response pathway of cells with high protein  

production it will induce apoptosis

v. Energy production in cancer cells by aerobic glycolysis

1. Cancer cells, even in the presence of oxygen preferentially  generate ATP via glycolysis: The Warburg effect: a 200-fold  increase in ATP production from glycolysis compared to normal  cells

a. This is how PET scans can identify tumors following  

consumption of radioactively labeled glucose

2. The glycolytic metabolites can be used by cancer cells for  macromolecules biosynthesis

3. Many cancers, like proliferating embryonic cells, abnormally  express the embryonic isoform of pyruvate kinase (PKM2). It has a  high Km, therefore does not turn over pyruvate quickly. Cell is  locked in glycolysis

4. Additionally, PKM2 promotes expression of a key transcription  factor that maintains the pluripotency and high proliferation rate of  embryonic stem cells, and thus converts adult cells into an  

embryonic stem cell-like state

vi. Hydroxyglutarate is a cancer-specific metabolite

1. Some cancer types produce novel metabolites

a. In normal cells the TCA cycle enzyme isocitrate  

dehydrogenase converts isocitrate into alpha-ketoglutarate

b. Mutant IDH forms (found in many brain cancers):

i. Convert isocitrate into 2-hydroxyglutarate (SHG)

ii. High 2HG inhibits proteins that require alpha

ketoglutarate for function, including enzymes that  

control the methylation state of histone proteins:

1. TET family of DNA hydroxylases

2. Histone methyl transferases

3. Histone demethylases

iii. Histone methylation is part of the epigenetic code  

that generates heterochromatin and shuts down gene  

expression. So, the absence of this activity open  

regions of chromosomes up for gene expression

vii. Uncontrolled proliferation is a hallmark of cancer

1. In addition to the uncoupling of controls of the cell cycle and lack  of dependence on growth signals, cancer cells must also evade the  exit to senescence

2. Chromosomes ends are protected by regions called telomeres,  repeated non-coding sequences of DNA that are shortened each  time DNA is replicated

BSC 300-001 Week 15 Notes

3. Normal cells can divide only about 50 times before the telomeres  are consumed and replication begins to erase coding portions of the  genome – such cells become senescent

4. During gamete formation telomere are lengthened by the action of  a germline-specific enzyme called telomerase, a reverse

transcriptase that contains an RNA template that adds the repeated  TTAGGG sequences

5. Most cancer cells are immortalized by mutations that activate  telomerase expression, thus preventing telomere shortening and  allowing such cell lines to live forever

viii. Cancer Cells escape the confines of tissues

1. Contact inhibition – normal somatic cells grown in culture arrest  as a monolayer when they make contact with other cells (anti mitogens are expressed upon crowding). This is true of cells in  vivo as well

2. Anchorage dependency – normal somatic cells require contact  with other cells or basement membrane (via cadherins and  

integrins). Lack of such contact promotes apoptosis

ix. Basic Properties of a Cancer Cell

1. Malignant cells are not responsive to the influences that cause  normal cells to stop growth and division

2. Malignant cells do not display:

a. Contact inhibition

b. Anchorage dependency

c. Have lost the requirement for stimulatory growth  


3. And are said to have undergone oncogenic transformation x. Tumors are heterogeneous organs that are sculpted by their environment 1. Mutation of the initial cells within a tumor leads to a genetically  heterogeneous population

2. Not all cells of a metastatic tumor are capable of metastasis; such  cells are called cancer stem cells and have acquired stem cell-like  properties. This source of cells may be:

a. A mutated normal tissue multipotent stem cell

b. Dedifferentiation of terminally differentiated cells

3. Like stem cell niches, the tumor microenvironment also  contributes to the heterogeneity of tumor cells, where neighboring  cells may be more, or less, conducing to tumor growth than others

4. CD8+ Cytoxic T Cells and Natural Killer Cells surround tumors  and migrate into them. Part of the evolutionary arms race is cancer  acquiring the ability to evade such detection

5. Additionally, our own immune system may help promote the onset  of cancer. Chronic inflammation promotes the immune system to  produce cytokines, including growth factors, that may help  

increase rates of tumor cell proliferation

xi. Tumor growth requires blood vessel formation

1. Without a blood supply tumors cannot grow larger than 2mm

BSC 300-001 Week 15 Notes

2. Most tumors induce angiogenesis by acquiring expression of key  signaling molecules, specifically:

a. Basis fibroblast growth factor (Beta-FGF)

b. Transforming growth factor (TGF)

c. Vascular endothelial growth factor (VEGF)

xii. Invasion and metastasis are late stages of tumorigenesis

1. Following the mutations that allow release from anchorage  

dependency, epithelial tumor cells must loose association with one  another to allow metastasis to occur

2. During normal development, a phenomenon called the epithelial  to mesenchymal transition (EMT) plays an important role in  

allowing cells to move and migrate in the embryo

3. Some tumor cells mimic this process, turning off the cadherins that promote their epithelial associations, activating expression of  genes involved in migration as well as expression of basement  

membrane digesting enzymes, like matrix metalloproteinases…

4. Two developmentally critical transcription factors appear to be  essential for developmental and tumor EMT, named snail and  

twist, which regulate the various genes promoting these processes b. The Origins and Development of Cancer

i. Carcinogens induce cancer by damaging DNA

1. Chemical carcinogens, aka mutagens, can be classified into two  broad categories:

a. Direct-acting carcinogens: Seek out and react with  

electron rich centers in other molecules. By chemically  

interacting with N and O in DNA these compounds can  

modify bases resulting in incorrect incorporation of  

nucleotides during subsequent replication. Relatively few  

of these compounds – including ethylmathane sulfonate  

(EMS) and nitrogen mustards

b. Indirect-acting carcinogens: Most chemical carcinogens  

have little effect until they have been modified by cellular  

enzymes. Such mutagens are water insoluble and to be  

cleared from our cells must be made soluble through the  

addition of an electrophilic center – usually an -OH group  

added by the P-450 family of enzymes. While this process  

solubilizes the molecules for elimination, it can also

convert harmless compounds into carcinogens

i. Basically makes them active by making them  


2. Chemical carcinogenesis by tobacco smoke

a. Benzo(a)pyrene was shown in 1933 to be a principle  

carcinogen found in coal tar

b. Later this chemical was shown to be a principle agent of  

carcinogen in cigarette smoke – it’s an indirect acting  

carcinogen modified by P450 to produce a byproduct that  

reacts with DNA – primarily at the N2 atom of guanines

BSC 300-001 Week 15 Notes

c. The conversion results in the replacement of G with T in  the next round of DNA replication – a transversion

mutation in which a primidine is replaced with a purine

d. A principle gene randomly hit by this process is p53 – the  major tumor suppressor involved in DNA repair – such  

mutations are found in about 1/3 of lung cancers

e. Other known carcinogens include:

i. Asbestos – mesothelioma

ii. Aflotixin – a fungal metabolite associated with liver  


iii. Heterocyclic amines – colon and breast cancer  

(found in the char or well-done meat)

ii. A multi-hit model explains cancer progression

1. Cancer is principally a disease associated with age because it takes  time and random change for a correct combination of mutations to  drive tumor formation and metastasis

2. This model is supported by lab experiments in which mice are  transgenically engineered to carry combinations of specific  

mutations found in many cancer types

3. For example gain of function mutations in the Ras G-protein  prevent it from hydrolyzing bound GTP, and promote increased cell proliferation

4. Similarly, gain of function mutations in the MYC transcription  factor, required for transition from G1 to S phase, increase cell  proliferation rates

5. In transgenic mice with either mutation expressed in mammary  tissue, less than 50% develop aging associated cancer

6. However, when these mutations are combined, by crossing the  mice, the offspring have a much higher incidence of cancer and it  is more aggressive and malignant

iii. Successive oncogenic mutations can be traced in colon cancer 1. CRC is most often a cancer found in elderly patients and highlights  the multi-hit hypothesis of cancer progression, with easily  

observable morphopological progression of precancerous to  cancerous growths

2. Polyps, benign adenomas and malignant carcinomas can be  surgically removed and genotyped to identify mutations associated  with each stage of progression

3. Excessive polyp generation can produce many benign growths each with the potential to develop into cancer and is found in  families with familial adenomatous polyposis (FAP), a  

predisposition for colon polyp formation

4. Families with FAP share loss of function mutations in Wnt  signaling pathways, usually in the regulatory protein  

Adenomatous Polyposis Coli – a key inhibitor of Wnt signaling 5. Random mutation in the second copy of APC promotes formation  of benign polyps

BSC 300-001 Week 15 Notes

6. Subsequent mutations in other driver genes promotes progression  from polyp to adenoma to malignant tumor, including:

a. Activating mutations in one of the three human Ras genes,  

followed by loss of p53

b. DNA from human colon carcinomas generally show  

mutations in all three: APC, Ras and p53 that are the  

principle drivers of tumor formation

c. The loss of p53 function then promotes increased mutation  

rates that hit other drivers of metastasis and invasion

c. The Genetic Basis of Cancer

i. Virtually all human tumors have inactivating mutations in cell cycle check  point genes – these are the principle tumor suppressor genes

ii. Likewise, virtually all cancers have gain of function mutations in genes  that promote cell proliferation

iii. Activating mutations in proto-oncogenes

1. Proto-oncogenes are usually involved in one of the many signal  transduction pathways that promote cell proliferation. They may  encode signaling molecules, receptors or components of the  

pathways that lead to activating proliferation

2. The gain of function mutations overrides the build-in control  mechanisms of regulated expression or activation of some  

component of the pathway

3. Gain of function mutations are genetically dominant, meaning it  takes only one mutation at one locus, rather than the homologous  loci of both chromosomes, to exert an effect

4. Three general types of gain of function mutations convert proto oncogenes to oncogenes:

a. Point mutations: that alter the activity of the encoded  


i. Ex: mutation that inhibits the GTPase activity of  

Ras prevent it from turning itself off. Always in  

glycine 12

b. Gene amplification: multiple copies of a gene lead to  

excessive production of a component of the pathway –

results from inaccurate DNA replication

c. Chromosome translocation:  

i. Places the proto-oncogene under control of different  

regulatory DNA sequences leading to constitutive


1. Ex: a signaling molecule is no longer  

regulated, but always expressed. Results  

from chromosomal breaking and improper  


ii. Or fuses two genes together to produce a chimeric  

protein with constitutive activity

iv. Cancer Causing viruses contain oncogenes or activate proto-oncogenes

BSC 300-001 Week 15 Notes

1. In 1911 Peyton Rous showed that certain viruses could cause  cancer when injected into suitable vertebrate hosts (birds and some  rodents, but fortunately not humans)

2. The virus called Rous Sarcoma Virus is a retrovirus: ssRNA  genome that is reverse transcribed and integrated into the genome.  It possesses one gene critical for tumorigenicity, an oncogene that  was named v-src (viral sarcoma)

3. In the 1970’s it was discovered that vertebrates possess a proto oncogene with a high degree of similarity to v-src, and it was  named c-src (cellular)

4. Current hypothesis is that an ancient virus took a copy of the c-src  gene with it after infection and new virus production

5. RSV is an acute retrovirus in that it can induce tumor formation  within days of infection

6. Other slow-acting retroviruses lack oncogenes, but rather  integrate into host genomes near proto-oncogenes and a portion of  the viral genome acts as an enhancer to increase expression of  specific proto-oncogenes

v. Loss-of-function mutations in tumor suppressor genes

1. Tumor suppressor genes normally function to regulate the cell  cycle and halt its progression if cell cycle checkpoints identify  defects (DNA damage, failure to produce chromosome  

biorientation, etc)

2. Intracellular proteins that regulate or inhibit entry into the cell  cycle – for example retinoblastoma (pRb)

3. Receptors or signal transducers for secreted hormones/signals that  inhibit cell proliferation

a. Ex: TGTF-Beta

4. Checkpoint pathway proteins that arrest the cell cycle following DNA damage

a. Ex: p53

5. Proteins that promote apoptosis

6. Enzymes that participate in DNA repair

7. In general, tumor suppressor mutations are loss of function and  require inactivating mutations in both copies of the affected gene a. The mutations are said to behave recessively

8. In some cases, however, reduction to half the normal amount of  protein is sufficient to produce an effect

a. Such genes are said to be haplo-insufficient

9. Some people inherit a loss of function copy of specific tumor  suppressors, which predisposes then to develop cancer, since it  would only take one random somatic mutation in any dividing cell  to completely shut off that gene

a. Ex: children with hereditary retinoblastoma inherit one  non-functional copy of the RB gene. Such children develop  retinal tumors early in life, generally in both eyes

10. Mutations to the RB gene to not only lead to retinal cancer

BSC 300-001 Week 15 Notes

a. pRB blocks the function of the S phase promoting  transcription facto E2F, until a go-ahead signal inactivates  pRB. Mutations in RB are therefore associated with many  cancers

11. Other examples include familial adenomatous popyposis and the  APC tumor suppressor

a. And breast cancer associated with mutation in the tumor  suppressor BRCA1, in which an inherited loss of function mutation increases a woman’s risk of cancer up to 50%

12. Three mechanisms for loss of heterozygosiy of a tumor suppressor  gene:

a. Point mutation

b. Aneuploidy

c. Mitotic recombination between nonsister chromatids. -most  common mechanism

13. Other mechanisms of tumorigenesis

a. Epigenetic changes

i. Altered covalent modifications to DNA and  

histones (methylation and acetylation) that either  

activate or inactivate chromosomal regions for  

transcription. This results from aberrant  

misexpression of DNA and histone modifying  

enzymes, as we saw earlier for the absence of alpha


b. Micro-RNAs

i. MiRNAs are small (20-22 nucleotide) RNAs  

encoded by nonprotein encoding genes. They bind  

the 3’ regions of transcripts with which they have  

sequence complementarity, and in doing so recruit a  

protein complex that prevents translation (either by  

degrading the transcript or inhibiting ribosomes  

from translating). Depending on the function of  

their target genes miRNAs may function in manners  

similar to either oncogenes or tumor suppressors

c. LOF mutations in specific miRNA genes are involved in  formation of some cancers, like chronic lymphocytic

leukemia (CLL) in which two miRNAs miR-5-a and miR 16 prevent cell proliferation by binding and targeting  

transcripts for destruction. In their absence there is  

increased proliferation of B cells

14. Identifying drivers of tumorigenesis

a. Rapid genomic sequencing ability has only recently been  developed (past 10 years) which now make identifying  mutations associated with cancer easier to identify

b. Before that, few mutations were identified. One notable  exception is the BCR-ABL mutation associated with  

Chronic Myelogenous Leukemia (CML)

BSC 300-001 Week 15 Notes

c. Many cases of CML result from chromosomal breakage  

and rearrangement between two regions of chromosomes 9  

and 22 – resulting in an easily identifiable hybrid  

chromosome called the Philadelphia Chromosome

d. The translocation occurs between two genes BCR and  

ABL, generating a hybrid gene and protein called BCR


i. BCR-ABL is a constitutively active kinase that  

phosphorylates numerous cell proliferation proteins  

like JAKs and STATs – promoting rapid  


1. It is also an example of a very successful  

screen for molecules that can bind and  

inhibit the hybrid protein only, and not  

normal BCR or ABL

e. The drug named Gleevec (imatinib) bind and blocks only  

BCR-ABL and effectively prevents its function

f. And has essentially made this type of CML as treatable as  

diabetes, with a regular dosage of the drug

g. At what cost? Up until its patent expired in 2017 it cost  

about $120,000 per year

i. Now the patent is expired and its annual cost is  

about $8800

h. Sequencing cancer genomes has revealed there are few  

driver mutations that are shared by diverse classes of  


i. Most are shared among only 2-10% of tumors in any given  

cancer type, so it is still not possible, for most types of  

cancer, to pinpoint the most critical mutations that could be  

the target of new drugs

j. However, as more and more cancers are sequenced patterns  

may emerge that lead to this type of tumor-type specific  


d. Misregulation of Cell Growth and Death Pathways

i. Effects of oncogenic mutations in proto-oncogenes that encode cell surface receptors

1. Receptors:

a. Oncogenic receptors do not require signaling molecules to  

be activated

b. Mutations that alter HER2 and EGF receptor

transmembrane domains causes dimerization and constitute  

activation of the receptor, even in the absence of thr normal  

EGF-related ligand

ii. RTK/RAS/MAP kinase pathway components are frequently mutated in  cancer

1. Signal-transducing proteins:

a. Ras is one of the most commonly mutated proto-oncogenes  

in human cancers

BSC 300-001 Week 15 Notes

b. RTK/RAS/MAP kinase pathway components:

i. Gain of function mutations in many components of  

this pathway generates an oncogene

ii. Recessive loss-of-function mutation in the NF1

GTPase-activating protein (GAP) leaves Ras in  

GTP on state. The NF GAP was named after a type  

of cancer associated with such LOF mutations  


iii. Chromosomal translocation in Burkitt’s lymphoma

1. Transcription factors:

a. In this example a translocation between chromosomes 8  

and 14 positions the MYC proto-oncogene adjacent to an  

antibody heavy chain (CH) gene, where MYC is regulated  

by the antibody-gene promoter/enhancer

b. This causes overproduction of MYC transcription factor in  

lymphocytes, which causes transformation into a  


iv. Loss of TGF-Beta antiproliferation signaling

1. Transforming growth factor Beta (TGF-Beta) normally inhibits  proliferation to control growth

2. Recall TGF-B binds to its receptor, leading to activation of Smad  transcription factors

3. So cancer can be initiated by mutations that:

a. Prevent receptor/ligand binding

b. Prevent receptor dimerization

c. LOF in SMADs

d. Or the principle genes regulated by the SMADs

v. Two notable and often mutated tumor suppressor genes are the  Retinoblastoma (Rb) and p53 genes

1. Rb blocks transcription of genes required for G1 to S transition. It  is inactivated by G1 and G1/S Cyclin/Cdks. Loss of Rb allows  

cells to progress into S phase without regulation

2. P53 – the guardian of the Genome – is activated in response to  widespread DNA damage and transcriptionally activates the CDK  inhibitor p21 in order to block entry into S phase, allowing time for  cells to repair DNA damage. If repair is unsuccessful, p53 triggers  apoptosis

3. With p53 absent cells proliferate with DNA damage leading to  genetic instability and more DNA damage

4. A subcategory of p53’s target genes are the proapoptotic BAX and  BAK encoding genes. Therefore in the absence of p53 cells cannot  initiate apoptosis, neither intrinsic nor extrinsic

e. Deregulation of the Cell Cycle and Genome Maintenance Pathways in Cancer i. Restriction point control: pRB and unregulated passage from G1 to S 1. pRB guards against uncontrolled proliferation

2. mutations that promote unregulated passage from G1 to S phase  are oncogenic in about 80% of human cancers

3. Not only due to mutations in RB

BSC 300-001 Week 15 Notes

4. Overproduction of Cyclin D

5. Expression of an inhibitory protein E7 – encoded by the Human  Papilloma Virus  

6. Loss of function mutations that prevent the inhibitory p16 from  binding the Cyclin/CDK complex

ii. Loss of p53 abolishes the DNA damage checkpoint

1. P53 guards against excessive DNA damage

2. Recall DNA (UV irradiation) activates ATM kinase activity  triggering three pathways leading to arrest in G1:

a. Phosphorylated Chk2 phosphorylates Cdc25A, marking it  for degradation and blocking its role in S phase cdk  


b. Phosphorylated p53 is stable and promotes transcription of  genes that arrest cells in G1, promote apoptosis, and repair  DNA

c. Phosphorylation of MDM2 releases p53 from targeted  


d. Another regulator p14ARF is activated and binds MDM2  preventing it from acting on p53

iii. The Most vulnerable locus in the human genome

1. A small 30kb region on human chromosome 9 is considered the  most vulnerable locus in the human genome to oncogenic change 2. Tt encodes both the p16 protein and p14ARF as well as another  cyclin D regulator p15

3. Therefore if this locus is deleted, both pRB and p53 functions are  disrupted

12/6 Review Session

I. Lecture 23 – slide 13 pic

a. Mechanism for activating inflammazone

i. Priming signal – synthesis of NaLP3

1. Caspase 1 is the active component


a. Light chains have constant copy of locus

b. In order for functional gene, each B cell has to undergo VJ recombination in light  and V(D)J in heavy chains

i. Loop out

1. Exposes OH group at end of gene itself

2. OH attack adjacent strand and cleaves intervening strand of DNA

a. Symmetric or asymmetric cleavage

i. Asymmetric – polymerase just adds correct nucleotides

b. Opening ends allows things like DNA polymerase to come in  

and add nucleotides

c. DNA Ligase 4 joins them back together

d. Now we have a new locus – added together one joiner and one  

variable segment

c. Junctional imprecision

d. C corresponds to constant region of light or heavy chain  

i. End terminal region is where hydrovariable loops are

ii. C portion identical for every light chain

iii. Also identical for heavy chain but switching occurs (M and D to A or G) III. Lec 24 – slide 15

a. Big pic of processing and presentation?

i. Class I and II found in different cells

1. Class I in all nucleated cells

a. Present self to immune system so it will recognize cell so that  

it won’t be killed

b. Introduces viral particles if that cell has been infected so it can  

be recognized by t cells and be destroyed

2. Class II used by antigen presenting cells (macrophages, B cells,  

dendritic cells)

a. Introduce antigens at cells following phagocytosis,  

endocytosis, etc

b. Mechanism inside cell is very different

IV. How do helper T cells help B cells?

a. Bind to through cell surface B cell receptor – initiates endocytosis and digest it i. Present peptides on cell surface

ii. Bind to T cell that has binding affinity through t cell receptor

iii. Physical contact – release cytokines

iv. Leads to activation (proliferation, class switching, etc)

V. Cancer Slide 32

a. This is one way that viruses can cause cancer:

i. Src viral and Src cellular phosphorylate target proteins

1. Viral has acquired mutations in B start, so it is always on and mimics  phosphorylation

a. So activation not necessary when it attaches bc its already on

12/6 Review Session

2. C src has to be phosphorylated

VI. Loss of function is losing that gene

VII. P53 becomes phosphorylated – MDM dissociates

a. P53 moves into nucleus and transcribe p21 – cell cycle checkpoint  

i. Puts pause on cell cycle til damage can be removed

b. P53 since it has loss of function cannot tell the damaged cell to stop replicating – replication of damaged cell causes even more damage

VIII. Cancer has both gain and loss of function

IX. Benign – gain of function mutation

a. Grows to a certain point

b. If the cells are healthy they will be able to recognize that they are doing something  wrong and put it in senescence (these are moles)

X. P16 and p15 turns on retinoblastoma

XI. P14 turns on p15

XII. Genes that encode these 3 are all within the same region of a chromo – tumor  suppressors

a. Lose region – lose activators on tumor suppressor pathway

XIII. MHC restriction – incompatibility of MHC complex to present antigen that can be  recognized by more than one type of T cell receptor and cell itself from being recognized  by genotypic variant of T cell

XIV. MHC are a fancy type of cell surface receptor – have antigen or ligand bound to them – present that to receptor on another cell

a. MHC complex presents antigen to T cell receptor

b. Only if there is a perfect fit can that T cell be activated by it (lock and key) XV. CD4 and CD8 are located on respective T cells

a. Half of a binding site – other half is antigen

XVI. Helper T cell vs cytoplasmic T cell

a. Immature T cells can go either way – controlled by transcription factors XVII. Dendritic cells- branch of white blood that are scavengers

a. Role of getting into peripheral tissues and looking for non-self pathogens like  bacteria/viruses

b. Migrate there in response to inflammation

c. Professional APCs

XVIII. Invariant chain

a. Involved with MHC class II – things that have been digested and ingested into  endosome

b. Prevent class II from binding to random peptides that they have affinity for

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