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TCU / Biology / BIOL 30603 / What is co-immunoprecipitation?

What is co-immunoprecipitation?

What is co-immunoprecipitation?


School: Texas Christian University
Department: Biology
Course: Molecular, Cellular, and Developmental Biology
Professor: Misamore dr. akkaraju
Term: Spring 2019
Cost: 50
Name: Cell Biology: Exam 2 Study Guide
Description: This covers PPT's 1-7: PPT 1: Cell Signaling I PPT 2: Cell Signaling II PPT 3: Protein Transport and Vesicular Trafficking PPT 4: Glycobiology I PPT 5: Glycobiology II PPT 6: Cytoskeleton I PPT 7: Cytoskeleton II Note: This study guide is about twice as long as the one for the first exam meaning about twice as much content was given in lecture/readings.
Uploaded: 03/05/2019
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Cell Biology: Exam 2 Study Guide

What is co-immunoprecipitation?

PPT 1: Cell Signaling I 

∙ Pre-Lecture Reading 1

o Why is signaling more complicated in multicellular organisms? o How are the signaling cell, target cell, extracellular signals, and  intracellular signals related?

o Types of signaling

 Endocrine

 Paracrine

∙ Autocrine

 Contact dependent

 Neuronal  

o What determines whether a cell responds to a signal molecule?  Has to have a receptor

o Why would the same signal molecule interacting with the same  receptor produce different responses to different target cells?  Effector proteins-have direct effect on the target cell

 Intracellular relay system and intracellular effector proteins vary from cell to cell

What are examples of common cellular responses to extracellular signals?

o There are both cell-surface and intracellular receptors. How will  the signal molecules that bind them differ?

 Cell-surface receptors

 Intracellular receptors

o Understand the connection between steroid hormones and  nuclear receptors. What function does the activated nuclear  receptor have on the cell? Don't forget about the age old question of What was the title of the book that charles scott sherrington wrote?

 Steroid hormones

 Nuclear receptors

∙ Act as transcription factors in the nucleus

o Besides steroid hormones, give another example of an  extracellular signal molecule that can pass through the plasma  membrane?

 NO gas

o How does Viagra work? Why is it effective in treating erectile  dysfunction?

 Blocks the enzyme that degrades cGMP

Why is signaling more complicated in multicellular organisms?

 Prolongs the NO signal  blood vessel dilation  more  

penile erection

o What are examples of common cellular responses to extracellular signals? What are effector proteins?

 Effector proteins-have a direct effect on the behavior of  the target cell

 Common cellular responses

∙ NO gas

∙ Testosterone

∙ Estradiol

∙ Cortisol

∙ Thyroid hormone (thyroxine)

o What are intracellular signaling pathways? Explain how these  pass information from the surface of the cell to the effector  proteins. In addition to relaying a signal, what are some of the  functions of intracellular signaling pathways? We also discuss several other topics like What is the testosterone in males?

 Message passed downstream from one intracellular  

signaling molecule to the next

 Functions of the intracellular signaling pathway

∙ Relay the signal

∙ Amplify the signal

∙ Detect signals from more than one pathway and  

integrate them together

∙ Distribute the signal to more than one effector  


o How do intracellular signaling proteins behave as molecular  switches? Understand the two major switches

phosphorylation/dephosphorylation and GTP binding/hydrolysis  Receiving a signal causes them to switch from inactive to  active state

 Phosphorylation/dephosphorylation

∙ Protein kinase-adds phosphates We also discuss several other topics like What is the additive rule of probability?

o Serine/threonine kinase

o Tyrosine kinase

∙ Protein phosphatase-removes phosphates

 GTP binding/hydrolysis

∙ Have intrinsic GTP-hydrolyzing (GTPase) activity

∙ Types of GTP binding proteins

o G proteins

o Small monomeric GTPase

o What is a phosphorylation cascade?

 Switch proteins controlled by phosphorylation are  

themselves kinases

∙ One protein kinase activated by phosphorylation  

goes on to phosphorylate the next protein kinase in  

the sequence and so on

o What is a small monomeric GTPase?

 Type of GTP-binding protein; cell-surface receptor

 Regulatory proteins

∙ GEF-promote exchange of GDP for GTP

∙ GAP-promotes exchange of GTP for GDP


∙ Cellular Communication

o Cells don’t exist in isolation

∙ Extracellular Receptors

o Extracellular signal Don't forget about the age old question of What can a newborn see most clearly before their vision develops?

o Intracellular signaling molecules

o Effector proteins

o Extracellular signal  extracellular receptor  intracellular  signaling molecules  effector protein  target cell responds ∙ Major Effects of Extracellular Signals

o Altered protein function

o Altered protein synthesis

∙ Major Effects of Extracellular Signals Continued

o Nuclear receptors

∙ Intracellular Receptors

o Small enough or hydrophobic enough to cross the plasma  membrane

∙ Intracellular Signaling Pathways Don't forget about the age old question of Do we have case morphology in english?

o Signal transduction

o Multiple functions

∙ Intracellular Receptors: Intracellular Enzymes

o Dissolved gases such as NO

 Cyclic GMP-second messenger

∙ Intracellular Receptors: Nuclear Receptors

o Bind to steroid hormones

o Huge impact on physiology and development

o Memorize cortisol, estradiol, and testosterone effects  (Table 16-1) 

∙ Intracellular Receptors: Nuclear Receptors

o Signal molecule

 Released  travel throughout entire body  passes into  plasma membrane  passes into the cells

 Cell expresses it  will meet its receptor

o Steroid hormones are nuclear receptors Don't forget about the age old question of What punishment can come with a capital felony?

o Where nuclear receptors bind, have specific DNA sequences  Consensus sequence in promoter

∙ Intracellular Receptors: Enzymes

o NO diffuses through membranes

∙ Viagra

o Treats erectile dysfunction

 cGMP accumulates  lots of relaxation of smooth muscle  increases blood flow  erection

∙ Molecular Switches

o Modifying a protein

 Phosphorylation/dephosphorylation

o Signaling by GTP binding proteins


 Hydrolysis of GTP to GDP

∙ Cellular Crosstalk

o Not just one signaling pathway happens at a time

∙ 3 Major Classes of Cell Surface Receptors

o Ion-channel coupled receptors

o G-protein coupled receptors

o Enzyme coupled receptors

∙ G-Protein-Coupled Receptors (GPCRs)

o Transmembrane receptor

o 7 passes; looks like squiggle

o Coupled to G proteins

o General components

 Receptor

 G protein

 Enzyme

o Signal binds to GPCR  conformational change  allows G protein to be activated  interacts with enzyme and activates signaling  pathways

∙ Receptors are coupled to G-proteins

o GPCR is complex of 3 subunits

 Alpha

∙ Inactive: GDP

∙ Active: GTP

 Beta

 Gamma

∙ How G-Protein-Coupled Receptors Work

o Inactive G protein with GDP  signal binds to G protein

o Binding causes dissociation of GDP  GTP can bind 

conformational change of alpha subunit  decreases affinity and  alpha subunit can dissociate from beta gamma subunit  effect  from alpha or beta gamma subunit

o Exchange GDP for GTP  activates G protein

o Can activate or inhibit target

o Intrinsic GTPase activity

 GTP is hydrolyzed to GDP  subunit dissociates from target protein  complex reforms

∙ G Protein Targets: Ion Channels

o Ion channels

 ACH (extracellular signaling molecule)  released  binds  to receptor  activation of alpha subunit  dissociation of  

alpha from beta gamma subunit (GQ)  beta gamma  

subunit interacts with ion channel causing them to open  lets K+ through and changes membrane potential  harder for cells to become activated  heartbeat slows  GTP is  

hydrolyzed to GDP


∙ G Protein Targets: Enzymes

o Enzymes

 Alpha subunit is active with GTP  interacts with enzyme  converting some molecule to another molecule (2nd 

messenger)  activate other things in the cell

o 2 major enzymes

 Adenylyl cyclase-cyclic AMP (cAMP)

 Phospholipase C-inositol triphosphate (IP3) and  

diacylglycerol (DAG)

∙ Adenylyl Cyclase & Cyclic AMP

o Adenylyl cyclase: AMP  cAMP

o Cyclic AMP phosphodiesterase: cAMP  AMP

o Has many targets

 Protein kinase A (PKA)  phosphorylates other proteins  o Amplifies a signal

∙ cAMP signaling

o cAMP signaling 1

 Binding of adrenaline  activates G protein (alpha subunit;  stimulatory)  adenylyl cyclase activated (brown protein)  AMP to cAMP  binds and activates PKA  activates other  

proteins such as phosphorylase kinase  activates other  

proteins such as glycogen phosphorylase  causes  

glycogen to break down  increase of glucose in cell 

helps muscles do work and provides energy

∙ 1st messenger-adrenaline

∙ 2nd messenger-cAMP

 GTPase property of G proteins shuts pathway off

∙ cAMP Signaling Continued

o cAMP signaling 2

 Adrenaline  G protein  activates adenylyl cyclase  AMP  to cAMP  activates PKA  goes to nucleus 

phosphorylates transcription regulator  activated and  

regulates gene expression by turning genes on and off  

with transcription factor

o Glycogen breakdown will be faster because just modifying  proteins

∙ Pre-Lecture Reading II

o What is co-immunoprecipitation? How can this be used to study  protein-protein interactions?

 Co-immunoprecipitation

∙ How it works

o Attach antibodies specific to one member of a  

suspected protein: protein binding pair to  



o Beads are incubated with a mixture of proteins  

often from a cell lysate

o Protein of interest binds to the antibody

o Is separated from all other proteins

o Other proteins that bind to this protein of  

interest also may be precipitated

o Binding partners of the antigen will be co


o Pelted beads can be physically or chemically  

treated to disrupt all weak protein-binding  


o Released proteins bound to the  

immunoprecipitated proteins can be identified

∙ Used to identify which proteins interact when an  

extracellular signal molecule stimulates cells

o Why would a mutant form of Ras, that can’t hydrolyze bound GTP result in constant activation of the Ras protein? Why would this  result in cell proliferation even in the absence of a proliferation  signal?

 Constantly active because it has lost its ability to hydrolyze bound GTP keeping Ras protein switched on

∙ Can’t turn Ras off

 Mimics effect of extracellular signal

o When discussing signaling pathways, the terms “upstream” and  “downstream” are often used. This refers to the order in which  proteins are activated. If there is a signaling pathway that is  activated in the order X  Y  Z, then Y is upstream of Z and  downstream of X. In the example described in this reading  assignment, understand how a mutant form of protein X/Y and  overactive Ras were used to determine the order of the signaling  proteins (X  Ras  Y)

 See if overactive Ras can “rescue” the cell and restore  


 With inactive X

∙ Overactive Ras can restore signaling

∙ Thus downstream of X; encounters Ras as X’s signal  

is passed down pathway and has time to restore it

 With inactive Y

∙ Overactive Ras can’t restore signaling

∙ Thus upstream of Y; doesn’t encounter Ras as Y’s  

signal is passed down pathway and thus there is not  

time to restore it


o The western blot is a common technique used to detect a  specific protein in a mix of many proteins. Focus on  

understanding how the technique works.

 Western blot

∙ Protein samples are mixed with the detergent SDS

∙ Protein/SDS mixture is boiled

∙ Samples analyzed by SDS-polyacrylamide gel  

electrophoresis (SDS-PAGE)

o Electric current applied to gel

o Proteins are negatively coated and migrate  

towards positive electrode

o Proteins separated based on size

 Small proteins migrate faster than large  


∙ Proteins transferred from the gel to a nitrocellulose of

polyvidone fluoride (PVDF) membrane

∙ Actual western blotting step

o Antibodies are used

 Primary antibody-specific antibody  

binds to a specific protein

 Secondary antibody-attaches to the  

primary antibody

 Add substrate

∙ Interaction between enzyme and  

substrate allows us to see protein  

of interest

∙ Cholera

o Caused by bacteria

o Spread through contaminated water  gets into body and  intestines  causes massive watery diarrhea

 Diarrhea allows for transmission of bacteria

o Very deadly for children

o Cures/prevention

 Oral rehydration therapy

 Clean drinking water

∙ Cholera Signaling

o Secretes cholera toxin  binds to receptor on intestine epithelial  cells  bring toxin into cell through endocytosis  once inside it  interacts with G-protein (Gs alpha)  modifies it to where it can’t  hydrolyze GTP  constantly on and activating adenylyl cyclase  lots of cAMP  opens chloride channel  chloride leaves cell in  large amounts because signal can’t turn off  negative chloride  ion draws water out  water into lumen of intestine  constant  watery diarrhea


∙ G Protein Targets: Phospholipase C

o GPCR  G protein  GTP is activated  beta gamma subunit  dissociates (GQ)  activates/interacts with phospholipase C (PLC)  acts on target of inositol phospholipid  cleaves it  creates  inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG)  activates PKC  phosphorylates target  other targets

 IP3  ER  opens Ca2+ channel  Ca2+ leaves ER 

activated PKC (protein kinase C)

∙ Calcium is normally low and sequestered in ER

 DAG  stays in plasma membrane  activates PKC

o Ca2+  calmodulin  CaM kinases (Ca2+ calmodulin dependent  protein kinases)  go on to phosphorylate targets

∙ Ion-Channel Coupled Receptors

o When bound to ligand, they open and allow ions to go through o Alters membrane potential

∙ Enzyme Coupled Receptors

o Receptor tyrosine kinases (RTKs)

 Must dimerize to be active

 Autophosphorylate each other

 Receptor  makes a dimer/heterodimerizes (2 halves of  receptor come together to be active); can only dimerize in  presence of their ligand; signal molecule is often also in a  dimer  ligand is bound  they can phosphorylate each  

other on tyrosines  stems that are in cytosol have kinase  activity  serve as docking scaffold  many proteins can  

now interact with them  adaptive proteins can bind to  

other things and have multiple signaling paths

∙ Receptor Tyrosine Kinases

o Different RTKs  recruit different signaling proteins  different  responses

o Allows many signaling molecules to bind to receptor

o 3 common pathways

 Phospholipase C-can be activated by GPCR; RTK can also  activate it

 Ras

 PI-3-kinase

∙ Ras Monomeric GTPases

o 2 classes of GTP binding proteins

 G protein-couples with GPCR; heterotrimeric

 Ras-monomeric, small GTPases

o How GTPases work

 Active-bound to GTP

 Inactive-bound to GDP

o 2 other enzymes key to regulation

 GEP: GDP  GTP; activates Ras


 GAP: GTP  GDP; inactivates Ras

∙ RTK and Ras Signaling

o RTK  dimerizes with presence of signal molecule  adaptor  protein  interacts with Ras-GEF  GDP is exchanged to GTP  Ras protein is activated  activates downstream targets

∙ Ras and MAPK Signaling

o Ras protein is activated  activates downstream product which is Map Kinase Kinase Kinase  phosphorylates Map Kinase Kinase  phosphorylates Map Kinase  phosphorylates target proteins  causes changes in protein activity or gene expression by  

transcription factors

o Common target is changes in cellular survival and/or proliferation  30% of cancers have mutation in Ras

 inactivating GTPase activity  Ras is always active and  can’t turn off by hydrolyzing GTP to GDP 

survival/proliferation  cancer

∙ RTK and PI-3-Kinase Signaling

o RTK  phosphorylates inositol phospholipid (IP3) at 3 position  phosphate is added  interacts with target molecules such as  protein kinase 1  Akt  cell survival  will go and phosphorylate  Bad

∙ AKT Promotes Cell Survival

o Akt  phosphorylates Bad  inactivates Bad  releases active  Bcl2  suppresses apoptosis and promotes cell survival

 Bad normally stimulates apoptosis by inactivating Bcl2

∙ Summary

o Be able to draw all pathways from memory! 

PPT 2: Cell Signaling II 

∙ Why study signal transduction?

o Real world applications

∙ PPAR-gamma is expressed in Huntington’s disease article o Looking to see if they can develop therapeutics to help treat  Huntington’s disease

∙ Huntington’s Disease (HD)

o Dominant genetic disease

o Onset later in life and thus genes get passed on

o Neurodegenerative disease

 Problems with speech, coordination, motor function, and  cognition

o Lethal and no cure

 Treatments only help relieve symptoms

o Shana Martin and her mom

 Mom was adopted so it was a surprise she had HD

 10 when mom was diagnosed


∙ Huntingtin Gene (HTT)

o Caused by trinucleotide CAG repeat in coding region

 Excess of glutamines

 More repeats = earlier onset and quicker progression

o Repeats of CAG  excess glutamine  incorrect protein  folding/conformation  resistant to degradation and accumulates in cell over time

o Gene we all have; has normal function in cell

o Main target is brain; accumulates in nucleus of neurons ∙ Molecular Impacts

o Transcriptional dysregulation

o Impaired protein degradation

o Altered protein folding

o Disrupted neuronal circuitry

o Mitochondrial dysfunction

∙ What proteins does HTT interact with?

o Knew that transcription was disrupted

o Wanted to identify transcription factors that interact with HTT  Both in nucleus of cells

o Transfected HEK293 cells

 HEK293 cells-cell line of human cells

∙ Immortalized cells

∙ Easy to work

∙ Work in the same way over time

∙ Mechanisms that stop cell division are absent

o Transfect cell line with construct

 Recombinant DNA construct-splice together different  things into a plasmid to express them

∙ Combined GFP (fluorescing protein) with HTT

o Produces 1 protein

 For transfection

∙ Promoter drives transcription and translation to make

GFP-HTT protein

o HTT-25Q-normal version of HTT gene

∙ What proteins does HTT interact with? Continued o GFP fused to HTT-25Q

o Recombinant DNA construct used

 HTT-25Q-normal version

o Did immunoprecipitation

 Used antibody

∙ What proteins does HTT interact with? Continued o Lyse open the cells and collect the proteins

o Use antibodies to separate out/tag only the GFP-HTT-25Q  proteins


o Co-immunoprecipitation (IP)-add molecular beads to  antibodies which makes complex heavier

 Use a GFP specific antibody to pull down GFP-HTT-25Q and  whatever is bound to it

o Centrifuge tube to immunoprecipitate

o Discard supernatant

∙ Pre-Lecture Reading I

o Recombinant DNA

 Joining together of DNA molecules from 2 different species  that are inserted into a host organism to produce new  

genetic information

 Makes it possible to isolate one gene or any other segment of DNA

o Transfection

 Virus-mediated gene transfers into eukaryotic cells

 Results in unexpected morphologies and abnormalities in  target cells

o Immunoprecipitation

 Antibody: antigen interactions

 Helps identify proteins of interest using techniques like the  Western blot

 Can be used to isolate a specific protein from a mixture  and to identify proteins that bind to each other

 Can isolate a specific protein from a mixture of  


∙ Antibodies to the protein of interest are chemically  

attached to small beads

∙ Beads are incubated with the mixture of proteins

∙ Specificity of antibody: antigen binding causes only  

the proteins in the mixture that interact with the  

antibodies to attach to the immobilized antibodies

∙ Centrifuge the mixture

∙ Precipitate the antibody/antigen/bead complex

∙ All other proteins remain in the supernatant

 Protein of interest separated from all other proteins

o Co-immunoprecipitation

 Pull-down assay

 Helps identify protein: protein binding interactions

 How it works

∙ Attach antibodies specific to one member of a  

suspected protein: protein binding pair to beads

∙ Beads are incubated with a mixture of proteins

∙ Protein of interest will bind to the antibody and can  

be immunoprecipitated


∙ Other proteins that bind to this protein may also be  


∙ The beads can be physically or chemically treated to  

disrupt all weak protein-binding interactions

o HD Abstract

 PPAR-delta is…

∙ Repressed in HD

∙ Required for normal neuronal function

∙ Targeted therapeutically

 HD is progressive neurodegenerative disorder

∙ Caused by accumulation of CAG repeats that code for


 PPAR-delta interacts with HTT

∙ Mutant HTT represses PPAR-delta

 Increased PPAR-delta fixed mitochondrial dysfunction and  improved cell survival of neurons in mice

 Expression of dominant negative PPAR-delta in CNS of mice showed phenotypes that are typically associated with HD

 In mouse models of HD, activation of PPAR-delta using  

agonist KD3010 improved motor function, reduced  

neurodegeneration, and increased cell survival

 PPAR-delta activation also reduced HTT-induced  

neurotoxicity in vitro and in stem cells from individuals with HD

 PPAR-delta activation may be beneficial for HD and related  disorders

∙ What proteins does HTT interact with? Continued o Add GFP antibody

o Centrifuge tube

o GFP-HTT-25Q creates one giant protein

 Unknown protein should only be interacting with HTT

∙ What proteins does HTT interact with? Continued o Transcription factor binding site array

 Fusion protein (GFP-HTT-25Q) and anything it is bound to  Mystery protein is likely a transcription factor

o What is this array?

 Tiny chips bound with strands of DNA

 Each strand has a different sequence which is a different  consensus site for a transcription factor

 Look and see which DNA consensus sequence binds with  the mystery protein

∙ Proteins should only interact with one consensus  


o Mystery protein is PPAR-delta (transcription factor) 12

∙ Peroxisome Proliferator Activate Receptor (PPAR) o 3 parts

 Alpha

 Delta

 Gamma

o Involved in lipid metabolism

o Members have different gene targets in nucleus (transcription  factor)

o Will heterodimerize with retinoid X receptor (RXR)  Binds with PPAR-delta

 X can be any amino acid

 Type of intracellular nuclear receptor

∙ Ligand  nucleus  activates transcription

∙ Confirm HTT-PPAR-delta Interaction

o Make sure that PPAR-delta isn’t interacting with GFP

o 1st experiment

 Take GFP and transfect into cells

 Take PPAR-delta and added flag tag

 No HTT present

 Both transfected into cells  green and red proteins  

expressed  collected cell lysates  immunoprecipitated  

with GFP antibody  

∙ Only GFP left in tube, why?

o IP with GFP and pull down GFP

o Don’t pull down PPAR-delta because only HTT  

binds to PPAR-delta, not GFP

o Confirms that HTT is only interacting with PPAR


o 2nd experiment

 Overexpressing GFP-HTT protein and PPAR-delta with flag  collect lysates  immunoprecipitate with GFP antibody

 Would see everything in tube

∙ If you pull down GFP fused to HTT, it should also pull  

down PPAR-delta because GFP has HTT with it and  

HTT should bind to PPAR-delta

∙ Confirm Interaction Continued

o How would you actually see this?

 Western Blot

∙ Uses gel electrophoresis

∙ Takes proteins before or after IP (GFP-HTT and PPAR


∙ Load on well of gel

∙ Apply current and let proteins run

o Separate by size


 Larger proteins are slower

o Won’t see anything because it is a clear gel

∙ Take gel and transfer to nylon membrane

o Stack weight on it which forces water from  

aqueous gel into nylon membrane underneath  


o Migrates out of gel into membrane

o Lot easier to work with membrane

∙ Detect presence of a specific protein using an antibody (i.e.  immunoblot)

o Transferred to nylon membrane

o Western blots are dependent on antibodies that determine  proteins of interest

o IB done after western to see if protein is there

 Immunoprecipitated with GFP  detect presence of PPAR delta so have to have antibody against PPAR-delta

∙ Primary antibody-recognizes the protein (PPAR


∙ Secondary antibody-recognizes primary antibody;  

enzyme substrate coupled to it causes florescence

o Add chemical to cause fluorescence to see if protein of interest is present

 No protein of interest  primary antibody can’t bind 

secondary antibody can’t bind  nothing fluoresces

∙ Western Blot

o Run proteins on gel  transfer to membrane  use immunoblot  incubate/wash  add detection system  expose nylon  

membrane to detection system (camera)  see or don’t see  bands (usually only 1 band)

o Only see proteins that bind to antibodies

o Autoradiography

o Separate by size

 Higher on gel = bigger protein

o Can run protein ladders just like DNA ladders

 Looking for protein band that is right size for protein of  interest

∙ Experimental Set Up

o 3 batches of cells with different tags

 Transfecting with flagged PPAR-delta and GFP constructs ∙ GFP (control)

∙ GFP with normal HTT (25Q)

∙ GFP with abnormal HTT (104Q)

o Allow proteins to express

o Both HTT ones should interact with PPAR-delta


o Overexpress proteins  collect proteins  lyse cells  IP with GFP   western blot with antibody for PPAR-delta (flag antibody)  IP with GFP-make sure that HTT is what interacts with  PPAR-delta

 IB with flag antibody-it specifically recognizes PPAR


o Only ones with HTT produced a band

∙ Experimental Set Up and Actual Data from Paper

o Input-all cellular proteins before IP

o 3 different experiments (see groups above)

o Will find bands for all 3 when input is run because PPAR-delta  present in all 3 groups originally

 Make sure going in that you have everything

∙ Data from Paper

o Input-all cellular proteins before IP

o Lyse cells and run everything on the gel

o After IP, only ones with HTT should have bands because PPAR delta doesn’t interact with just GFP

o Why is it important to include GFP alone as control?  Shows that HTT and PPAR-delta are actually interacting o Why did they IB for GFP? 

 Make sure that result isn’t false negative

 Make sure you have proteins that were run initially

∙ There are proteins in the control, but just not PPAR

delta proteins

 Constructs are present and the right size

∙ Band sizes: 104Q > 25Q > GFP

 Show that GFP was successfully immunoprecipitated, but  just doesn’t contain PPAR-delta and thus doesn’t pull down  when IB for PPAR-delta is used

∙ Hypothesis and how they tested it

o Hypothesis-mutant HTT binds to PPAR-delta and prevents it  from activating gene transcription

 If true, then PPAR-delta gene expression in mouse with HD  will cause gene expression to decrease

o Made knock-in mouse expressing HTT protein with different  polyQ lengths

 Mutant gene added exactly where normal gene is

o Transgenic-inserted gene into animal; randomly inserted  anywhere

o Compare expression of genes that are known to be regulated by  PPAR-delta

 WT = ST-Hdh Q7/Q7

 HD = ST-Hdh Q111/Q111

 Knew what genes are regulated from previous studies


∙ PPAR-delta gene expression in HD Mice

o Looked at genes/targets of PPAR-delta

o Normal mouse should have higher expression of PPAR-delta  genes because PPAR-delta is normal and will increase gene  expression

o HD inhibits gene expression of PPAR-delta

 Shows interaction of HTT with PPAR-delta has a functional  consequence

∙ Next Hypothesis and Experimental Set Up

o Hypothesis

 Mutant HTT is interacting with PPAR-delta and preventing  gene transcription (proved in previous study)

 Loss of PPAR-delta gene expression is contributing to  

mitochondrial abnormalities, neurodegeneration, and  

motor dysfunction

∙ Interfering with regulation of PPAR-delta produces  

similar phenotypes to HD

o Are not manipulating HTT

o Knock-in mouse

 Messed with dominant negative PPAR-delta

∙ Prevents interaction between PPAR-delta and RXR

∙ Masks normal effect of PPAR-delta

o Normal PPAR-delta expressed, but dominant  

negative binds and doesn’t interact with RXR 

decreased gene expression

∙ Results from this Study

o Loss of PPAR-delta gene expression contributes to  phenotypes similar to those seen in HD

 Mitochondria are smaller

 Less ATP produced

 Decreased neuron numbers

o Loss of PPAR-delta contributes to motor dysfunction  Weigh less

 Loss of balance

 Hind legs clasp up when held upside down

 Muscle control is inhibited (kyphosis/rounding of back)

o Conclusion

 Interfering with PPAR-delta activity and therefore gene  expression can cause the same phenotypes as HD mouse,  but this model had nothing to do with HD

∙ PPAR-delta dysregulation is key to HD pathogenesis

∙ Phenotypes similar to HD; but no mutation in HTT  


o Overall summary of paper figures so far 


 Figure 1-physical interaction between HD and PPAR-delta;  nobody had seen this before

 Figure 2-functional consequence of interaction between  HTT and PPAR-delta; PPAR-delta gene expression is  

dysregulated (decrease in gene expression)

 Figure 3-PPAR-delta dominant negative mouse leads to  similar phenotypes as HD; dysregulation of gene  

expression leads to similar phenotypes as HD independent  of HTT protein

∙ Creating a Treatment Option

o Effective treatment would be to increase signaling of PPAR-delta o Used KD3010 because it is potent, specific, and can cross blood  brain barrier

 HD pathology is in neurons of brain

o KD3010 is approved for use in humans in a Phase 1b  metabolic disease safety trial

 Already tested in humans

 No incidence of side effects

 Already approved in clinical trials

 Phase 2-effective with small patient group (maybe 100) o Agonists developed for PPAR-delta NOT HTT 

∙ KD3010 PPAR-delta Agonist Results

o 3 groups of mice looked at

 Non-Tg control-control mice (wild type)

 HD control mouse with a vehicle control (HD is  


∙ Vehicle control-control for whatever treatment  

worked, whatever drug is dissolved in (usually water  

or saline)

o Placebo control

o Just injecting the solvent doesn’t produce an  


 HD mouse with KD3010 (HD treated)

 Important to include both controls 

o Looked at neurological dysfunction score

 Higher number = more dysfunction

 Mice that got treated did better than HD mice, but not back to normal of WT

o Conclusions

 Control without HD survived the best

 Vehicle (HD untreated) survived the worst

 Treatment helped, but is not a cure because it didn’t bring  mice back to WT

∙ Helped them survive longer than vehicle


∙ Helped make symptoms less severe

∙ Summary of How PPAR-delta Works

o PPAR-delta ligand is intracellular signal  passes through  membrane of cell  binds to PPAR-delta receptor  moves to  nucleus  interacts with RXR  transcription factors initiated o Known prior to study

∙ What did these scientists discover that was novel? o Showed relationship between PPAR-delta and HTT  PPAR-delta ligand  passes through membrane  binds to  PPAR-delta receptor  interaction with HTT  decreases  

gene expression of PPAR-delta  causes phenotypes similar to HD (mitochondrial abnormalities, neurodegeneration,  

and motor dysfunction)

∙ Can’t interact with RXR

o Repurposed KD3010 as a treatment

∙ Shana Today

o Didn’t have HD from mom!

PPT 3: Protein Transport and Vesicular Trafficking 

∙ Pre-Lecture Reading I

o Why do cells need separate compartments in order to carry out  their reactions? Why not just have everything mixed together?  How do cells accomplish this?

 Different intracellular processes need to be separated to  avoid chemical chaos and degradation of enzymes and  


 Strategies for separation

∙ Aggregate enzymes needed for certain reactions

o Prokaryotes and eukaryotes

o Ex. synthesis of DNA, RNA, and proteins

∙ Organelles

o Eukaryotes only

o Cell membranes provide selectively permeable  


o Know the function of all the major organelles (Table 15-1). Which  organelle is the most common (at least in liver cells)? Which  membrane in the cell has the largest area? (hint: it is not the cell  membrane)

 Cytosol

 Nucleus

∙ Nuclear envelope

 Endoplasmic reticulum (ER)

∙ Rough ER-has ribosomes attached to its cytosolic  



o Ribosomes

∙ Smooth ER-lacks ribosomes

o Has some unique functions

 Steroid hormone synthesis

 Sequesters Ca2+

 Detoxifies organic molecules like alcohol

 Golgi apparatus

 Lysosomes

 Endosomes

 Mitochondria

 Chloroplasts (plant cell)

 Peroxisomes

 Which organelle has the largest area?

∙ Nucleus

 Which organelle is the most common (at least in  liver cells)?

∙ Smooth ER

o Why are bacteria able to get by with only their plasma  

membrane to carry out all membrane-dependent functions (such  as ATP and lipid synthesis)? Why don’t they need organelles like  eukaryotic cells do?

 Smaller size and volume

 Plasma membrane is sufficient to sustain all vital functions o How did membrane organelles evolve in eukaryotic cells? What is the endomembrane system? Why are mitochondria and  

chloroplasts isolated from the extensive vesicular traffic that  connects most of the other membrane-enclosed organelles?  How did organelles besides mitochondria and  

chloroplasts evolve?

∙ Invagination of the plasma membrane of bacteria

∙ Led to two-layered envelope

∙ Pinched off completely from the plasma membrane

 Endomembrane system-ER, Golgi apparatus,  

peroxisomes, endosomes, and lysosomes

∙ Interiors of these organelles are connected and  

communicate with each other

 Mitochondria and chloroplasts

∙ Mitochondria-aerobic prokaryote was engulfed by a

larger pre-eukaryotic cell

∙ Chloroplast-eukaryotic cell with mitochondria  

engulfed a photosynthetic prokaryote

∙ Both have own small genomes and can make some  

their own proteins


∙ Remain isolated from vesicular traffic that connects  

all other organelles

∙ Organelles

o Function same from cell to cell, but number of each varies  o Allows cell to specialize and do more functions than prokaryotic  cell

o Divide cellular functions and metabolic processes

∙ Protein Sorting

o Free ribosomes

 Made in cytosol  stay in cytosol or go to nucleus,  

mitochondria, chloroplasts, peroxisomes

o Membrane-bound ribosomes in ER

 Made in cytosol  signal indicates to go to ER  majority of  protein synthesis occurs when bound to ER  translocated  into ER  stay in ER or go to other organelles (Golgi,  

endosomes, lysosomes) or secreted outside of cell

∙ Signal Sequence Determines Location

o Gated transport

o Signal sequences-defined, particular sequences of different  organelles/locations; 15-60 aa long

 Physical properties of amino acids more important than  exact sequence

 Removed once protein is sorted

∙ Tends to stay in destination

 Necessary and sufficient for directing a protein to its  


∙ Necessary and Sufficient

o Signals are necessary for transport to correct destination  Won’t reach ER if signal sequence is gone; will remain in  cytosol

o Signals are necessary for movement to occur

o All you need is signal sequence; it is sufficient

 If you put signal sequence on protein that normally stays in cytosol, the protein will move to the ER even though it is  

supposed to be in the cytosol

∙ The signal alone is sufficient for movement to occur

o Mutations in the signal sequence can keep the protein from going to the correct location

∙ Protein Transport: 3 Mechanisms

o Gated transport-moves things in and out of the nucleus  Nuclear pores-proteins go into nucleus and mRNA comes  out

o Transmembrane transport-channels in the membranes  (protein translocators)

 Cytosol  chloroplast, mitochondria, peroxisomes, and ER 20

o Vesicular transport-vesicles bud off of ER  take proteins to  other organelles

 ER  Golgi  wherever

∙ Nuclear Transport

o Nuclear pores-large, complex, 60 or so different proteins, on  outside of nucleus

 Fast transport in and out of nucleus

o Proteins that make up interior of pore are large and  disordered

 Mesh

 Kelp forest

 Long strands of protein

 Proteins need to be small enough to diffuse through the  mesh

∙ If not, have to have active transporter to help it  


o Requires energy if protein is too large

o Cytosolic fibers-nuclear import receptors bind to them  Get into nuclear pore and push disordered chains aside to  bring in cargo

 Opens up passageway through the meshwork

∙ Nuclear Transport Continued

o Key thing is the presence of nuclear localization signal (NLS)  Nuclear localization signal (NLS)-tells the receptor that  the protein belongs in the nucleus

∙ Allows signal sequence to bind to the receptor

∙ Allows uptake of proteins

∙ Is sufficient by itself

∙ Still need import receptors though

∙ Nuclear Transport Continued

o Protein that is being transported to the nucleus

 Transported folded

o Recognizes NLS

o Import receptors-imports cargo

o RAN protein-monomeric GTPase

 Binds GTP or GDP

∙ Bound to GTP-can bind to cargo receptor

 Provides energy for transport

o Import/export system

 Protein with NLS (necessary and sufficient to be taken up  into the nucleus)  binds to cytosolic nuclear import  

receptor  passes through nuclear pore with or without  

cargo  leaves  picks up cargo on next run if didn’t  

initially pick up cargo


 Protein brought into nucleus  binding of RAN-GTP to the  import receptor  triggers release of cargo  import  

receptor leaves nucleus bound with RAN-GTP  outside of  nucleus  RAN-GTP hydrolyzed to RAN-GDP  RAN-GTP  

dissociates from receptor  receptor can take up another  

protein for transport

 Cargo can only get in by being bound to the receptor

 Energy for transport comes from RAN

o Takes energy to move things in/out of the nucleus  Hydrolysis of GTP causes conformation change  more  energetically favorable  energy is provided

o Why is nucleus essential?

 Keeps things organized

 Can control import/export

 Keeps RNA from leaving before it has been processed

∙ Allows for post-transcriptional modification

∙ Nuclear Transport Continued

o RAN GTPases

 Critical for regulating import/export

 Accessory enzymes help exchange GDP and GTP

∙ RAN-GEF: GDP  GTP; creates RAN-GTP; only in  


∙ RAN-GAP: GTP  GDP; creates RAN-GDP; only in  


 Causes directionality for import/export

∙ Nuclear Transport Continued

o Localization of RAN-GTP provides directionality for  import/export

 Import receptors-release cargo in presence of RAN-GTP  Export receptors-bind cargo in presence of RAN-GTP

o Import summary

 Protein with NLS  binds to cargo receptor  moves into  nucleus  RAN-GTP binds to import receptor  triggers  

release of cargo  cargo is delivered  import receptor can  go back to cytoplasm

o Export summary

 Export receptors bind cargo in nucleus in presence of RAN GTP  moves to cytosol  conversion of RAN-GTP to RAN

GDP  triggers release of cargo from export receptor

∙ Nuclear Envelope During Mitosis

o Nucleus breaks down during prophase

o During telophase

 Nuclear envelope and nuclear pores must reform


 Proteins must be reimported to nucleus from cytosol after  every division cycle

o Lots of energy used to do this process

o Proteins that are nuclear proteins don’t have their NLS cleaved ∙ Protein Transport: 3 Mechanisms: Transmembrane Transport o Transmembrane transport-important for mitochondria,  chloroplasts, ER, and peroxisomes

∙ Mitochondrial Transport

o Most protein imported from cytoplasm even though it has its own genome and can make proteins

 Most BUT not all are imported!

o Fully synthesized in cytoplasm, but transported unfolded o Signal sequence will have different combinations of amino acids  than NLS  can bind to specific import receptors on mitochondria membrane

o How it happens

 Precursor protein with signal sequence  binds to import  receptors  protein translocated through outer membrane  translocater  inner membrane translocater  inside  

mitochondrial matrix (lumen)  signal peptide is cleaved  

because protein won’t leave mitochondria  folds into  

proper formation  proper functional protein

o Requires energy

 Dependent on ATP and membrane potential in  


o Similar process occurs in chloroplast

∙ Mitochondrial Transport Continued

o Chaperone proteins will bind to proteins bound for the  

mitochondria and keep them unfolded

 Hsp70

 Requires ATP

o Signal sequence  binds to receptor  fed into the protein  translocater  release of Hsp70 requires ATP hydrolysis  inside  and between outer/inner membrane  signal sequence is  

positively charged

 Chaperones in lumen of ER

 Proton gradient allows protein to move through

∙ Lots of protons because of ATP synthase

∙ Signal sequence is positively charged and moves  

towards the more negative lumen

o Lumen is negatively because of proton gradient

with ATP synthase

∙ Positively charged signal sequence  lumen is more  

negatively charged  favorable for positively charged

signal to be going into the lumen  gradient drives  


translocation of signal sequence  signal gets inside  

 mitochondrial Hsp70 pulls protein through in ATP  

dependent manner

o Chloroplast behaves in similar way

∙ Peroxisomes

o Breaks down substances like fatty acids, toxins, etc.

o Uses different oxygens  removes hydrogen items from organic  substances  creates peroxide (H2O2)  oxidizes other molecules such as ethanol  creates water

 RH2 + O2  H2O2

 H2O2 + R’H2  R’ + 2H2O

o Most proteins are from the cytoplasm, but some come from the  ER

o Use protein translocators, but not well understood

o Proteins don’t need to unfold

o ATP required for transport

∙ Zellweger Syndrome

o Mutation in import receptor (PXR1 gene) on peroxisomes  Causes proteins to not be brought in like normal  “empty”  peroxisomes; membrane with no protein aggregates

∙ Can’t break down fatty acids

o Genetic disease

o Infants born with abnormalities in liver, brain, kidneys

 Rarely survive past a year

o No treatment

 Have transfected unmutated PXR1 into cells in dish, but  hard to do in humans

∙ Endoplasmic Reticulum

o One-way transport: cytoplasm  ER  maybe to other  organelles through vesicular transport

 Doesn't go back to cytoplasm

o Imports both soluble and transmembrane proteins to ER  Where signal sequence is located differs

o Smooth ER-lipid synthesis

 Steric hormones-made from cholesterol; major  

component of membranes, type of lipid

o Protein processing between Golgi and ER

 Post translational modification in form of glycosylation

o Sequesters Ca2+ in lumen which is crucial for signaling  Higher concentration in ER than cytoplasm

∙ The Ribosome Pool

o Rough ER-made from ribosomes binding to the ER membrane o Ribosomes in cytoplasm or bound to ER

 Only difference is what mRNA they are translating 24

∙ Protein with ER signal  ribosome goes to ER  

membrane  start translocating as the protein is  

being synthesized  ribosome goes back into  

ribosomal pool in cytosol

∙ mRNA with no ER signal remains free in cytosol

∙ Finding the ER

o Way protein is designated depends on presence of ER signaling  sequence

 Usually on N terminus of protein; translated first

∙ Protein is being translated as soon as ER signal  

sequence is translated

o Signal recognition particle (SRP)-binds to ER signal sequence  interacts with receptor  protein translocater imbedded in ER  membrane

 Pauses translation

 Moves it to ER

o Proteins are translocated as they are synthesized  Partially translated before moved to ER

 Energy is already required for protein synthesis

∙ No additional energy need for translocation besides  

addition of amino acids which push protein through  


 Translation (protein synthesis) and translocation are  

coupled in ER

∙ Entering the ER: Soluble Proteins

o Soluble protein-in lumen of ER and not inserted into the  membrane

o One signal sequence on N terminal side

o Protein translocated into ER as being synthesized  entire  protein translocated  peptidase next to translocater protein  cleaves off signal sequence  signal sequence degraded  protein released  can fold in the ER

o Signal is hydrophobic

∙ Entering the ER: Transmembrane Proteins

o C terminal end in cytosol and N terminal end in ER

o 2 signal sequences

 1st signal sequence

∙ Signal of N-terminus (start-transfer sequence) 

initiates transfer  binds to receptor  opens up the  

translocater  protein is synthesized and pushed  

through the protein translocater

 2nd signal sequence

∙ Stop-transfer sequence  makes it into membrane 

stops translocation  protein released into  


translocater  signal peptidase still cleaves start

transfer sequence off  N-terminal released into  

lumen side of ER, but remainder of protein (C

terminal) is in cytosol  hydrophobic region that  

stays inserted into the membrane (1 insertion point  

in membrane)

 Protein fully translates, but isn’t pushed through the  

translocater into the ER

 Both signals are hydrophobic

∙ Entering the ER: Transmembrane Proteins Continued o Multipass transmembrane proteins-imbedded in the  membrane in more than one location

o Simple multipass example

 2 transfer sequences

∙ 1st-initiates transfer

∙ 2nd-stops transfer

 Can have multiple start/stop transfer sequences

∙ Ex. GPCR

 Difference from soluble proteins is the location of start  transfer sequence

∙ Multipass protein-start sequence isn’t at N

terminal end; in middle of protein

o Process

 Start codon (AUG) is translated  protein begins to be  translated  receptors uncover start-transfer signal 

protein is directed to ER with ribosome  inserted into  

protein translocater  protein is synthesized  protein  

pushed through translocater  stop transfer sequence  

uncovered  stops protein from being pushed through  

translocater  no more transfer sequences  finish  

synthesizing protein  protein released

o No cleavage of initial signal because imbedded in middle of  protein; stays imbedded

 N-terminal and C-terminal ends up in cytosol

 Middle of protein in lumen of ER

∙ Transmembrane Proteins

o Complex multipass protein-multiple times looped through  membrane; multiple start/stop sequences

 Ex. GPCR = 7 times through membrane

o Can be predicted by looking for multiple hydrophobic regions  indicating start/stop sequences on protein sequence

∙ Transmembrane Proteins Continued

o Can’t go back to cytosol


o Once protein is imbedded in the membrane, the orientation  doesn’t change wherever it ends up after ER

∙ Protein Transport: 3 Mechanisms: Vesicular Transport o Vesicles bud off of ER

 Proteins made in ER, in lumen, in membrane, on surface of ER in vesicles  Golgi  different locations or secreted from cell entirely

∙ Vesicular Transport

o ER  vesicles  Golgi  other locations or out of the cell o Bud from one compartment and fuse with another; contents are  transferred

∙ Coated Vesicles

o Coats have 2 roles

 Helps shape the membrane

 Helps capture molecules for transport

o Cargo receptors-pick up cargo

o Adaptin-recognizes cargo receptors and binds them

o Clathrin-binds to adaptor molecule

 Coats molecule

o Dynamin-causes vesicle to bud; cleaves narrow channel  connecting vesicle to parent organelle

o Will lose coat once vesicle is formed

 Transport vesicles are “naked”

∙ Distinguishing Membranes: Phosphoinositides

o Phosphatidylinositol (PI)-no phosphates on ring

o Inositol head group-hydroxyl groups can be modified with  phosphates

 PIP-1 phosphate added

 PIP2-2 phosphates added

 PIP3-3 phosphates added

∙ PI Kinases and Phosphatases

o Kinases and phosphatases add/remove phosphates on PI o Found in different membranes

∙ Phosphoinositides and Membrane Specificity

o Helps proteins that do vesicle formation recognize which  membrane vesicle goes to

 Recognize certain head groups to get specificity

o When identifying phosphoinositides, count numbers on  ring counterclockwise from attachment point 

∙ Vesicle Formation and Fusion

o Vesicles need to know where to go

o Rab proteins-vesicle targeting

o SNARE-vesicle fusion

∙ Vesicle Docking: Rab & SNARE Proteins

o Different Rab proteins target different things


o Tethering proteins also differ and create specificity

o Types of SNAREs

 T-SNARE-on target membrane

 V-SNARE-on vesicle membrane

o Tethering-each Rab binds to a specific tethering protein o Docking-V-SNARE and T-SNARE bind together very tightly which  brings vesicle to membrane

∙ Vesicle Docking: SNARE Proteins

o Need SNARE proteins for docking and fusion to occur

o Fusion-lipid bilayers fuse and cargo protein is released ∙ SNARE Disruption

o Tetanus

 Caused by SNARE disruption

 Spastic paralysis-can’t relax muscles

∙ Can’t expand diaphragm which leads to death

o Botulism

 Cause by SNARE disruption

 Flaccid paralysis-can’t contract muscles

∙ Can effect diaphragm leading to death

 Dangerous in infants

 Botox

∙ Used to prevent wrinkles

∙ Used to get rid of tension headaches because it  

relaxes the muscles (same as botulism)

o Interact specifically with mammalian cells and interfere with  signaling

∙ Normal Neurotransmitter Release

o Toxins effect neurons and neurotransmitter release

o How neurons work

 Neuromuscular junction or neuron-neuron junction

 Vesicles released into synapse

 Neurotransmitter can be an activating or inhibiting signal o VAMP and SNAP-25



 Have to interact to release neurotransmitters

∙ Disrupted Neurotransmitter

o Toxins bind to neuronal cells

 Brought in by receptor mediated endocytosis

o Subunits cleaved

 One forms pore in endosomal membrane

 Other enters the cytoplasm

o Toxin-protease that cleaves VAMP

 Results in no vesicle fusion because can’t interact with  SNAP-25


 No exocytosis

 Neurotransmitter signaling is blocked because SNAREs  don’t interact

∙ Toxins

o Botulism

 Affects neuromuscular junction

 ACH is inhibited

∙ Normally stimulates muscle contraction

∙ When blocked, it causes flaccid paralysis where  

muscles can’t contract

o Tetanus

 Affects neuron-neuron junction

 Blocks GABA release

∙ Inhibitory neurotransmitter

∙ Normally counteracts ACH

∙ When it is blocked, it can’t counteract ACH and too  

much muscle contraction occurs (spastic paralysis)

∙ Vesicular Transport

o Proteins transported through endomembrane system

 ER  stays in ER

 ER  Golgi

∙ Proteins have to be properly folded to leave

o Assisted by chaperone proteins

∙ Leaving the ER

o Chaperone proteins-make sure proteins are folded when they  leave the ER

o ER’s controls may be too stringent at times

 If not completely folded, can’t leave ER

∙ Cystic Fibrosis

o Caused by lack of functional CTFR channel

 Multiple mutations

∙ Channel present, but defective

∙ Channel not present and misfolded

o Blocking chloride production keeps water from leaving and  mucus that protects cell becomes too thick

 Becomes like cement in the lungs causing bacteria to be  trapped in the lungs

o Protein can’t leave ER

o Treatment

 Loosen mucus

 Drugs

∙ Kalydeco-improves function of existing receptors

o Won’t bring back to WT level, but some  



∙ Symdeco-ER releases more receptors resulting in  

improved lung function

∙ Super expensive!

∙ Going to the Golgi

o ER  Golgi  plasma/cell membrane (transmembrane proteins),  exocytosis, other organelles in endomembrane system

o Protein modification through glycosylation occurs in the golgi ∙ Exocytosis

o 3 options (depending on signal)

 Signal-mediated diversion to lysosomes

 Signal-mediated diversion to secretory vesicles (for  

regulated secretion)

∙ Ex. neurotransmitters such as ACH

∙ In vesicle until there is a signal to release the  

contents of the vesicle

 Constitutive secretory pathway

∙ Default pathway

∙ Goes to outside of cell

∙ Secretory Cargo Concentrated

o ER  Golgi  concentrates in vesicles  bud off (clathrin coated  vesicles)  merge with immature secretory vesicle  multiple  vesicles fuse with immature secretory vesicle  mature secretory vesicle

o Concentration of cargo increases in mature secretory vesicle ∙ Exocytosis Examples

o Pancreatic cell-insulin

o Mast cell-histamine

o Cargo is...

 Densely packed

 Released all at once

∙ Summary of PPT

o Proteins in cytosol can end up in nucleus, mitochondria,  chloroplast, ER, or peroxisomes

 Nucleus-bidirectional transport

∙ Proteins enter

∙ mRNA exits

 Rest of transport systems are one way out of cytosol

∙ Can’t go back to the cytosol 

o Look at this figure! 

PPT 4: Glycobiology I 

∙ Pre-Lecture Reading I


o Understand the differences between: monosaccharides,  disaccharides, oligosaccharides, polysaccharides, and complex  oligosaccharides

 Monosaccharides

∙ Ex. glucose

∙ Additional subgroups

o Aldoses

o Ketoses

o Can form a and B links

 Disaccharides

∙ Ex. sucrose, maltose, lactose

 Oligosaccharides

 Complex oligosaccharides

 Polysaccharides

∙ Ex. glycogen

o What is an isomer? What is the difference between glucose and  galactose and mannose? What is a sugar derivative? Name some examples of sugar derivatives.

 Isomer-sets of molecules with the same chemical formula, but different structures

∙ Optimal isomers-mirror-image pairs

∙ Glucose, galactose, and mannose are isomers

 Sugar derivative-the hydroxyl groups of a simple  

monosaccharide such as glucose can be replaced by other  groups

∙ Ex. glucose  glucuronic acid, glucosamine, N


o Why is it more difficult to determine the arrangement of sugars  in a complex polysaccharide than it is to determine the sequence of DNA?

 The number of possible polysaccharide structures is  

extremely large

∙ Free hydroxyl groups that can form a link to another  

monosaccharide or some other compound

∙ Can be highly branched

o What are some functions of sugars besides serving as an energy  source for cells?

 Mechanical supports

∙ Ex. cellulose, chitin

 Components of mucus, slime, and gristle

 Forming glycoproteins and glycolipids

∙ Help protect cell surface

∙ Help cells adhere to one another

o Key Terms


 Condensation reaction-a molecule of water is expelled  as a bond is formed

∙ How sugars, nucleic acids, and proteins are linked  


 Hydrolysis-a molecule of water is consumed to break a  bond

o What is the central paradigm of molecular biology?

 DNA  RNA  protein

o What are glycans? What types of interactions can they mediate?  Glycans-array of covalently attached sugars  

(monosaccharides) or sugar chains (oligosaccharides)

 What interactions can they mediate?

∙ Cell  cell

∙ Cell  matrix

∙ Cell  molecule

∙ Interactions between different organisms

o Host  parasite

o Host  symbiont

∙ Glycoconjugates such as glycoproteins and  


o Why did the study of glycobiology lag behind other areas of  molecular biology in the 1970s and 1980s?

 Believed to lack other biological activities; only seen as  energy source

 Also due to…

∙ Being structurally complex

∙ Difficult to determine their sequences

∙ Biosynthesis couldn’t be directly predicted from a  

DNA template

o What does the study of glycobiology encompass?

 Coming together of traditional disciplines such as  

biochemistry, molecular biology, and understanding the  


 Glycobiology-the study of the structure, biosynthesis,  biology, and evolution of saccharides (sugar chains or  

glycans) that are widely distributed in nature, and the  

proteins that recognize them

∙ Central Dogma

o DNA  RNA  protein

o Proteins are modified

 Glycosylation-adding sugars to proteins

∙ Changes function of protein

∙ Most common type of protein modification


o Glycobiology-study of structure and function of glycoconjugates (proteins with sugars attached) and proteins that specifically  interact with them

∙ Glycoconjugates (Glycans)

o Glycoconjugates-covalent addition of one or more sugars to a  protein or lipid

 Ex. glycoprotein or glycolipid; glycan

 Originate in ER and modified in Golgi

∙ Glycosylated Proteins

o Glycoprotein-complex oligosaccharides added to a protein  Much smaller

 Little bit of sugar on protein

∙ Protein > sugar

 Ribonuclease

o Proteoglycan-contains polysaccharide chains called GAG chains  Much bigger

 Little bit of protein on sugar

∙ Protein < sugar

 Aggrecan

∙ Glycosaminoglycan (GAG) Chains

o GAG-long, unbranched sugar chains composed of repeating  disaccharide units one of which is an amino sugar

 Specific type of polysaccharide

 Often sulfated

 Highly negatively charged and attracts water

 Helps cell withstand compressive forces

∙ In extracellular spaces, facial tissues, joints

∙ Proteoglycans

o Huge GAG chains added covalently to a protein

o Assembled on core protein in Golgi

∙ Hyaluronan/Hyaluronic Acid

o GAG chain is not linked to a core protein

o Found in skin

 Gives skin smoothness

 Naturally lose it as you get older, thus causing wrinkles  Restoring hyaluronic acid

∙ Restylane-injections of it

∙ Night creams-have hyaluronic acid in them

∙ More effective if it has been injected

∙ Some Glycoprotein Functions

o Any protein that leaves the ER has sugars

o Protect protein from degradation

o Holds protein in ER until properly folded

o Transport signal for protein trafficking


o Forms glycocalyx-sugar coating involved in cell to cell recognition and protection

o Exist on surface of cell and interact with other cells

o Not polysaccharides, but still negatively charged and attract  water

 Has slimy coating

 Helps ease friction between cells

∙ Protein Glycosylation in ER

o Most cases of glycosylation begin in the ER

 No variety to it-same sugar sequence is added to every  protein that is glycosylated

∙ 3 glucoses

∙ 9 mannoses

∙ 2 N-acetylglucosamines (GlcNAc)

o Pre-formed sugar complex-oligosaccharide; always added to  very defined locations on the protein sequences

 Transferred to protein as translated and translocated in ER  Linked to asparagines

o N-linked glycosylation-sugar complex transferred to side chain NH2 (amine group) on asparagine

 Will link to asparagine that follows a certain sequence

∙ Asn – X – Ser/Thr  

o X = any amino acid besides proline

∙ MUST have this sequence!! 

∙ Formation of Precursor Oligosaccharide

o Synthesized in cytosol  assembled sugar by sugar onto the  carrier lipid dolichol in membrane of ER

o First sugar linked by high energy phosphate bond  addition of 2  GlcNAc and 5 mannoses (Man) in cytosol  structure flipped  across the membrane by carrier protein  additional 4 Man and 3 glucose (Glc) added  final complex with Man, GlcNAc, and Glc  hangs out in lumen of ER

o Final structure has 3 Glc, 9 Man, and 2 GlcNAc

∙ Protein Glycosylation in ER

o Oligosaccharyl transferase-scanning for amino acid  sequences as protein is passed through

 Scans growing peptide chain for Asn sequence

 Adds preformed sugar complex (oligosaccharide) to Asn  sequence

 Energy in pyrophosphate bond provides energy for transfer ∙ Protein Folding in the ER

o Protein folds in ER because it can’t be transferred to other  organelles until properly folded


o Unfolded protein (glycosylated and oligosaccharide has been  added)  3 terminal sugars are 3 glucoses  protein starts to fold  last 2 glucoses trimmed  chaperone proteins (Calnexin) bind  to partially folded protein’s sugar chain (1 glucose)  last sugar  can be cleaved by glucosidase  properly folded  can leave ER  because it isn’t bound to glucosyl transferase

o If protein isn’t properly folded  bound by glucosyl transferase  adds glucose back on  bound again by chaperone proteins  (Calnexin)

o Enzymes/components

 Glucosidase-removes glucose

 Glucosyl transferase-adds glucose

 Calnexin-holds protein as it is ready to be  

folded/completely folded properly; chaperon protein

o Has to be properly folded to leave the ER

 Marker of proper folding-3 glucoses removed from  


∙ Glycosylation Trimming in the ER

o Trimming of last 3 glucoses during protein folding

o 1 mannose trimmed

o No diversity in protein glycosylation while in the ER!!  All proteins that are glycosylated in the ER will look  the same! 

o ER  Golgi

∙ Glycosylation in the Golgi

o Diversity in glycosylation

o Acts on terminal sugar only

o Lots of different enzymes

 Glycosidases-remove sugar groups

 Glycosyl transferases-add sugar groups

o Different enzymes organized sequentially in the Golgi; doesn’t  look the same throughout the whole complex

 Different enzymes present in different cisternas of the  


∙ Two N-Linked Oligosaccharide Classes

o High-mannose Oligosaccharide-lots of mannoses; hardly any  modification in the Golgi

 Only trimmed in the Golgi

 No new sugars added

 Very similar to original sugar added in ER

o Complex oligosaccharide-lots of sugars, not repetitive, highly  branched

 Will look different from ER oligosaccharide

 More sugars added in Golgi

 Lots of variety


∙ Assembly of Complex Oligosaccharides

o Enzymes

 Glucosidase I-removes 1 glucose

 Glucosidase II-removes 2 glucoses

 ER mannosidase-removes 1 mannose in ER

 Golgi mannosidase I-removes 3 mannoses in Golgi

 N-acetylglucosamine transferase I-transfers a N

acetylglucosamine (GlcNAc) onto the oligosaccharide

 Golgi mannosidase II-removes 2 mannoses in Golgi

o Initial assembly for both types

 Sugar complex added to Asn sequence in ER  trimming of  3 glucoses by glucosidases in ER  trimming of 1 Man by  

mannosidase in ER  sugar chain transported to golgi  

during vesicular transport

o Assembly of high-mannose oligosaccharide

 Sugar chain transported to golgi during vesicular transport   mannoses trimmed with Golgi mannosidase I  no more  modification  high-mannose oligosaccharide is complete

o Assembly of complex oligosaccharide

 Sugar chain transported to golgi during vesicular transport   mannoses trimmed with Golgi mannosidase I  additional sugars added (N-acetylglucosamine transferase/GlcNAc)  mannoses further removed by Golgi mannosidase II  more sugars added by various processes  complex  

oligosaccharide complete

o How do we study this?

 Determine what type of oligosaccharide something is by  seeing how it reacts with Endo H in the lab

∙ Endo H-enzyme that degrades anything that has lots

of mannoses

o High-mannose oligosaccharide-sensitive to  

Endo H; glycosylation isn’t preserved when  

exposed to it

o Complex oligosaccharide-not sensitive to  

Endo H; glycosylation is preserved when  

exposed to it

∙ Assembly of Complex Oligosaccharides Continued o Starts in ER  Asn-X-Ser/Thr  folded and exported  trimming of  3 glucoses and 1 mannose  additional trimming and/or  

additional processing in Golgi

∙ O-Linked Glycosylation

o N-linked glycosylation-added to NH2 group

 Asparagine chain

 Started in ER


 90% of proteins

o O-linked glycosylation-chains added to protein are completely  synthesized in the Golgi; serine or threonine

 Added to OH group

∙ Glycosylation Summary

o Glycosylation (proteins with sugars added) happens in the ER  and Golgi

o Pre-formed oligosaccharide begins in the ER and sometimes  further modified in the Golgi

o Some protein glycosylation (i.e. O-linked) starts in the Golgi ∙ Proteoglycans

o Contain O-linked glycosylation

o Huge polysaccharides added on to core protein

o Started in the Golgi

o Long sugar chains

o GAG chain-long repeating disaccharide units

 Ex. mucins found in mucus

∙ Importance of Glycosylation

o Most common post-translational modification of proteins! o 3 examples of the role of glycosylation

 Lysosome function

 Blood typing

 Influenza treatment

∙ Glycosylation and the Lysosome

o Lysosome-degrades proteins

 Lysosomal hydrolases-enzymes that degrade  


∙ Ex. nucleases, lipases, etc.

o Start synthesis in cytosol  ER signal sequence  ER  Golgi  lysosome

o Have to have correct signal sequence to be moved to the  lysosome

 Mannose 6-phosphate tag (H6P)-added to lysosomal  hydrolases in the Golgi; tags proteins so that they know to  go to the lysosome

 ER signal sequence that originally gets proteins to ER  cleaved because soluble protein  ER  Golgi  M6P tag  

added  lysosome

 Important thing is to add phosphate onto the mannose

∙ Mannose 6-Phosphate Tag

o Directs protein to lysosome

o How is it put on a protein?

 Lysosomal hydrolase enzyme has sugar added with  

terminal mannose  lysosomal enzyme recognized by  

GlcNAc phosphotransferase  adds GlcNAc with phosphate  37

to mannose that is bound to lysosomal hydrolase  1  

phosphate leaves  mannose, phosphate, and GlcNAc are  left attached  GlcNAc phosphotransferase can release 

lysosomal hydrolase with GlcNAc and phosphate attached  to mannose in oligosaccharide  GlcNAc removed by  

another enzyme  mannose 6-phosphate added to protein ∙ GlcNAc phosphotransferase-adds GlcNAc with  

phosphate to mannose on lysosomal hydrolase

o Recognizes proteins that have particular signal  

sequence when protein is folded correctly

∙ Addition of phosphate happened in golgi with specific

recognition signal that binds to GlcNAc  


∙ Initial sugar complex is oligosaccharide that was  

added in ER

∙ Transport of Lysosomal Hydrolases

o Lysosomal hydrolase precursor and mannose from ER  P-GlcNAc added  GlcNAc removed  M6P added  recognized by receptors in vesicles (M6P receptor in membrane of Golgi)  binds M6P tag   buds off  vesicle receptor and lysosomal hydrolase  leaves  

Golgi  lysosomal hydrolase dissociates from receptor 

phosphate removed in endosome  lysosomal hydrolase  

precursor in early endosome

o M6P receptors are recycled back to golgi

o Dissociated only at acidic pH in endosome

o Addition of M6P is just to get the hydrolase to the  lysosome 

o Addition of phosphate tag is getting hydrolyzed where it  needs to be in the lysosome 

 Doesn’t need to be added to bind to the M6P  


∙ I-Cell Disease

o Lysosomal storage disease

o Defective GlcNAc phosphotransferase gene

 Lysosomal enzymes don't get to lysosome and  

macromolecules can’t be broken down

o Inclusion bodies with protein aggregates that aren’t getting  degraded

o Lysosomal enzymes secreted into the blood instead of going to  the lysosome

 Fully functional enzymes, but not in the right place

o Fatal by 6 years of age

o No treatment

o Shows importance of glycosylation and protein formation 38

 Not enough to just make the protein; have to get it  where it needs to be so that it can be effective 

∙ Blood Types

o Example of glycosylation

 Whether you have certain sugar strands on surface of RBCs o Blood types

 Blood type A-possesses GalNAc transferase

∙ Adds GalNAc

∙ Recognizes O antigen

 Blood type B-possesses Gal transferase

∙ Adds galactose

∙ Recognizes O antigen

 Blood type AB-possesses both enzymes

 Blood type O-possesses neither enzymes

∙ Bird Flu H5N1

o Severe flu with high mortality rate (55%)

o People in close contact with birds (generally chickens) are  infected

 General population wasn’t as severely affected

o Flu normally kills old and weak; this one killed young and healthy o Jumps from birds to humans, but never transferred from human  to human

∙ Influenza and Sugar Linkages

o Receptors that virus binds to on mammalian cells is glycosylated  Oligosaccharide bound to receptor

 Transmembrane protein

 In order to infect, flu has to attach to receptor

o Sialic acid and galactose are terminal sugars

∙ Influenza and Sugar Linkages Continued

o 2 terminal sugars (sialic acid/Neu5AC and galactose) are linked  to each other at 6 or 3 position

o Different flus recognize different linkages

 Human flu-recognizes a2, 6 linkages

 Bird flu-recognizes a2, 3 linkages

∙ Human vs. Bird flu

o Humans-have a2, 6 linkages in upper respiratory tract and a2, 3  linkages in lower respiratory tract

o Birds-have a2, 3 linkages everywhere in respiratory tract ∙ Bird Flu H5N1

o Bird flu binds to a2, 3 linkages in respiratory tract

o Humans exposed to flu virus

 Will come into contact with upper respiratory tract which  binds a2, 6 linkages, but can only infect lower respiratory  



o H5N1 virus mutated allowing it to infect humans, but it never  recognized the a2, 6 linkages

 Could infect humans, but harder because has to infect  

bottom respiratory tract

o High morality, but not high infectability

o Worry for the future is that it could mutate and have high  morality and high infectability

 Mutate to be able to travel from human to human

PPT 5: Glycobiology II 

∙ Sialic Acids and Enzymes

o Sialic acids-most common terminal sugar in mammalian cells  NOT in plants, invertebrates, or prokaryotes 

∙ Exception is some human pathogens that use it as  

decoy mechanism

 Highly negatively charged like most sugars

 Essential for embryonic development

∙ Sialic Acids

o Many kinds

 Neu5AC-most common in humans

 Neu5GC-most common in all other mammals

∙ Difference is addition of hydroxyl group

∙ Loss of Neu5GC in humans

o Neu5AC is precursor to Neu5GC

o Enzyme encoded by CMAH gene-converts Neu5AC to Neu5GC  Humans lost this gene 2-3 million years ago

 Almost all other mammals still have CMAH gene

∙ Why did we lose the CMAH gene?

o One theory

 We lost it as a way to avoid infection by Plasmodium  

species that causes malaria

∙ Malaria

o #3 cause of death due to infectious disease in the world o Mainly infects tropical areas

o Caused by parasite (Plasmodium sp)

 Infects RBCs  burst out of RBCs  go and affect more  


o Synchronized lysing of RBCs causes chills/fever

o Spread by Anopheles mosquito only found in tropical regions o We continue to try to escape it in many ways

 Presence of sickle cell anemia

 Loss of CMAH gene

∙ Malaria Lifecycle

o Plasmodium enters through mosquito bite (resides in salivary  glands of mosquito)  human liver  human bloodstream 


infects human RBCs (feel sick)  parasite replicates in human  RBCs  grow and divide  burst out of RBCs and can go infect  more RBCs (amplification of parasite)  Plasmodium  

gametocytes ingested by mosquitos when it bites human  lifecycle completed in mosquito  goes and bites another human o Critical point in lifecycle is binding to RBC

o Symptoms related to lysing of RBC

 Causes anemia

∙ Not always life threating; depends on strain of  

Plasmodium and overall health including blood count

∙ Kids are more susceptible than adults

o No human to human transmission!! 

∙ Plasmodium Species

o Has to be able to bind to RBC to infect it

o Hallmark symptoms are chills/fever

o Two types

 Plasmodium falciparum-infects humans; most deadly  Plasmodium reichenowi-infects apes and other  


o No cross-infectivity even though Plasmodium species seem  nearly identical

 HUMAN and ape cross infection studies showed no side  effects when injected with opposite type of Plasmodium  


∙ Plasmodium Species Continued

o Aotus monkey-one of few mammals susceptible to Plasmodium  falciparum like humans

o Presence of Neu5AC/Neu5GC on RBC

 Other mammals have both because Neu5AC is precursor to Neu5GC

∙ Majority is Neu5GC

o Looked at Aotus monkey’s RBCs

 Has primarily Neu5AC like humans

∙ Malaria and RBC Binding

o Reason Plasmodium falciparum could infect the Aotus  

monkey/humans was because of its ability to bind to RBCs with  Neu5AC

 Focused on Plasmodium protein EBA-175

∙ Binds to glycoprotein on RBCS

∙ Types

o Pf-EBA175-comes from Plasmodium  

falciparum; binds with Neu5AC RBCs

o Pr-EBA175-comes from Plasmodium  

reichenowi; binds with Neu5GC RBCs

o Plasmodium can’t cause damage if it can’t bind to RBCs 41

o Looked at how well each protein binds to RBCs of Neu5GC or Neu5AC

 Results

∙ Pf-binds better to Neu5AC

∙ Pr-binds better to Neu5GC

∙ Hypothesis confirmed!

o Cross-infectivity doesn’t occur because the terminal sialic acid  can only recognize one form (Neu5AC or Neu5GC)

∙ Loss of CMAH

o Humans lost the CMAH gene as an attempt to avoid infection by  Plasmodium reichenowi with Malaria

o All mammals recognized Neu5GC  malaria became prevalent  put pressure on humans due to high mortality rate  2-3 million  years ago humans lost CMAH gene  no longer able to be  infected by Plasmodium reichenowi  population becomes  resistant to Plasmodium reichenowi that caused malaria 

Plasmodium evolved into 2 species  Plasmodium can now bind  to Neu5AC  current plasmodium falciparum  malaria still  occurs in humans

∙ Consequences

o We consume animal products which contain Neu5GC (beef, pork,  milk, etc.)

o Our enzymes that incorporate sugars into our proteins can’t tell  the difference between Neu5AC and Neu5GC (super similar)  Our glycosylated proteins contain Neu5GC even though we don’t make it

∙ Thus on surface of our cells

o Immune system can tell the difference between Neu5AC  and Neu5GC

 Develop antibodies against Neu5GC because we can’t  

make it and it is considered foreign

o Experiment: Measure Neu5GC antibodies in moms and  newborns

 Amount of antibodies produced that recognizes Neu5GC  varies from person to person

 Looked at levels in children before, during, and after birth

∙ At 3 months-have hardly any Neu5GC antibodies  

because drinking mostly breast milk

∙ At 6-12 months-have more Neu5GC antibodies  

because babies are introduced to solid food  

containing animal products

∙ Immunology 101


o Antibody-protein produced by B-cells in immune system that  binds/recognizes “foreign” molecules

 Doesn't directly kill antigen

 Creates neon flag/kill signal

 Binds “foreign” materials

o Antigen-molecule that induces immune response

 What an antibody binds

 Should be foreign material

 Most common is a type of protein, but can also be a sugar o What happens if an antibody binds its antigen?

 Bacterial infection with antigens  immune system  

recognizes that molecules are foreign  antibodies  

produced by B-cells  antibodies bind to antigens  signals  antigen for destruction (neon flag)  recruits macrophages, neutrophils, etc.  destroy foreign agent

∙ Inflammatory response

o Tissue injury/infection (paper cut)  recruitment of WBC’s  (microphages and neutrophils) and serum proteins (antibodies,  complement)  attack invader with reactive oxygen species  (shown in example with hydrogen peroxide), reactive nitrogen  species  kills microbes

o Microbial destruction through phagocytosis  complement  mediated killing  release of antimicrobial chemicals

o Chemicals can damage your own cells with prolonged  inflammation

 Acute inflammation-initial inflammatory response; paper  cut

 Chronic inflammation-prolonged inflammation;  

chemicals constantly released, causes damage to our own  cells

∙ Chronic Inflammation

o Can damage host cell

 Neu5GC antibodies recognize Neu5GC incorporated in our  own cells as foreign

o Cancer and atherosclerosis (cardiovascular disease)

 Other causes

 Definitely makes it worse

 Tumors grow faster at sites of chronic inflammation

∙ Anti-Neu5GC Antibodies

o Xeno-autoantibodies-recognize a “non-self” animal-derived  antigen in the context of “self”

 Autoantibodies-recognize own cells as antigens

∙ Happens in autoimmune disease


 Antibodies created against foreign substance that has been incorporated into our own cells

o Summary Questions

 Why don’t our cells make Neu5GC?

∙ Because we don’t have CMAH gene

 Where is Neu5GC coming from?

∙ Foods we eat

 Why do our cells incorporate Neu5GC?

∙ Can’t tell the difference; just see terminal sialic acid

 Why do we make antibodies against Neu5GC?

∙ Foreign and immune system recognizes foreign  


∙ Anti-Neu5GC Antibodies Continued

o What is potential consequence of “xeno-autoantibodies”?  Recruiting own immune system to attack your own cells  chronic inflammation  possibly cancer

∙ Cancer Correlation

o WT mouse-has CMAH gene; makes Neu5GC

o CMAH -/- mouse-lacks both copies of CMAH gene; homozygous  knockout; doesn’t make Neu5GC and makes antibodies against  Neu5GC

o Is there really a cancer correlation?

 Antibodies  chronic inflammation  cancer

o Which type of mouse is more likely to develop inflammation and  eventually cancer after exposure to Neu5GC?

 CMAH -/- knockout mouse

∙ Cancer Correlation Continued

o 2008 experiment

 Injected WT and CMAH -/- mice with tumor cells expressing  Neu5GC

 In which mouse would the tumor grow more and  


∙ CMAH -/- knockout mice would have increased tumor

growth (increased volume) because have antibodies  

that recognizes Neu5GC  increase chronic  

inflammation  increased tumor growth  cancer

∙ Supported by data

o This doesn’t prove that eating Neu5GC rich foods causes  cancer because gave them tumor directly 

o This doesn’t prove that exposure to Neu5GC can cause  spontaneous cancer because gave them cancer and then  saw which one was worse 

∙ Neu5GC Rich Foods: Inflammation and Cancer

o Hypothesis


 In CMAH -/- knockout mice (no CMAH gene), exposure to  Neu5GC rich foods in the presence of Neu5GC antibodies  increased inflammation  development of spontaneous  


 Knocked out both copies of CMAH making mice like  


∙ Experiment 1: Does it result in increased inflammation? o Step 1: Give CMAH -/- mice antibodies to Neu5GC  Inject control antibodies

 Inject Neu5GC antibodies

 Animals would have developed them naturally, but would  have taken too long

o Step 2: Feed the animals

 PCM = pork mucus (Neu5GC rich)

 Soy = vegetable (no Neu5GC)

o Step 3: Monitor levels of inflammatory markers

 Mouse fed pork mucus should have higher levels of  

inflammation because Neu5GC antibodies are present 

higher levels of inflammation

∙ Results from Experiment 1: Levels of Inflammatory Markers in  CMAH -/- Mice

o Looked at levels of inflammatory markers

 Serum, HP, IL-6 are all measures of inflammatory  


o Diet

 PCM or Soy

o Sera

 Control-control antibodies

 Immune-Neu5GC antibodies

o Data

 Soy with control-no change; negative control

 PCM with control-identical to negative control; just  

feeding pork mucus doesn’t change inflammation levels

 PCM with immune-inflammatory marker levels increased ∙ Have to have been fed pork mucus and given  

Neu5GC antibodies

∙ P value was less than 0.05 = statistical significance

o Data supported hypothesis

∙ Experiment 2: Does it lead to development of spontaneous  cancer?

o Step 1: Create Neu5GC antibodies by immunizing CMAH  -/- mice with Neu5GC

 Inject mice with something that should help mice generate  own antibodies


∙ Inject RBC membranes from humans or chimps

o Human-Neu5AC

o Chimp-Neu5GC

o Step 2: Feed animals a Neu5GC rich diet for 85 weeks (all  given PCM)

o Step 3: Harvest livers and examine pathology

∙ Experiment 2 Results: Incidence of Hepatocellular Cancer  (HCC)

o Looking at incidence of liver cancer

o Results

 WT immunized with Neu5GC-won’t make antibodies for  Neu5GC

∙ Has CMAH gene and able to naturally make Neu5GC;  

Neu5GC isn’t considered foreign

 WT immunized with Neu5AC-won’t make antibodies for  Neu5GC

∙ Has CMAH

∙ Naturally makes Neu5GC; thus Neu5GC isn’t  

considered foreign

∙ Not even targeting Neu5GC here; targeting Neu5AC  

which is made naturally and not considered foreign  


 CMAH -/- immunized with Neu5AC-won’t make  

antibodies for Neu5GC

∙ Not exposed to Neu5GC RBC membranes; thus no  

antigen seen

 CMAH -/- immunized with Neu5GC-have antibodies for  Neu5GC

∙ Doesn’t have CMAH and can’t naturally make  


∙ Neu5GC isn’t self, thus creates antibodies

∙ Greatest level of liver cancer (almost 50%)

o Not entirely naturally system

o WT and CMAH -/- immunized with Neu5AC could potentially  develop lots of tumors over time

o Results are statistically significant (P value < 0.05)

∙ Was hypothesis confirmed?

o Yes! 

 Animals lack CMAH gene  exposed to Neu5GC rich foods  presence of Neu5GC antibodies  increased inflammation  development of spontaneous cancer

∙ Experiment 1: Exposure to Neu5GC rich food in  

presence of Neu5GC antibodies  increased  



∙ Experiment 2: Neu5GC rich food in presence of  

antibodies the organism creates from RBC  

membranes  spontaneous cancer

o Is this a risk in humans?

 Maybe…

 Only did experiment with mice

 Neu5GC rich foods  Neu5GC antibodies  increased  

chronic inflammation  higher risk for cancer

∙ Humans vs. Chimps

o Chimps are closest relatives genetically

 1-2% genetic difference

∙ Chimps have CMAH gene

∙ Chimps don’t get cancer or cardiovascular disease

o Could be attributed to making Neu5GC

 Doesn’t recognize Neu5GC in Neu5GC  

rich foods as foreign

∙ Big Questions

o Could our attempt to escape infection by Plasmodium and loss of CMAH gene led to current trouble with diseases such as cancer  and cardiovascular disease?

 Maybe!

 Cancer in humans has multiple causes! 

o Would it be beneficial to eliminate Neu5GC from our cells?  Over time it could get rid of Neu5GC in our cells

 Have to stop eating meat and meat products

∙ Food for Thought

o Neu5GC is highest in red meats and goat cheese

o Less Neu5GC in byproducts such as milk and butter

PPT 6: Cytoskeleton I 

∙ Pre-Reading I (see Week 7 notes for more details!) o What are the 3 major families of cytoskeletal filaments? What are the subunits that make up each filament? Which subunits bind a  nucleoside triphosphate (i.e. ATP/GTP)? Which do not?  

Understand how the subunits come together to form each  filament. Understand the properties of each filament (Fig. 17.2 is  a good overview).

 Intermediate filaments

∙ Formed by fibrous protein

∙ Flexible

∙ Toughest and most durable filament

∙ Form nuclear lamina

∙ Formation


o Rod  coiled-coil dimers  staggered tetramer  

 final structure

∙ Doesn’t bind to nucleoside triphosphate

∙ No polarity

∙ Provides cell with mechanical strength

 Microtubules

∙ Formed by tubulin (dimer subunit)

∙ Hollow cylinders

∙ Rupture when stretched

∙ Rapidly disassembles and reassembles

∙ Binds to GTP

∙ Formation

o Tubulin (dimer subunit)  protofilament  13  

parallel protofilaments

o Grows out from centrosome

 Y-tubulin (ring shaped)

 aB-tubulin dimers

∙ Plus end-B-tubulin; where growth  


∙ Has plus and minus ends

∙ Forms mitotic spindle, cilia, flagella

∙ Intracellular transport

∙ Drugs that affect them

o Taxol

o Colchicine/colcemid

o Vinblastine/vincristine

 Actin filaments/microfilaments

∙ Formed by actin

∙ Helical polymers

∙ Organized in linear bundles, 2-d networks, and 3-d  


∙ Binds to ATP

∙ Formation

o Twisted chains of globular actins  all point in  

same direction

∙ Has plus and minus ends

∙ Supports the cell

∙ Allows for muscle contraction

∙ Cytokinesis

∙ Crawling of the cell

∙ Drugs that affect them

o Phalloidin

o Cytochlasin

o Latrunculin


o Both actin and microtubules display polarity but intermediate  filaments do not. Why not? What is the “plus” and “minus” end  of actin and microtubules? How does that affect the addition of  new monomers?

 Intermediate filaments don’t have polarity because ends of the tetramer are identical

 Actin and microtubules have plus and minus ends ∙ Microtubules

o Plus end-has B-tubulin

 Add subunits more rapidly to this end

o Minus end-has a-tubulin

o Aids in assembly and function once formed

∙ Actin

o Plus end-add monomers at a rate faster than  

bound ATP can be hydrolyzed causing growth

o Minus end-ATP is hydrolyzed faster than new  

monomers can be added

o Important for treadmilling

o What are dynamic instability and treadmilling? Be able to answer question 17-3 on p. 577 (answers are in the back of the  


 Dynamic instability-microtubules switch back and forth  between polymerization and depolymerization allowing  

them to undergo rapid remodeling; crucial for function

∙ Polymerization-add aB-tubulin dimers to plus end  

and grow outward from centrosome

∙ Depolymerization-lose tubulin dimers from its free  

plus end

∙ Driven by GTP hydrolysis

o Polymerization-results in GTP cap that allows  

microtubule to grow

o Depolymerization-GDP causes dimers to  

dissociate and peel off of microtubule

∙ Only occurs at plus end

 Treadmilling

∙ Dependent on ATP

o Plus end-actin monomers are added at a rate  

faster than bound ATP can be hydrolyzed  

causing growth

 Gains subunits

o Minus end-ATP is hydrolyzed faster than the  

new monomers can be added

 Loses subunits


∙ Simultaneous gain and loss of monomers causes  

filament to stay the same size

o Key terms

 Cytoskeleton-intricate network of protein filaments that  extends throughout the cytoplasm

∙ Mostly in eukaryotic cells

∙ Controls location of organelles

∙ Dynamic and consistently reorganized

∙ Cytoskeleton Filament Functions

o Microtubules-intracellular transport, move organelles and  vesicles, mitosis

o Actin-cell shape, crawling locomotion, cytokinesis, contraction o Intermediate filaments-mechanical strength, push/pull cells,  withstand shearing forces

∙ Cytoskeleton Organization

o Organized like an ant trail

 Quickly changes and adapts

 Structure may persist, but individual components are  

constantly changing

 Analogous to how ant trail continues to move forward when an obstacle is in its path, but individual ants move around  the obstacle

o Advantages

 Rapid diffusion of subunits

 Rapid structural reorganization

 Quick response to environment

o Ex. actin

 Signal such as nutrient source  disassembly of filaments  rapid diffusion of subunits  assembly of new filaments at  a new site

 Move in a particular direction very quickly

∙ Ex. neutrophil chasing down bacteria

∙ Filament Growth

o Polarity matters for growth and development in actin and  microtubules

o Plus end has faster growth than minus end

o Subunits have to be in GTP or ATP form to be added  T form-ATP/GTP bound; squares on diagram

∙ ATP/GTP cap favors growth

 D form-ADP/GDP bound; circles on diagram

∙ Microtubule Growth

o GTP is only hydrolyzed on B-tubulin subunit

o Losing GTP cap weakens microtubule and causes it to shrink  Catastrophe-filaments peel away from each other


o Continuously grows and shrinks very easily

 Allows for spatial and temporal flexibility in filament  


∙ Filament Growth Summary

o Microtubules-dynamic instability; filament grows and shrinks o Actin-treadmilling; filament stays same size

∙ Spindle Formation

o Microtubules create spindles

o Not permanent

 Assembly/disassemble quickly in specific stages of the cell  cycle

o 3 classes of dynamic microtubules

 Kinetochore microtubules

 Interpolar microtubules

 Astral microtubules

∙ Polarization of the Cell: Transport with Microtubules o Highway in cells

o Directional transport because of polarity

 Plus end-towards axon terminal

 Minus end-towards cell body

o Have to be rigid and strong to have such a long path

∙ Bacterial Homologues

o FtsZ-tubulin like homologue; cell division

o Actin homologues-determine cell shape (circular, rod, etc.) o Some elements of cytoskeleton like eukaryotes, but doesn’t  function the same way

∙ Intermediate filaments

o Formed from tetramers

o No polarity

o No nucleotide triphosphate

o Important for strength

 Strong rope-like properties due to strong lateral bonds

 Easy to bend, but hard to break

o Good for mechanical stress

 Epithelial cells such as skin and intestine

∙ Types of Intermediate Filaments

o From cytoplasm

 Keratin filaments-epithelial cells

 Vimentin and vimentin-related filaments-in connective tissue cells, muscle cells, and glial cells

 Neurofilaments-in nerve cells

o From nucleus

 Nuclear lamins-in all animal cells

∙ Intermediate Filaments Form Nuclear Lamina


o Nuclear lamina-intermediate filaments that line nuclear  envelope; between nuclear envelope and chromatins in nucleus  Attachment point for chromosomes

 Increases structural integrity of inner nuclear  


 Phosphorylation of lamina causes disassembly during  


∙ Nuclear Envelope During Mitosis

o Nuclear pores must reform and be reimported from cytosol after  every cycle of cell division

o Interphase  phosphorylation of lamins  nuclear envelope  fragments in prophase  dephosphorylation of lamins  fusion of  nuclear envelope fragments in telophase  fusion of enveloped  chromatins  interphase

∙ Progeria

o Rapid aging disorder

o By 1-2 years old, children show signs of things from old age  Loss of hair, wrinkles, loss of teeth, etc.

o Defect in lamin processing

 Nuclear lamina doesn’t properly form

 Nuclear envelope doesn’t have enough structural support  and is abnormally shaped

 Causes impaired cell division, increased cell death,  

diminished tissue repair

o No cure

o Very rare

 Isn’t passed on because children don’t live past their teens  and don’t reproduce

 Caused by spontaneous mutations

∙ Intermediate Filaments: Keratin

o Found in epithelial cells

o Bundles of keratin

o Linked to cells with junction of keratin

 Filaments connect from cell to cell (desmosome)

o Can distribute force to cells in tissues so don’t damage the cell  that is getting immediate force

∙ Epidermolysis Bullosa

o Mutations in keratin (most common), laminin, or collagen o “butterfly skin”

 Skin is extremely fragile

 Can’t withstand stress because connections between  

epithelial cells are absent

 Touching skin can cause damage or bruising

 Skin is like tissue paper

 Often wheelchair bound for life


o Genetic disease

o Don’t live past 20s

∙ Junctional Epidermolysis Bullosa

o Study conducted in November of 2017

o Caused by mutation in laminin

 Part of extracellular matrix proteins that distribute  

mechanical stress

o Were able to regenerate the entire human epidermis using  transgenic stem cells for this 7-year-old and saved his life

o Used autologous transgenic keratinocyte cultures and  gene therapy

 Autologous-own cells that are part of self; won’t reject  them

 Transgenic-inserted gene (wild-type lamb3); gene  

inserted randomly

∙ Mutated gene is still there, but that’s ok because it is

the absence of a normal gen which causes the  


∙ Inserting randomly is a problem because it could  

cause cancer

 Keratinocyte-skin cells that make keratin

o Regenerated epidermis remained intact and could withstand  mechanical stress; didn’t develop blisters or erosions

 Entirely sustained by limited number of transgenic  

epidermal stem cells that can self-renew in vitro and in vivo ∙ Gene Therapy in Stem Cells

o Added wild type version of laminin gene to a portion of his skin  cells

o Experimental therapy that was not without risks

 Death vs. chance of getting cancer

 Risk of inserting gene randomly into genome

o Grew cells in the lab in a dish

o Used viral vector to insert genes

 Retrovirus-will insert genome inside virus into host cell  DNA

∙ DNA from nucleus inside actual genome

∙ Permanently part of genome as long as cells survive

o Infected autologous epidermal cells

 Let cells from his skin replicate in a dish

∙ Undifferentiated and can continue to divide

 Used retroviral vector to add unmutated lamb3 gene

 Found 3 cell types

∙ Holoclone cells-undifferentiated cells; stem cells


∙ Meroclone & paraclone cells-partially  

differentiated stem cells

∙ Results in mosaic of cells in dish

o Formed transgenial epidermis-sheet of skin; all have  transgenic gene

 derived from stem cells and now have unmutated lamb3 o Did 3 skin graft operations and restored 80% of his skin  Grew skin in lab and then grafted it back on to his body ∙ Results

o After skin grafts, skin is fully functional and takes over entire  population of skin cells from initial holoclone cells

 Other types stopped growing

 Holoclone were able to divide and replicated and replace  all the skin

o Can self-renew in the lab (in vitro) and on his body (in-vivo)  New skin cells replaced by holoclone cells

∙ Results of Therapy

o New skin is robust and resistant to mechanical stress and didn’t  develop blisters/erosions during 21 months’ after

o Saved his life and dramatically improved the quality and length  of his life

∙ Zellweger’s Syndrome Revisited

o Mutation in PXR1 gene, import receptor

o Results in “empty” peroxisomes

 Can’t bring protein into peroxisomes to break down  


o Recessive genetic disorder

o Abnormalities of brain, liver, and kidneys

o Rarely survive longer than 1 year

o Why can’t doctors fix it like they fixed EB?

 They can’t because you need peroxisomes for all the cells  and not just a particular organ like skin

∙ Have to replace genes in every single cell in your  

body which is hard to reach

∙ Can’t grow every single cell type in lab

o Gene therapy-ability to bring in wild type of gene into cells/host  Only been effective with cells you can easily reach (skin,  blood cells)

 Hard to find one virus that targets all cells or successfully  targets one type of cell

PPT 7: Cytoskeleton II 

∙ Accessory Proteins

o Interact with filaments

o Regulate length, stability, and function of cytoskeletal filaments 54

o Roles

 Nucleation

 Elongation

 Stabilization

 Disassembly

 Cross-linking to form larger structures

 Motor proteins to generate movement

o Many different kinds

∙ Nucleation

o Initial process of filament formation when subunits form initial  aggregate or “nucleus” that can elongate

o Rate limiting step-regulates how quickly a filament can form o Actin can grow in just solution without accessory proteins  Actin subunits  form filaments  grow  treadmilling

 Lag phase occurs

∙ Actin subunits come together and fall apart very  


∙ Have to get enough coming together at once to  

create a stable structure

o 3 stages of growth

 Nucleation-lag; actin subunits briefly come into contact  with each other, but don’t form a stable form

 Elongation-rapid growth phase

 Steady state-equilibrium phase, treadmilling (growth and  disassembly at equal rates)

o Use accessory proteins because don’t want to rely on random  chance for filaments to form

 Accessory proteins-allow cells to skip nucleation/lag  

phase; provide stable base for filament to grow

∙ Need to have at least 3 subunits to be stable

∙ Microtubule Nucleation

o Gamma (y)-tubulin-forms a ring complex that nucleates  microtubules

 Uses microtubule organizing center/centrosome

 Structurally similar to alpha/beta subunit and thus can bind to alpha/beta subunits

 Filament is immediately stable when it binds to gamma tubulin and skips nucleation/lag phase  

∙ Microtubule Formation

o Grows outward from centrosome

 Only get addition on positive end

 Gamma-tubulin blocks negative end

o Important for spindle formation during mitosis and  

orientation/directionality of the cell


∙ Actin Nucleation: Formin

o Formin-binds and elongates from plus end; exists as a dimer  Increases addition and makes it go even faster

 Allows formation of straight, unbranched actin filaments ∙ Stabilizes actin and allows it to interact with other  

accessory proteins

 Minus end isn’t blocked

∙ Filament Binding Proteins: Fimbrin

o Formin-produces straight, unbranched actin filaments; microvilli o Fimbrin-stabilizes and connects all the actin filaments together ∙ Actin Nucleation: Arp 2/3

o Arp 2/3-subunits which are very similar to actin at plus end  Similar to how y-tubulin works

∙ Filament grows from plus end

o Skips lag period

∙ Blocks minus end

 Can be active or inactive

∙ Arp 2/3 inactive (2/3 are separated a little bit)  cell  

signaling pathway activates it  something binds to  

Arp 2/3  causes 2/3 to come together  allows actin  

to bind to it  addition only on plus end because  

blocks minus end

o Tends to nucleate on the edge of filament or on the side of pre existing filament

 Growing/branching actin chains

 Creates tree-like web

 Pushes membrane out

o Usually found near plasma membrane

 Creates cell crawling motion

∙ Regulating Free Subunits

o Actin free subunits bound to accessory proteins thymosin and  profilin

 Profilin-interacts with formin and Arp 2/3 to speed up  

actin elongation

∙ Keeps actin monomers in reserve until they are  


 Thymosin-binds subunits and prevents assembly

o Is important because there is a lot of free actin in the cell and the cell doesn’t want filaments to be forming when  they aren’t needed 

∙ Filament Binding Proteins

o Important for assembling large structures through cross-linking  Ex. microvilli

o Important for stabilization


 Ex. prevent catastrophe

o Contractile bundles-loose packing allows myosin-II to enter  bundle

 A-actinin-binding protein; lot more space between actin  filaments; seen in muscles

o Parallel bundles-tight packing prevents myosin-II from entering bundle

 Fimbrin-short protein; binds to actin filaments, but not a  lot of space between them

∙ Filament Binding Proteins: Filamin

o Filamin-gel-like, 3-d structure; used to construct lamellipodia,  binds and stabilizes branching of actin

 Cells that lack it can’t form lamellipodia and crawl poorly;  can’t spread out and look like circular blobs

∙ Ex. melanoma cells, cancerous cells

o How lamellipodia works

 Protrusion-actin polymerization at plus ends protrudes  lamellipodia and pushes membrane forward

 Attachment to substrate

 Contraction-myosin and actin interactions push  

lamellipodia forward through contraction at back end

 Results in crawling motion

∙ Filament Binding Proteins

o Plectin-filament binding protein that links intermediate filaments with microtubules; stabilizes cytoskeleton, cell to cell contact  Mutation causes autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD)

∙ Results in muscle degeneration over time

∙ Membrane Protrusion

o Lamellipodia at leading edge involved in wound healing  Pushes membrane forward  Arp 2/3 helps form actin  

filaments  older actin depolymerized  actin recycled

o Similar mechanisms drive membrane invaginations during  phagocytosis

o Filopodia-slender cytoplasmic projections; long bundled actin  filaments

 Might help determine attachment points and direction for  crawling

 Ex. microvilli

o Lamellipodia-flat 2-d sheet like structure, cross-linked mesh of  actin

 Needed for cell crawling

∙ Coordination of Actin


o Actin cytoskeleton is regulated by Rho family of  

monomeric GTPases

 Includes Cdc42, Rac, and Rho 

∙ Active-bound to GTP

∙ Inactive-bound to GDP

∙ Regulated by GEF and GAP



∙ Rho Family

o Rho-forms stress fibers; actin bundles, can interact with myosin o Rac-lamellipodial extension; activated at edges of cell and form  sheets that will spread out and allow cell to crawl

o Cdc42-forms filopodia; parallel bundles of actin form projects to  cell

o Different GTPases that we have learned about 

 Ran-involved in nucleus for transport

 Ras-cell signaling; Map Kinase pathway

 Rho family (Cdc42, Rac, and Rho)-cytoskeleton

∙ Neutrophil Movement

o N-formyl methionine on bacteria that neutrophil chemosenses o What is happening?

 External stimuli  receptor  monomer GTPases (Rac)  activated GTPases  WASP/Scar (docking protein)  Arp 2/3 and accessory proteins activated  elongation of actin  

filaments  membrane pushed forward  capping protein  

terminates elongation  aging filaments are broken up 

ADF/cofilin severs and depolymerizes ADP-actin filaments  profilin catalyzes exchange of ADP for ATP  pool of ATP

actin profilin compounds hanging out in cytoplasm

∙ Arp 2/3 and profilin work together to elongate actin  

filaments so that movement of the cell can occur  


∙ Molecular Motors

o Proteins that bind and move along cytoskeletal filaments using  ATP

o Move unidirectionally in the cell

o Motor domains-binds filament and hydrolyzes ATP

o Tail-identifies and binds cargo; varies from protein to protein  Cargo is usually vesicles and pulled by molecular motors  along microtubules (highways of the cell)

o Myosin-binds actin

o Kinesin-moves toward positive end; binds microtubules o Dynein-moves toward negative end; binds microtubules o Prokaryotes don’t have them!!! 


 Have homologues to cytoskeleton proteins (tubulin and  actin), but don’t have motor proteins

∙ Kinesins and Dyneins

o Motor proteins that bind and “walk” down microtubules; have 2  feet

 Kinesins-move towards positive end

 Dyneins-move towards negative end

∙ Cargo Transport

o Motor domain (globular head)-binds filament and hydrolyzes  ATP; conserved because binds with microtubules

o Tail-identifies and binds cargo; varies from protein to protein  depending on cargo

∙ Kinesin

o High processivity-travels for hundreds of ATPase cycles without dissociation from microtubules

 Very deliberate movement

∙ Always have one “head” attached to microtubules so  

that they don’t dissociate from microtubules

o Leading and lagging heads

 Leading head-bound to ADP at the beginning of the cycle  Lagging head-bound to ATP at the beginning of the cycle o How it works 

 Leading and lagging heads with specific bound molecules  ATP on lagging head is hydrolyzed to ADP  phosphate  

released from lagging head  ADP on leading head is  

exchanged for ATP and keeps it stable on the microtubule   linker protein comes forward and throws lagging head  

over the leading head  lagging head binds to the  

microtubule and becomes leading head

 One foot/head is always in contact with microtubule

o 10x slower than myosin

∙ Pre-Lecture Reading I: Pg. 592-599

o Muscle contractions

 Involuntary movements-cardiac muscle and smooth  muscle

∙ Ex. heart pumping, gut peristalsis

 Voluntary movements-skeletal muscle

∙ Ex. running, jumping, swimming

o Muscle contraction depends on interacting filaments of  actin and myosin

 Myosin-II-muscle myosin

∙ 2 globular ATPase heads

∙ Single coiled-coil tail

∙ Dimers


 Myosin filament-cluster of myosin II molecules bound to  each other through their coiled tails

∙ Double headed arrow

∙ Each set binds to actin filaments and pulls the  

filaments in opposite directions

 Myosin actin bundle generates a strong contractile force o Actin filaments slide against myosin filaments during  muscle contraction

 Skeletal muscle fibers

∙ Huge

∙ Multinucleated

∙ Formed by fusion of many separate smaller cells

 Myofibrils-contractile elements of the muscle cell

∙ Cylindrical structures

∙ Consists of sarcomeres

 Sarcomeres-identical tiny contractile units

∙ Repeating pattern gives it a stripped appearance

o Actin filaments-thin filaments, extend inward  

from each end of the sarcomere

 Anchored by their plus ends to the Z disc

 Minus end overlaps the ends of the  

myosin filaments

o Myosin filaments-myosin-II; thick filaments,  

centrally positioned in each sarcomere

 Contraction of a muscle cell

∙ Simultaneous shortening of all the cell’s sarcomeres

o Caused by actin filaments sliding past the  

myosin filaments

 No change in the length of either type of  


∙ Sliding motion generated by myosin heads that  

interact with adjacent actin filaments

∙ Contraction process (will be discussed later on)

 All the sarcomeres are coupled together and  

triggered simultaneously by the signaling system  

causing the entire muscle to contract almost  


o Muscle contraction is triggered by a sudden rise in  cytosolic Ca2+

 Contraction only occurs when skeletal muscle cells receive  a signal from a motor nerve

∙ Neurotransmitter triggers an action potential 

electrical signal spreads through transverse tubules  


(T-tubules) that extend inward from the plasma  

membrane  relayed to sarcoplasmic reticulum  

o Sarcoplasmic reticulum-adjacent sheath of  

interconnected flattened vesicles that surround

each myofibril

 Specialized region of the ER

 Contains high concentration of Ca2+

∙ Relayed to SR  releases Ca2+ through ion channels  

 activates molecular switch of accessory proteins  

associated with actin filaments

o Tropomyosin-rigid, rod-shaped molecule that  

binds in the groove of the actin helix; prevents  

myosin heads from associating with the actin  


o Troponin-protein complex that includes a  

Ca2+ sensitive protein associated with the end

of a tropomyosin molecule

∙ Ca2+ binds to troponin and causes a conformational  

change  tropomyosin molecule shifts position 

myosin heads can bind to the actin filaments 

muscle contraction occurs

∙ Once done, Ca2+ goes back to SR and troponin and  

tropomyosin resume original positions

∙ Polarization of the Cell

o Directional transport

 Kinesins-walk towards positive end; highly processive

 Dyneins-walk towards negative end

o Exocytosis-things exported by cell; mainly vesicles

o Endocytosis-things taken up by cell; binds to receptor o Kinesins-take neurotransmitter to synapse; walk towards cell  membrane

o Dyneins-take neurotransmitter to cell body

∙ Cilia and Flagella

o Not in all cells! 

o Useful in movement

o Cilia

 Can be in single celled protozoa

 Moves fluid across respiratory tract cells

∙ Inhale something  cilia in lungs help sweep toxins  

out  sneeze or cough it out

 Obedex-helps move egg down fallopian tubes

∙ Both Made of Microtubules

o Flagella-undulating wave-like motion, enables cells to swim  through liquid, longer than cilia


o Cilia-beat with whip-like motion, propel single cells or move fluid  over the surface of cells (ciliary elevator), shorter

 Power stroke motion

∙ Microtubule Organization

o Cilia/flagella axoneme-composed of microtubules and  associated proteins

 Forms doublets-9+2 arrangement (9 in circle with 2 in  center)

∙ Characteristic of almost all cilia/flagella

∙ Dynein motors connected to doublets

∙ Nexin-doesn’t move; binds doublets together  


∙ Microtubule Bending

o No nexin

 Initially parallel

 In presence of dynein/ATP, they slide away from each other o With nexin

 Causes binding of microtubule

 In presence of dynein/ATP, bends to release pressure from  the forces causing motion of the cilia/flagella

o Kartagener’s syndrome-ciliary dynein is nonfunctional  Respiratory infection because cilia can’t beat and mucus is  trapped

 Complete infertility in males because sperm don’t move  Reduced fertility in females because eggs don’t move as  quickly down the fallopian tubes

∙ Bacterial Flagella

o Bacteria also have flagella, but don’t contain  


 Made up of flagella proteins

 Turned by rotary motor in bacterial wall related to ATP  


∙ Uneven distribution of protons causes motor to turn  

and move flagella

∙ Myosin Interacts with Actin

o Myosin

 Interacts with actin

 ATP involved

 Binding and release of filament

 Low processivity-myosin-II only binds actin for a fraction  of a second

o How does muscle contraction happen? 

 Myosin head lacking a bound ATP or ADP is bound to actin  in a rigor configuration  molecule of ATP binds to the  


“back” of the myosin head  conformational change in the  part that binds to actin  reduces affinity for myosin head  to actin  release of myosin to move along the filament 

cleft closes around the ATP molecule  large shape change  that causes the head to be displaced by a distance of  

about 5nm  ATP is hydrolyzed  ADP and phosphate  

remain tightly bound to myosin head  weak binding of  

myosin head to a new site on the actin filament  release  of phosphate  triggers power stroke  generates a shape  change and loses ADP  head regains its original  

conformation  initial strong interaction between myosin  

head and actin filament regained

o Rigor mortis-stiff muscles after death; myosin can’t release  from actin because no more ATP being made; permanent  

contraction of muscles

∙ Multiple Myosins

o Myosin-II-functional in muscles; best studies

o 9 different myosins-exclusively or primarily in steria cilia of the  ear; sound waves

o Motor domain is highly conserved across all type because it binds with actin 

∙ Myosin I

o Simplest myosin

o Head group always moves towards the positive end of actin  filament

 True for ALL myosins 

∙ Muscular Myosin: Myosin-II

o Found in muscles

o Best studies

o Organization

 2 myosin heads that come together

 Coiled coil tail

 Myosin filaments

∙ Myosin and Muscles

o Muscle cells-long thin cells that form during development by  the fusion of many separate cells; multinucleated

 Myofibrils-long cylindrical structures in cytoplasm

∙ Sarcomeres-repeated chains of contractile units;  

gives striated appearance

∙ Sarcomeres: Contractile Unit

o Striated due to composition

 Middle part/dark region-thick filaments; myosin heads  lumped together

 Light region-thin filament; actin

 Z disc-accessory protein, binds end of actin filaments


o One sarcomere is from Z disc to Z disc

o Contract/shorten very quickly

∙ Muscle Contraction

o Thick filaments-myosin heads

o Thin filaments-actin

o Relaxation-no overlap between thick and thin filaments; a few  myosin heads may touch actin filaments

o Contraction

 Signal causes myosin heads to grab on to actin

 Binding and release through power strokes

 Causes overlap of thick and thin filaments

∙ Actin filaments moved over myosin heads

 Sarcomere/Z disc distance shortens

o Actin and myosin filaments ALWAYS stay the same length  Contraction/relaxation determined by amount of overlap ∙ Controlling Contraction


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