61 Class Note for MICRB 251 at PSU
61 Class Note for MICRB 251 at PSU
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MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh CHAPTER 15 CELL COMMUNICATION Cells communicate with each other through signaling molecules Fig 15 1 Cells sense signaling molecules with receptor proteins These are intracellular receptors There are several classes of cell surface receptors 1 kinases 2 phosphatases 3 GTP7binding proteins 4 others Each class is evolutionarily and structurally related Each class has a type of activity associated with it eg kinases phosphorylate other proteins Each receptor even Within a class is specific for a particular signal Some receptors are located on the cell surface Others are located inside the cell Receptor proteins often transduce their signal to another intracellular signal transduction protein How do they know which protein to relay their signal to Ultimately the signal transduction cascade ends by phosphorylating a particular target protein which alters its activity Examples gene regulatory proteins ion channel proteins metabolic enzymes cytoskeleton Thus signaling molecules change What cells can do MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh GENERAL PRINCIPLES OF CELL COMMUNICATION Cells do not need to be part of a multi cellular organism to communicate Yeast cells used to make bread and beer normally are single cells Fig 152 There are boy yeast cells and girl yeast cells Each sends out boy and girl pheromones Boy meets girl they mate fuse their cells and become diploid Extracellular Signal Molecules Bind to Specific Receptors Signaling molecules are also generically called ligands Types of signaling molecules proteins small peptides amino acids nucleotides steroids retinoids fatty acid derivatives nitric oxide carbon monoxide How do they get out of the signaling cell Diffusion if very small Exocytosis if big How do signaling molecules move away from the signaling cell Some don t They remain bound to the cell surface and act locally Some just diffuse away Some are transported by carrier proteins How do signaling molecules find their target cell Do they move directly to their target cell Nope They diffuse or are transported to all cells Only those cells having a matching receptor have the potential to respond to the signal Appropriate signal transduction apparatus needs to be in place Hydrophobic signaling molecules can diffuse through the membrane Why Hydrophilic signaling molecules require cell surface receptors MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 3 Extracellular Signal Molecules Can Act Over Either Short or Long Distances Short distance acting upon nearby cells Contact dependent signaling Fig 15 4a Signaling molecules that remain bound to the surface of the signaling cell must act locally right So the target cell must come in contact with the signaling cell Paracrine signaling Fig 15 4b Signaling molecules move away from the signaling cell but nearby cells have a matching receptor Long distance acting upon cells on the other side of the body Synaptic signaling Fig 15 4c The cell reaches out to the other side of the body Nerve cells do this The signaling molecules secreted at the end of the nerves axon are called neurotransmitters A nerve axon can deliver its signal to a single specific target cell Endocrine signaling The signal is sent into the blood stream which allows it to permeate the entire body Endocrine cells do this The signaling molecule is called a hormone While all cells are bathed in the hormone only cells having a matching receptor will respond to the hormone Autocrine Signaling Can Coordinate Decisions by Groups of Identical Cells Fig 156 Ever hear of Horton Hears A Who Well the autocrine system is a bit like that Nearby cells cannot hear a few cells shouting but can hear a concerted blast from a group of cells This becomes self reinforcing Early stages of animal development behave this way giving rise to patterns of cell types that ultimately give rise to different body parts Gap Junctions Allow Signaling Information to Be Shared by Neighboring Cells Rather than shouting so that all your neighbors can hear you could try whispering in your friends ear This is what gap junctions do They are channels between two adjacent abutted cells Fig 15 7 The charmels are constructed of protein But proteins are too big to pass through so only small molecules pass through Calcium ions Cyclic AMP or CAMP This type of direct and directed signaling is also important for development of multicellular organisms MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh Each Cell Is Programmed to Respond to Specific Combinations of Extracellular Signal Molecules All cells in an organism are constantly bathed in hundreds of different signaling molecules However groups of cells are programmed to hear and respond to only a subset of signals What are they hearing Fig 15 8 A minimum subset of signals is required for the cell stay alive No signal 9 I m all alone suicide cell death scientifically we call this apoptosis laypoptosis Apoptosis is a cellular program designed to destroy and recycle cellular components more on this later Certain signals cause cells to proliferate Other signals cause cells to differentiate or engage in a specialized function Example red blood cells make hemoglobin undifferentiated stem cells can turn into brain cells Different Cells Can Respond Differently to the Same Extracellular Signal Molecule Not all cells respond to a particular signal or set of signals Cells that do respond might respond differently Cells within the same group will respond the same Cells that are different and thus programmed differently will respond differently to the same signal Example Fig 1579 Muscle cells contract in response to acetylcholine a neurotransmitter Heart muscle cells relax in response to acetylcholine Different receptors to the same signal can elicit a different response Alternatively same receptors but different internal signal transduction machinery Heart muscle cell vs salivary gland cell The Concentration of a Molecule Can Be Adjusted Quickly Only If the Lifetime of the Molecule Is Short Rapid control requires rapid turnover of signaling molecules Imagine driving a car in which your ability to take your foot off the accelerator is very slow Nitric Oxide Gas Signals by Binding Directly to an Enzyme Inside the Target Cell Nitric oxide NO is a very small molecule that is produced by cells and is used as a diffusible signaling molecule Nitric oxide readily diffuses across membranes and binds to a nitric oxide receptor MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 5 Nuclear Receptors Are Ligand activated Gene Regulatory Proteins Small hydrophobic signaling molecules hormones Fig 15 12 Steroids 7 made from cholesterol examples testosterone estradiol cortisol estrogen progesterone glucocorticoids Vitamin D 7 made from cholesterol Thyroid hormones Example thyroxine Retinoids 7 made from Vitamin A Example retinoic acid Hydrophobic means water fearing so they need carrier proteins when moving around the blood They tend to last a long time and thus are involved in long duration responses like tissue development They freely diffuse across the hydrophobic cell membrane Require intracellular receptors Li ganded receptors bind to certain promoter DNA in the nucleus and regulate gene expression Fig 15 13bc Certain unliganded receptors lie dormant in the cytoplasm until bound by the ligand Others reside in the nucleus bound to their target DNA but are inactive until liganded Called nuclear receptors Nuclear receptors form a large gene family They are all structurally similar Each recognizes a different ligand and a different promoter DNA sequence Some ligands can be quite similar Glucocorticoid receptor binds glucocorticoids Vitamin D receptor binds Vitamin D Orphan nuclear receptors have not had their signaling ligands determined yet Maybe some day you will figure out What the ligands are Promoter bound liganded receptors activate transcription of the adjacent gene by recruiting component of the transcription machinery The Three Largest Classes of Cell Surface Receptor Proteins Are lon Channel linked G Protein linked and Enzyme linked Receptors All three classes are transmembrane proteins Domain external to the cell binds the ligand Hydrophobic transmembrane domain keeps it in the membrane Cytosolic domain inside the cell is the business end Ion channellinked receptors Fig 15 15 Involve nerve cells and neurotransmitter signaling molecules Gproteinlinked receptors Receptor transmits its signal to a G protein trimeric GTPbinding protein G7protein is turned on which then activates a target protein also located at the cell membrane Target protein then opens up an ion channel or catalyzes the production of a second messenger signaling molecule Enzymelinked receptors The receptor itself is an enzyme or associates with an enzyme Ligand binding activates the enzyme which catalyzes the production of a second messenger MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh Most Activated Cell Surf ace Receptors Relay Signals Via Small Molecules and a Network of Intracellular Signaling Proteins Ligand binding on the cell surface receptor triggers the production of second messengers which diffuse throughout the cell These are small molecules not protein Some are water soluble cAMP Calcium Some are hydrophobic and thus diffuse throughout the membranes only Diacyl glycerol Other receptors transduce their signals to other proteins which transduce their signal to other proteins and so on until the final target is reached You do not need to know the different functional classes of proteins described in Fig 15 16 and on pp 844 845 But do get a sense of the range different activities involved in the signal transduction process Some Intracellular Signaling Proteins Act as Molecular Switches Fig 15 17 Molecular switches are inactive proteins that become active in response to a signal Important factor for a switch how long to stay on Two important classes Protein kinases GTPbinding proteins Protein phosphorylationdephosphorylation is a major switching mechanism Protein kinases put phosphates on proteins Two major classes Serinethreonine protein kinases 7 guess what amino acid gets phosphorylated Tyrosine protein kinase Protein phosphatases take phosphates off of proteins Each kinase phosphorylates its target amino acid serthr tyr at a specific location on a specific protein GTP binding proteins Active when bound to GTP GTP 9 GDP Pi Inactive when bound to GDP Two types Fig 15 18 Trimeric Gproteins Monomeric Gproteins Multiple different signaling events must be integrated into a coordinated response MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 7 Intracellular Signaling Complexes Enhance the Speed Efficiency and Specificity of the Response A single type of extracellular signal can initiate multiple distinct signal transduction cascades leading to multiple outcomes change in cell shape and cell movement Fig 15 19 Interactions Between Intracellular Signaling Proteins Are Mediated by Modular Binding Domains Fig 1520 SH2 domains 39 Src homology domain 2 SH2 domain 39 A family of related protein domains that have the common property of binding to phospho tyrosine on certain proteins 0 The tertiary protein structure surrounding the phosphoityrosine determines whether or not a SH2 domain will dock with it 39 Found on many oncoproteins Cells Can Respond Abruptly to a Gradually Increasing Concentration of an Extracellular Signal 39 Cooperative assembly of an intracellular signaling complex by signaling molecules can lead to all or none responses Fig 15 23 A Cell Can Remember The Effect of Some Signals Cells Can Adjust Their Sensitivity to a Signal 39 Cells respond to changes in signal levels rather than absolute concentration 39 Called adaptation not to be confused with genetic adaptation during evolution 39 This allows a signal transduction cascade to work over a wide range of concentrations which cannot always be maintained at the same level throughout the body 39 Through a variety of different negative feedback mechanisms cells become desensitized to the signaling molecule Fig 15 25 MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh S ummury All cells in all multicellular organisms are programmed to respond to specific extracellular signals Different signals and different combinations of signals elicit different cellular response changes in gene expression typically Signals are typically small molecules that bind to cell surface receptors or intracellular receptors Three types of cell surface receptors ion channel G protein linked Enzyme linked Intracellular signal transduction pathways relay cell surface signals throughout the cell and typically involve protein phosphorylation or GTP binding as molecular switches A variety of mechanisms exist to turn gradients of signal concentrations that exist throughout an organism into onoff switches MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh SIGNALING THROUGH GPROTEINLINKED CEL LSURFACE RECEPTORS G protein linked receptors form the largest family of cell surface receptors found in eukaryotes Odors constitute signaling molecules There are about a thousand different G protein linked receptors that respond to particular odor molecules giving us the ability to smell lots of different things Other signals that activate G protein linked receptors include hormones and neurotransmitters All G proteins have similar structure Fig 15 26 I The polypeptide snakes through the membrane 7 times Most drugs legal and illegal work by binding to G protein linked receptors Trimeric G Proteins Disassemble to Relay Signals from G Protein linked Receptors Trimeric G protein transduce signals from G protein linked receptors They are attached to the cytoplasmic side of the cell membrane Fig 15 27 When a signaling ligand binds to the G protein linked receptor a conformational change in the protein is induced through plasma membrane The altered conformation of the receptor activates the trimeric G protein There are a lot of related G proteins that are specific for certain receptors G proteins are composed of three subunits 0L 5 y The default state is inactive and has a GDP guanosine diphosphate bound Interactions with an activated receptor cause the alpha subunit to release its bound GDP Fig 15 28 GTP then binds the alpha subunit which induces it to change conformation so that it can no longer bind y Alpha then is activate to transduce its signal to a different protein molecule Contact of alpha with its target protein induces alpha to hydrolyze GTP to GDP returning it to the inactive state Fig 15 29 Other proteins generically called GAPs GTPase activating proteins also cause GTP binding protein to hydrolyze GTP O GAPS are often specific for a certain Giproteins MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 10 Some G Proteins Signal By Regulating the Production of Cyclic AMP 39 Activated alpha subunit of G protein turns on membrane bound adenylyl cyclase 39 cAMP acts as second messenger by binding to and activating other proteins 39 cAMP phosphodiesterase destroys cAMP 39 Cholera toxin causes modification of G protein alpha subunit so it can t hydrolyzed GTP o Caused by bacterial infection 7 bacteria usually present in bad drinking water 0 Signal is always on causing ion channels to open 0 Leads to efflux of chloride ions dehydration diarrhea death 39 Many hormone induced cell responses mediated by cAMP 0 Look over Table 1571 Cyclic AMP dependent Protein Kinase PKA Mediates Most of the Effects of Cyclic AMP 39 What does cAMP do 39 cAMP dependent protein kinases PKA bind cAMP causing dissociation of inhibitory subunits Fi g 15 32 0 PKA phosphorylate target proteins at specific serine and threonine amino acids 39 What are these target proteins 0 One example is CREB cAMPiresponse element binding protein 0 CREE is a geneispecific transcriptional activator o PhOSPhOACREB recruits CBP CREE binding protein which is a chromatin remodeling factor 39 So where are we Fig 15 33 Specific example 0 Signal binds receptor which binds G protein which activates adenylyl cyclase which makes cAMP which binds PKA which phosphorylates promoteribound CREE which recruits CEP which acetylates histones which makes the promoter more accessible for transcription complex assembly ie activates transcription I Protein Phosphatases Make the Effects of PKA and Other Protein Kinases Transitory 39 Phosphatases remove phosphates from proteins MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 11 Some G Proteins Activate the lnositol Phospholipid Signaling Pathway by Activating Phospholipase C S I When certain G proteins get activated rather than activating adenylyl cyclase they activate another membrane bound enzyme called phospholipase C I Phospholipase C S cleaves PIP2 into DAG IP3 O PI45P2 phosphatidyl inositol bisphosphate O DAG diacylglycerol 0 P3 inositol trisphosphate I DAG activates Protein Kinase C PKC Ca2 Functions as a Ubiquitous Intracellular Messenger I Some events triggered by a change in intracellular Ca2 levels 0 Fertilization of an egg by sperm initiates a wave of calcium in ux that ultimately establishes the body plan Fig 15737 0 Muscle contraction I Cytosolic calcium levels are kept low while levels in the endoplasmic reticulum ER and outside the cell are high 0 Calcium ion pumps are transmembrane protein the kick calcium out of the cytoplasm I Opening of calcium ion channels in these membranes result in an in ux of calcium I IP3 is one signaling molecule that opens calcium channels Ca2Calmodulin dependent Protein Kinases CaM Kinases Mediate Many of the Actions of Ca2 in Animal Cells I Intracellular calcium receptors examples I Troponin C muscle contraction I Calmodulin I Ca2 liganded calmodulin binds target proteins thereby altering their activity I Targets include calmodulin dependent protein kinases CaM kinases 0 Examples I myosin light chain kinase 9 muscle contraction I phosphorylase kinase 9 glycogen breakdown MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 12 Smell and Vision Depend on G Protein linked Receptors That Regulate Cyclic Nucleotide gated Ion Channels How are humans able to discern over 10000 different smells Inside the nose there are specialized odor smelling cells called olfactory neurons On the surface of these neurons are olfactory receptors Fig 15 43 Specific olfactory receptors bind to specific odor molecules 0 Each olfactory neuron displays only one kind of olfactory receptor but in large quantities This triggers a special G protein to activate adenylyl cyclase cAMP opens cAMP gated ion channels resulting in a wave of ion flux nerve impulse Vision works in a similar way except that rhodopsin is the light sensor a G protein linked receptor and changes in cGMP rather than cAMP create the nerve impulse G Protein linked Receptor Desensitization Depends on Receptor Phosphorylation S ummary 0 Many cell surface receptors transduce their signals to trimeric Gproteins o Gproteins are activated by GTP and dissociate into subunits that become regulators of other proteins such as adenylyl cyclase or phospholipase C o cAMP cGMP 1P3 DAG and Ca2 are second messengers MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 13 SIGNALING THROUGH ENZYMELINKED CELL SURFACE RECEPTORS Enzyme linked receptors are often involved in cell growth and movement 39 Ligands are typically growth factors short peptides 39 Some ligands are bound to the cell surface over which the target cell is crawling 39 Cytosolic domain is either an enzyme or directly associates with an enzyme 39 Genetic defects in these receptors can lead to uncontrolled cell proliferation cancer 39 Six classes of enzymelinked receptors 0 Tyrosine kinase Tyrosine kinaseassociated Tyrosine phosphatases Serinethreonine kinase Histidjneassociate kinase Guanylyl cyclase OOOOO MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 14 Activated Receptor Tyrosine Kinases Phosphorylate Themselves 39 Ligands are secreted peptide growth factors and peptide hormones o EGF Epidermal growth factor 0 NGF Nerve growth factor 0 PDGF Platelet derived growth factor 0 FGF Fibroblast growth factor 0 M CSF Macrophage colony stimulatory factor 39 Other ligands are bound to the cell surface upon which the target cell crawls o Ephrins o Ephrin receptors are tyrosine kinases 0 Important for development including brain development 39 Often the name of the receptor is the name of the ligand plus the word receptor 39 Receptor tyrosine kinases have their kinase domain inside the cell and ligand binding domain outside of the cell 39 Li gand bound receptor tyrosine kinases phosphorylate themselves and other proteins that bind to the phosphorylated receptor 39 Since enzyme linked receptors only have a single transmembrane pass in contrast to the seven for G protein associated receptors ligand binding cannot transmit a conformational change across the membrane 39 Enzyme linked receptors often oligomerize form dimers or bigger aggregates upon ligand binding allowing their cytosolic kinase domains to phosphorylate each other Fig 15 50 0 Sometimes the ligand itself simultaneously binds two receptors causing dimerization 39 Autophosphorylation does two things 0 It stimulates its own kinase activity 0 It provides docking sites for other proteins Fig 15 52 MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 15 Phosphorylated Tyrosines Serve as Docking Sites For Proteins With SHZ Domains 39 Phospholipase Cy PLC v is one enzyme that binds certain phosphorylated receptors Fig 15 53a 39 Src is a certain protein kinase that also binds phosphorylated receptors 0 Certain defective versions of Src cause cancer and thus is an oncogene 39 Phosphatidylinositol 3 kinase PI3 kinase 39 These target proteins that bind phosphotyrosine are structurally very different but they all possess a similar domain called an SH2 domain Src homology domain which binds to phosphotyrosine Fig 15 53c 0 Docking specificity is achieved through interactions with surrounding amino acids MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 16 Ras ls Activated by a Guanine Nucleotide Exchange Factor I Remember trimeric G proteins O Activated GTPibound a subunit dissociates from 5y allowing them to find their targets I There are also monomeric G proteins the most notable is called Ras I There are three major subfamilies related in structure and function 0 Ras O Rho 7 regulates cytoskeleton function 0 Rab 7 intracellular transport of vesicles I There are many different versions each with different targets I These proteins are attached to the inside of the plasma membrane 0 Covalently attached to a membrane lipid I Ras is activated when it binds GTP I It then engages its target like a protein kinase 0 These targets are often involved in processes like cell proliferation o Ras mutants that fail to hydrolyze GTP always on are oncogenic I How is Ras regulated Fig 15 54 0 Ras is a molecular switch I GTPibound 9 active I GDPibound 9 inactive o Guanine nucleotide exchange factors GEFs promote GDP dissociation so that GTP can bind 39 GEFs therefore activate Ras o GTPaseactivating proteins GAPS promoter GTP hydrolysis 39 GAPS therefore inactivate Ras o Ras is regulated mostly by a GEF call Sos I How is Sos regulated Fig 15 55 0 Certain activated receptor tyrosine kinases are bound directly by Grb 2 39 Do you think Grb72 has an SH2 domain 0 Other activated receptor tyrosine kinases do not present their phosphotyrosines in a way that Grb 2 can bind I They require an adaptor protein called Shc I Shc has an SH2 domain 0 Grb 2 binds and activates Sos MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 17 Ras Activates a Downstream SerineThreonine Phosphorylation Cascade That Includes a MAP Kinase Fig 1556 Ras 9 MAP kinase kinase kinase Raf 9 MAP kinase kinase MEK 9 MAP kinase So remember Ras 9 Raf 9 MEK 9 MAP kinase 9 lots of targets Targets include transcription factors cell proliferation factors other protein kinases MAP kinase moves into the nucleus upon phosphorylation MAP kinase activation requires two different phosphorylation events 0 One on tyrosine 0 One on threonine What turns off these kinases Different signaling molecules might employ distinct but parallel kinase cascades Others might share some of the same kinases How is cross talk prevented O A 9 B 9 C O X 9 B 9 Z Cross talk is prevented by physically attaching members of a pathway together Fig 5 57 Stress activates MAP kinase pathways 0 Ultraviolet light Heat shock Osmotic shock high external salt concentration Infection Starvation OOOO MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 18 P1 3 Kinase Produces 1nositol Phospholipid Docking Sites in the Plasma Membrane 39 Cell proliferation needs two things 0 Cell growth increase in mass induced by growth factors 39 Driven by P13 kinase 39 Many different P13 kinases 0 Cell division 1 9 2 induced by mitogens 39 Driven by Ras 7 MAP kinase pathway 39 P13 kinase phosphorylates a certain lipid located only on the inside of the plasma membrane Fig 15 58 0 The lipid is called phosphatidylinositol PI 0 The phosphorylated products are called PI34P2 and PI345P3 I Contains 2 and 3 phosphates respectively 39 These P1Ps remain in the membrane and become dockingactivation sites for downstream target proteins Fig 15 59a Fig 15 59b 0 They do not get cleaved O Eventually they do get dephosphorylated which shuts down the mitogenic signal 0 What would happen if the phosphatase was inactivated I P39TEN is one such phosphatase 39 What are the downstream targets Fi g 15 59c O Tyrosine kinases eg BTK in B lymphocytes 0 PLCA 39 BTK activatesphosphorylates PLCA 39 PLCA cleaves the P145P3 to make 1P3 O A conserved domain on these downstream targets binds the P1Ps I The conserved domain is called a PH domain 39 Remember P145P3 0 Its different 0 Gets cleaved into DAG 1P 0 DAG 9 activates protein kinase C PKC 1P3 9 intracellular calcium release MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 19 The PI 3 KinaseProtein Kinase B Signaling Pathway Can Stimulate Cells to Survive and Grow Fig 1560 PI3 kinase 9 PI345P 9 binds PK B protein kinase B 39 Do you think PKVB has 21 PH domain PI3 kinase 9 PI345P 9 binds PDKl phosphatidylinositolidependent protein kinase PDKl phosphorylates PK B PK B moves into the cytoplasm away from the plasma membrane PK B phosphorylates BAD o Unphosphorylated BAD induces cell death apoptosis 0 Phospho BAD is inactive So if a cell is not receiving a continuous signal that is propagated through PI3 kinase it will die 0 So death is the default state for many cells 0 Constant and proper signaling is a prerequisite for survival 0 So if a cell finds itself in the wrong place in your body it will die Summary Growth factors 9 9 PI3 kinase 9 9 PK B 9 cell growth survival 9 S6 kinase 9 stimulates ribosomes 39 S6 is a ribosomal subunit Know Fig 1561 MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 20 Tyrosine Kinase associated Receptors Depend on Cytoplasmic Tyrosine Kinases for Their Activity 39 The cytoplasmic domain of the receptor associates with a tyrosine kinase rather than being a kinase itself 39 These tyrosine kinases come in many avors each working in a different pathway 0 Examples Src Yes Fyn Lck Lyn 39 They have SH2 domains 0 What do SH2 domains bind MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 21 Cytokine Receptors Activate the Jak STAT Signaling Pathway Providing a Fast Track to the Nucleus 39 Cytokines are locally acting peptide signaling molecules 39 Here are some names of cytokines that you should know 0 interferon 7 activates macrophages a type of phagocytic immune cell winter feron 7 activates neighboring cells to resist viral infection Erythropoietin 7 stimulates production of red blood cells Prolactin 7 stimulates milk production Growth Hormone 7 stimulates growth Interleukins 7 stimulate blood cell development I 1L3 IL76 etc 39 Cytokine receptors are cell surface enzyme linked receptors 39 Janus tyrosine kinases J aks associate with the cytosolic side of these OOOOO receptors 0 Examples of Jaks 39 Jakl 39 Jak2 39 Tyk2 0 Certain J aks work with certain receptors 39 Jaks phosphorylate themselves the receptor and STATs Fig 15 63 39 STATS signal transducers and activators of transcription associate with the phosphorylated receptor to become phosphorylated 0 Do STATs have an SH2 domain 0 STATs are direct transcriptional activators 0 Examples of STATs 39 STATl 39 STAT2 39 STATS 0 Certain STATs work with certain receptors 39 The phosphorylation of the STAT causes it to dimerize o Dimerization occurs Via SH2 domains and phosphotyrosine 39 Homodimers and heterodimers can form 0 If SH2 domains interact with phosphotyrosines then will all proteins that contain these features dimerize with each other 39 How is specificity achieved 39 Dimerized STAT then moves into the nucleus and binds to specific promoter elements 0 In combination with other transcriptional regulators certain genes are turned on 39 Summary example Prolactin 9 Prolactin receptor 9 JaklJak2 9 STATS 9 target genes target genes whey acidic protein lactoglobulin casein other milk proteins 39 The J ak stat pathway is turned on by phosphorylation kinases and off by dephosphorylation phosphatases MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 22 Some Protein Tyrosine Phosphatases May Act as Cell Surface Receptors 39 Protein tyrosine phosphatases PTPs are very specific Fig 15 64 0 So there are lots of them 0 Some are membraneibound receptors 0 Others are cytoplasmic o In contrast serthr specific phosphatases act on a Wide variety of proteins 39 They function to down regulated J ak STAT signaling 0 Examples SHPil SHP72 o PTPs bind to phosphotyrosines so What must they have Signal Proteins of the TGF S Superfamily Act Through Receptor SerineThreonine Kinases and Smads Fig 1565 TGFS is a signaling peptide 39 Transforming growth factor beta 0 There are many different homologous classes of TGFi TGFif Activins BMPs 7 bone morphogenic proteins 0 Each doing different things Body patterning during development Cell proliferation Extracellular matrix production Apoptosis Tissue repair and immune regulation TGF3 receptor is a transmembrane receptor linked to a serthr kinase 0 The kinase phosphorylates latent cytoplasmic transcription factors called Smads o Smads move into the nucleus and bind to target promoters 39 Other regulatory proteins also needed 0 Examples of Smads 39 Smadl 39 Smad2 39 SmadS TGF S 9 TGF S receptor 9 serthr kinase 9 Smad 9 target genes Receptor Gnarlylyl Cyclases GQI iGt alE Cyclic ny Directly Bacterial Cl39remotaxis Depends on a Two Component Signalin Pathway MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 23 39 Must know 3 of the 5 classes of enzyme linked receptors 0 Receptor tyrosine kinases o Tyrosine kinase associated receptors 0 Receptor serinethreonine kinases 39 Ligand binding induces receptors to dimerize and cross phosphorylate each other 39 Kinases and GTPases bind to phospho receptors 39 A protein phosphorylation internal signaling cascade ensues 39 Ultimately transcriptional activators that bind promoter elements become active by this cascade MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 24 SIGNALING PATHWAYS THAT DEPEND ON REGULATED PROTEOLYSIS I Signaling pathways to be covered here Notch Wnt Hedgehog NF KB I Ever wonder where they come up with these names 0 Many like notch and hedgehog are names associated with a phenotype typically in Drosophila when the gene is mutated I Thatis the result of genetic investigation I Often the phenotype is observed first then responsible gene is isolated 0 Names like NF KB are derived from biochemical activities then the responsible gene is isolated I So NFiKB was identified as an activity from fractionated cell extracts that stimulated transcription of the immunoglobulin lgappa light gene I It was found in the r1uclear fraction rather than the cytoplasmic fraction I It was originally isolated from Eilymphocytes I Can you now see where the name came from MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 25 The Receptor Protein Notch ls Activated by Cleavage This is a major signaling pathway in animal development 0 Particularly in nerve cell development Delta is the signaling protein Notch is the receptor I Quick is this a cell surface receptor or an internal receptor Delta is presented on the cell surface of the nerve cell 0 Actually it s a transmembrane protein Nearby cells contain Notch When the nerve cells grows and moves around adjacent cells Delta binds to Notch This activation of Notch in the neighboring cells prevents these cells from developing into neurons Called lateral inhibition What are the down stream events upon Notch activation Fig 15 71 0 An intracellular protease cleaves off a cytoplasmic domain of Notch o This domain is a transcriptional co activator o The Notch tail binds to a promoter bound repressor called CSL turning it into a transcriptional activator of genes that code for transcriptional inhibitors of neural genes Notch and Alzheimer s disease 0 Notch is subjected to proteolytic cleavage at both its intra7 and extracellular domain by a protease call presenilinil o Presenilinil cleaves other membrane proteins as well such as iarnyloid precursor protein APP 0 Too much cleavage of APP results in accumulation of protein fragments outside of the cell 0 The protein fragments aggregate into plaques which are thought to kill the nerve cell 0 This nerve cell death cause senility and Alzheimer s disease MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 26 Wnt Proteins Bind to Frizzled Receptors and Inhibit the Degradation of 5 Catenin I Wnt signaling controls many aspects of animal development 0 Wnt is a local acting signaling peptide 0 So is its receptor going to be located on the cell surface I The Wnt receptor is called Frizzled Fig 15 72 I The way Wnt works is to set up a cascade of events that protects a co activator of Wnt responsive genes from getting degraded by proteolysis o Proteolysis I Proteolysis is the enzymatic removal of amino acids from proteins 0 The coactivator of Wntiresponsive genes is called Bcatenin or Armadillo in ies I icatenin normally associated with cadherins which reside at cellicell junctions I Cellicell junctions are where cells attach to each other I icatenin attaches cadherins to the cell s actin cytoskeleton 0 Any icatenin not part of this structural framework of the cell is rapidly degraded I When Wnt signaling is not present S catenin is phosphorylated by a protein complex having the following components 0 The kinase component is GSK3I3 glycogen synthase kinase 3b I Wonder where it got that name I Only Secatenin that is not part of cellular cytoskeleton gets phosphorylated 0 APC adenomous polyposis goli I APC helps GSKIS bind to icatenin I Defects in APC prevent icatenin degradation which leads to activation of Wntiresponsive genes even in the absence of Wnt I When this happens at the wrong place at the wrong time cell proliferation ensues I In the colon intestines polyps outgrowths arise that could be come cancerous I 80 of all colon cancer is due to defects in APC o AXin a scaffolding protein that hold GSK 3f5 APC and S catenin together I Phosphorylated S catenin is then rapidly degraded by the proteosome o Phosphorylated S catenin is rapidly ubiquitinated o The proteosome recognizes the ubiquitin and degrades S catenin I When Wnt signaling is present the bound Frizzled receptor sends an intracellular signal via a protein called Dishevelled to inhibit GSK 3f5 I Free S catenin migrates into the nucleus where it displaces a repressor protein called Groucho from the LEF lTCF transcriptional activator which is bound at Wnt responsive genes I Wnt 9 Frizzled 9 9 Dishevelled axinAPCGSK 3f5 S catenin S catenin LEF lGroucho Wnt responsive genes S catenin LEF l 9 Wnt responsive genes means inhibit whereas 9 means stimulates I c myc is a Wnt responsive gene MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 27 o c myc is a potent transcriptional activator of genes involved in cell proliferation 0 So can you explain how defects in APC lead to cancer 0 How is Wnt signaling and colon cancer related Hedgehog Proteins Act Through a Receptor Complex of Patched and Smoothened Which Oppose Each Other Fig 1573 39 Hedgehog proteins are signaling molecules I Drosophila larvae having mutations in Hedgehog reminded the student working on this of a hedgehog 39 They act locally 39 Patched is the receptor that binds Hedgehog 39 Like the Wnt receptor Frizzled liganded Patch prevents the degradation of a transcriptional activator of Hedgehog target genes MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 28 Multiple Stressful and Proinflammatory Stimuli Act Through an NFKB Dependent Signaling Pathway Upon infection or tissue damage certain cells of the immune system called macrophages secrete the cytokines TNF 0L Tumour Necrosis Factor 0L and IL 1 interleukin 1 0 These are peptides so they have cell surface receptors I Nearby cells respond by expressing genes involved in wound repair and fighting infection I These cells express the TNF 0L receptor and bind TNF0L I In normal healthy cells the transcriptional activator of these in ammatory response genes is normally sequestered in the cytoplasm I This activator is called NFKB Fig 15 74 0 There are 5 NF KB proteins in mammals I RelA I RelB I ciRel I NFiKBl I NFiKBZ I Did you ask what sequesters NF KB in the cytoplasm o IKB sequesters NF kB in the cytoplasm by blocking its nuclear localization signal I How does TNF 0L signaling release IKB from NF KB o Glad you asked 0 Upon TNF 0L signaling IKB gets I phosphorylated by IKB kinase IKK I then ubiquitinated I then degraded o NF KB is then free to translocate to the nucleus I Well then how does TNF signaling lead to IKK activation 0 You probably don t want to know but I ll tell you anyway 0 Phosphorylation of IKK by IKKK makes IKK active 0 IKKK is made active via a TNF 0L receptor associated kinase I Summary TNF 0L 9 TNF 0L receptor 9 RIP kinase 9 IKKK 9 IKK IKB NF KB NF KB 9 in ammatory response genes S ummary I Many transcriptional activators are held in the cytoplasm by an inhibitor protein I Signalinginducedproteolysis of the inhibitor releases the transcriptional activator I In other signaling systems the transcriptional activator is the direct target of proteolysis I Signaling event release the activator from proteolysis MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 29 SllV39 N XlllNl39 EN PlkN l S Multicellulai ity and Cell Communication Evolved Independently in Plants and Animals Receptor SerineThreonine Kinases Function as Cell Surf ace Receptors in Plants Ethylene Activates a Two Component Signaling Pathway Phytochromes Detect Red Light and Cryptochi omes Detect Blue Light S ummary MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 30 CHAPTER 16 THE CYTOSKELETON 39 Cells need to move be structurally robust and adopt certain shapes to conduct their function 0 Think of 39 muscle fiber contraction in muscle cells 39 a nerve cell that extends from your foot to your head 39 a sperm cell trying to find its way 39 a macrophage crawling throughout your body 39 skin cells that protect your body 39 Large work projects like moving chromosomes vesicles and protein complexes requires large structural machines 39 The cytoskeleton provides this THE SELFASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS 39 There are three major types of cytoskeleton filaments 0 Intermediate filaments provides mechanical strength to the cell 0 Actin filaments important for cell shape and motility o Microtubules made up of tubulin Tracks for intracellular transport 39 Motor proteins move vesicles and other large complexes along actin filaments and microtubules 39 Cytoskeletal filaments are dynamic constantly assembling and disassembling Fig 16 2 39 Proteins must regulate these dynamics MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 31 Each Type of Cytoskeletal Filament ls Constructed from Smaller Protein Subunits Structure Subunit Actin filament actin Microtubules tubulin Intermediate lamins filaments Vimentin keratins 39 Each subunit is a protein 39 Thousands millions of subunits line up head to tail Via noncovalent interactions 0 Remember from BMBMicrb 251 what these noncovalent interactions are 39 Also side to side interactions make for a very stable structure Fig 16 3 Filaments Formed from Multiple Protofilaments Have Advantageous Properties Nucleation Is the Rate limiting Step in the Formation of a Cytoskeletal Polymer The Tubulin and Actin Subunits Assemble Head to Tail Creating Filaments that Are Polar Fig 166 39 The repeating unit in microtubules is a heterodimer of oc tubulin and 5 tubulin 0 Both are structurally related 0 Arrangement is headitoitail 39 Both subunit bind GTP but only one can hydrolyze its GTP to GDP 39 GTP hydrolysis regulates microtubule stability 39 Microtubules are cylinders hollow tubes 39 Structurally they are very stiff 39 The repeating unit in actin filaments is a monomer of actin Fig 16 7 39 Each actin monomer binds ATP not GTP 39 ATP hydrolysis controls the dynamics 39 Actin filaments are not hollow but have a helical twist 39 They are quite exible 39 Actin filaments can be lashed together to make strong actin bundles MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 32 The Two Ends of a Microtubule and of an Actin Filament Are Distinct and Grow at Different Rates Filament Treadmilling and Dynamic Instability Are Consequences of Nucleotide Hydrolysis by Tubulin and Actin A growing microtubule involves addition of GTP bound tubulin to one end Within the microtubule GTP is hydrolyzed to GDP GDP tubulin dissociates from the microtubule end more rapidly 0 However once GDPitubulin is internal to the filament it no longer dissociates Net polymerizationdepolymerization is a race between GTP hydrolysis at the end and addition of new GTP tubulin subunits This is called dynamic instability Fig 16 1 1b 0 Allows filaments to grow and contract What factors contribute to net polymerization Net depolymerization Same with actin but with ATP Also rather than assembling and disassembling from one end actin assembles from one end and disassembles at the opposite end of the filament ADP actin dissociates faster than ATP actin Called treadmilling Treadmilling allows actin filaments to move which allows cells to move So what is the purpose of ATP and GTP hydrolysis with respect to filament dynamics Treadmilling and Dynamic Instability Require Energy but Are Useful Other Polymeric Proteins Also Use Nucleotide Hydrolysis to Couple a Conformational Change to Cell Movements Tubulin and Actin Have Been Highly Conserved During Eukaryotic Evolution MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 33 Intermediate Filament Structure Depends on The Lateral Bundling and Twisting of Coiled Coils Fig 1616 39 Intermediate filaments impart structural strength to cells 39 Things that tend to undergo mechanical stress tend to be anchored to the intermediate filaments 39 Unlike actin and tubulin intermediate filaments have subunits that are elongated and alpha helical 39 Monomers dimerize Via coiled coils 39 Dimer come together to form tetramers and so on to form long cables 39 There is no head tail arrangement no dynamic instability no treadmilling no nucleotide binding 39 Assemblydisassembly is regulated by protein phosphorylation Intermediate Filaments Impart Mechanical Stability to Animal Cells 39 Keratins 0 Finger nails hair scales and skin are made up of keratins 0 There are many different kinds of keratin intermediate filaments 39 Nuclear lamina o Lamins A B and C 0 Make up the inter lining and structure of the nucleus 39 Neurofilaments 0 Give structural stability to neuronal axons 0 ALS Lou Gehrig s disease I Results from accumulation and abnormal assembly of neurofilaments Filament Polymerization Can Be Altered by Drugs 39 Plants and fungi make toxins that alter the assemblydisassembly of the cytoskeleton 39 Fungal phalloidins stabilizes actin filaments 39 Calchicine from a crocus cause tubulin depolymerization 39 Taxol from the yew tree stabilizes microtubules 0 Used to treat cancer 39 Acrylamide disassembles neurofilaments o Neurotoxin 0 Used in the laboratory to make polyacrylamide gels MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 34 S ummary 39 Microtubules are made up of globular tubulin subunits and form long structural cylinders 39 GTP hydrolysis controls rates of net assembly and d isassembly 39 Microtubulesjunction in intracellular transport 39 Actin laments are made up of globular actin subunits 39 ATP hydrolysis controls the dynamics of assembly and disassembly 39 Actin laments provide cell shape and is the treadmill for cell movement 39 Neurofilaments provide mechanical strength to the cell 39 Subunit structure of neurofilaments are unlike actin and tubulin 39 Drugs can a ect cytoskeleton assemblydisassembly MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 35 HOW CELLS REGULATE THEIR CYTOSKELETAL FILAMENTS Microtubules Are Nucleated by a Protein Complex Containing y tubulin Fig 16 22a Microtubule assembly is initiated at the microtubule organizing center MTOC A number of proteins are part of MTOC One in particular is y tubulin y tubulin is related to 0L and S tubulin but serves only to anchor one end of the growing microtubule filament Microtubules Emanate from the Centrosome in Animal Cells Fig 1623 Centrosomes and MTOC are essentially the same thing in animals 0 Plants lack centrosomes but have numerous locations corresponding to MTOC Within the centrosome are many copies of the ytubulin ring complex which are responsible for nucleating each microtubule Also embedded in the centrosome is a pair of centrioles The centrioles are orientated at right angles to each other Centrioles organize the centrosome matrix During cell division they duplicate move to opposite sides of the cell and help pull apart duplicated chromosomes at mitosis Fig 18 18 What are centrioles made up of o Modified microtubules and other proteins Actin Filaments Are Often Nucleated at the Plasma Membrane Actin filament nucleation occurs at the plasma membrane So the highest density of actin filaments is just under the cell surface where it determines cell shape plasticity and movement Nucleation of actin is regulated by external signals in response to changing environments Nucleation is catalyzed by ARPs actin related proteins Fig 16 28 0 Analogous to the yitubulin ring complex Directional polymerization results in cell movement Fig 16 90 MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 36 Filament Elongation ls Modified by Proteins That Bind to the Free Subunits About half of the actin in the cell is in filaments The rest is free monomer Proteins like thymosin are inhibitory to monomer incorporation and thus regulate actin assembly Profilin competes with thymosin binding and enhances monomer incorporation into the end of the filament Fig 16 30 Profilin is activated by phosphorylation and inositol phospholipids Profilin is localized to the plasma membrane Extracellular signaling molecules bind to a section of the cell exterior can lead to local profilin activation and a burst of actin filament assembly resulting in filopodialamellipodia formation and cell movement Similar processes different protein and different location happen with tubulin Proteins That Bind Along the Sides of Filaments Can Either Stabilize or Destabilize Them These proteins tend to bind throughout the filaments Protein that bind microtubules are generically called MAPS microtubule associated proteins 0 Do not confuse with MAP kinase described earlier they are very different Some MAPs stabilize microtubules including the formation of large microtubule bundles Fig 16 33 Other MAPS link microtubules with other cellular components Tropomyosin holds together actin bundles Cofilin depolymerizes actin Cofilin preferentially interacts with the ADP form of actin which tends to be near the minus end in the treadmilling process Proteins That Interact with Filament Ends Can Dramatically Change Filament Dynamics Capping proteins cap the plus end of actin filaments 0 Subject to regulation by signaling molecules via PIPZ ARPs cap the minus end Microtubules have their own set of capping proteins as well MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 37 Filaments Are Organized into Hi gher order Structures in Cells Intermediate Filaments Are Cross linked and Bundled Into Strong Arrays Cross linking Proteins with Distinct Properties Organize Different Actin Assemblies 39 Actin filaments can be arranged in bundles or in a weblike network 39 Specific proteins are designed to crosslink actin filaments in different ways 0 aactinin and vimentin make bundles Fig 16 40 Fig 16 41 0 Spectrin makes weblike networks in blood cells Fig 10 31 0 Filamen also makes networks Fig 16 42 I Important for making at lamellipodia for crawling along surfaces I Fig 1647bcd Severing Proteins Regulate the Length and Kinetic Behavior of Actin Filaments and Microtubules Cytoskeletal Elements Can Attach to the Plasma Membrane 39 ERM proteins attach actin filaments to the plasma membrane Fig 16 48 39 Actually the ERM proteins attach to transmembrane glycoproteins such as CD44 39 Unlike the permanent attachment that occurs in red blood cells and muscle cells the ERM attachment must be dynamic and regulated 39 Phosphorylation and PIP2 regulate this in response to external signals MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 38 Special Bundles of Cytoskeletal Filaments Form Strong Attachments Across the Plasma Membrane Focal Contacts Adhesion Belts and Desmosomes When cells are slithering across a surface they need to grab on to things Cells do this through focal contacts Integrins are transmembrane proteins that bind to the extracellular matrix Cadherins allow cells to attach to each other 0 Cadherins are attached to catenins which attach to actin filaments Desmosomes are places of cell cell contact that have attached intermediate filaments not actin filaments Extracellular Signals Can Induce Major Cytoskeletal Rearrangements S ummary Monomeric GTPase Rho control actin filaments Fig 16 50 Microtubules nucleate at centrosomes Actin nucleates at the plasma membrane Microtubule and actin filament can be lashed into strong bundles by crossl inking proteins Microtubule and actin filament assembly and disassembly is dynamic and is controlled by capping proteins Extracellular signaling molecules can control actin filament assemblydisassembly Actin laments are anchored to the cell membrane by integral membrane proteins that also attach to extracellular matrix and other cells MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 39 MOLECULAR MOTORS Motor proteins 0 One end attaches to a particular type of cytoskeleton filament o The other end attaches to a particular cargo I organelles like mitochondria I chromosomes I muscle contraction 0 ATP hydrolysis moves the motor protein relative to the filament Actin based Motor Proteins Are Members of the Myosin Superfamily Myosin is a protein responsible for force generation during muscle contraction Myosin structure is important in force generation Fig 16 51 0 Long extended alpha helical region that dimerizes tails o Globular head that hydrolyzes ATP 0 Called myosin II heavy chain 0 Bound to each heavy chain is another subunit called myosin light chain 0 Two copies of myosin light chain are bound to each heavy chain Heavy chain tails form bundles that are symmetric Fig 1652 Bundles get together to form myosin filaments There are many different kinds of myosins each having different functions but all have similar structural arrangement particularly in the head region Myosin moves along the end of the actin filament upon ATP hydrolysis one step at a time There Are Two Types of Microtubule Motor Proteins Kinesins and Dyneins Kinesin is a motor protein that moves along microtubules Kinesin is structurally similar to myosin Fig 16 55 Fig 16 57 Kinesin is end directed Dyneins are motor proteins that move toward the end of microtubules They are unrelated to kinesin and are very fast Used to move cilia and flagella MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 40 The Structural Similarity of Myosin and Kinesin Indicates a Common Evolutionary Origin Motor Proteins Generate Force by Coupling ATP Hydrolysis to Conformational Changes 39 The myosin cycle is like paddling a canoe well work with me on this one Fig 16 58 step ATP binding site What happens to the myosin head 1 empty Bound to actin lament in a state of rigor cause of rigor nwrtis in death 2 ATP Detaches from actin filament at position n 3 ADP Pi Cocks forward conformational change induced by ATP hydrolysis 4 ADP Reattaches to actin filament at n1 position 1 empty Shifts back to original conformation power stroke Be able to identify what happens at each of these steps 0 ATP binding 0 ATP hydrolysis 0 Release of Pi inorganic phosphate 0 Release of ADP 39 The kinesin cycle is like walking well walk with me on this one Fig 16 59a step Head A Head B What happens to the kinesin heads A1 ADP Release of A from tubulin free A1 empty B bound to tubulin A2 ADP ATP Throws free rear head A forward past attached leading head B B1 empty A bound to tubulin B1 H ADP Release of B from tubulin free B2 ATP ADP Throws free rear head B forward past attached leading head A Be able to identify what happens at each of these steps Motor Protein Kinetics Are Adapted to Cell Functions MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 41 Motor Proteins Mediate the Intracellular Transport of Membrane enclosed Organelles 39 Microtubules emanate from the cell center minus end to the periphery end 39 Kinesin moves organelles toward the plus end periphery o Endoplasmic reticulum ER splays out toward the periphery due to Kinesin 39 Dynein moves things toward the minus end toward cell center Fig 16 63 0 The golgi stays toward the cell center due to dynein 0 Interestingly the structural attachment to the golgi is very similar to the underlying cytoskeletal structure of red blood cells Fig 10 3 1a 0 Golgi shape and red blood cell shape are similar Fig 10 27 vs Fig 1322 Motor Protein Function Can Be Regulated Muscle Contraction Depends on the Sliding of Myosin H and Actin Filaments 39 Muscles are derived from the fusion of many muscle cells 39 Myosin filaments are a strung out between two scaffolds Z disc by a massive protein called titin Fig 16 72 0 Titin is the largest known polypeptide chain I 3000000 daltons 25000 amino acids I Most proteins are 50000 daltons 500 amino acids 0 It acts as a bungee cord helping to recover overstretched muscles 39 The myosin filament is bipolar 39 Encircling each myosin filament are actin filaments which are also attached to the Z disc scaffold Fig 16 71 0 A separate set of actin filaments encircles each end of the myosin filament o The actin filaments are capped at one end by tropomodulin 39 Myo brils are composed of actinmyosin filaments 39 Myofibrils are striated due to the repeating nature of the filament arrangement Fig 16 69 0 Z disk actin myosin actin Z diskn sarcomere o Myofibrils are as long as the muscle itself 39 Lots of myofibrils form a muscle ber and are the product of many fused muscle cells Fig 16 68 39 Muscle contraction occurs when myosin fibers slide past the actin via the power strokes described above 0 So sarcomeres get smaller when a muscle contracts 0 Each myosin filament has about 300 heads each hydrolyzing ATP without coordinating it with the others MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 42 Muscle Contraction ls Initiated by a Sudden Rise in Cytosolic Ca2 Concentration 39 What happens when you decide to contract a muscle 39 A nerve impulse travels from the brain to the muscle 39 The wave of depolarization moves along the plasma membrane to invaginations called Ttubules Fig 16 73a 39 T tubules wrap around the myofibrils 0 But they are kind of one dimensional phone lines 39 The signal is transmitted from the T tubule to the sarcoplasmic reticulum o The sarcoplasmic reticulum is more like a two dimensional endoplasmic reticulum lattice wrapping around the myofibrils 39 The T tubules and the sarcoplasmic reticulum are connected by voltage gated calcium Ca2 channels 0 Calcium is stored in the sarcoplasmic reticulum and is released into the cytoplasm of the muscle cells by the wave of depolarization Fig 16 73c 39 Calcium binds to the Troponin complex causing it to dissociate from Tropomyosin o Tropomyosin is a filamentous protein that normally interacts along the actin filament groove in a way that does NOT interfere with myosin binding to actin Fig 16 74ab 0 However the troponin complex pulls tropomyosin over the region where myosin binds thereby preventing muscle contraction 0 So the key is to get the Troponin complex to release Tropomyosin so that Tropomyosin can move out of the way 0 The Troponin complex binds calcium causing it to dissociate from Tropomyosin o This now exposes the myosin binding sites on actin 39 Summary Nerve impulse 9 target muscle 9 T tubules 9 Ca2 gated ion channels 9 Ca2 release from SR 9 Ca2 Troponin 9 Tropomyosin myosinactin ATP hydrolysis Heart Muscle Is a Precisely Engineered Machine Cilia and Flagella Are Motile Structures Built from Microtubules and MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh Dyneins S ammary 43 The coordinated movement of tubulin within a microtubule filament relative to other parts of the filament can cause bending o Requires microtubules to be tethered at the end This occurs in sperm agella and protist agella Fig 16 77 and Fig 16 79 But not in bacteria 0 They use a different kind of protein filament that rotates like a propeller rather than undulate Motor proteins use ATP hydrolysis to move things along microtubules and actin laments but not intermediate filaments Myosin moves along actin filaments by rowing Kinesin moves along microtubules by walking MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh CHAPTER 19 CELL JUNCTIONS CELL ADHESION AND THE EXTRACELLULAR MATRIX I Major tissue types in vertebrates O O O O Nerve Blood Lymphoid Connective I Make lots of extracellular matrix including collagen I Cellicell junctions are rare I Provides a lot of mechanical stress resistance Epithelial skin Fig 19 1 I Very little extracellular matrix made I Many strong cellicell junctions make a strong epithelial sheet I Junctions are attached to the cytoskeleton providing resistance to mechanical stress I Epithelial sheets include the skin and the lining of your gut I Form barriers to water other molecules and cells I Molecules must move through the cell I Epithelial sheets rest on a bed of connective tissue I Extracellular matrix is formed from secreted macromolecules that provide the substratum upon which cells can attach and move around on I Cells are also bound to each other via cell cell junctions I Groups of tissue that have specific purposes are called organs MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 45 CELL J UNCTIONS I Points of cell cell attachment and cell matrix attachment I Highly abundant in epithelial cells I Three functional groups 0 Occluding junctions I Like zip lock bags nothing gets past 0 Anchoring junctions I How cells hold on to each other and the matrix I Are attached to the cytoskeleton o Communicating junctions I Provides a passage for communication between cells I Small molecules pass through the junctions Occluding Junctions Form a Selective Permeability Barrier Across Epithelial Cell Sheets I Also called tight junctions in vertebrates I The zip lock seal prevent molecules and cells from permeating between cells from the gut of an animal Fig 19 2 0 Actually cells regulate this too to let certain molecules like amino acids certain ions past I The tight junctions also prevent transmembrane carrier proteins on basal and lateral side of the cell from migrating over to the luminal side apical surface I Model for tight junctions Fig 19 4a Fig 19 5 0 Claudjn proteins are integral membrane proteins that form the tight junctions Anchoring Junctions Connect the Cytoskeleton of a Cell Either to the Cytoskeleton of Its Neighbors or to the Extracellular Matrix I They attach to each other across membranes between cells Fig 19 7 I Inside the cell they are attached to the cytoskeleton I Note the cell membrane does not provide any major structural stability to cells I Two major functional forms 0 Cadher39ins form adherens junctions and desmosomes Fig 19 9 I Adherens junctions usually attaching to actin filaments via anchoring proteins such as catenins vinculin and aiactinin I Desmosomes attach to intermediate filaments like keratin filaments Fig 19 1 1 o Integrins from focal adhesions and hemidesmosome attachments to the extracellular matrix Fig 19 12b I Focal adhesions attach to actin filaments via anchoring proteins I Hemidesmosomes attach to intermediate filaments MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 46 Summary cell cell Adherens junctions cadherins attach to actin filaments Desmosomes cadherins attach to intermediate filaments cell matrix Focal adhesions integrins attach to actin filaments Hemidesmosomes integrins attach to intermediate filaments Adherens Junctions Connect Bundles of Actin Filaments from Cell to Cell Desrnosomes Connect Intermediate Filaments from Cell to Cell Anchoring Junctions Formed by lntegrins Bind Cells to the Extracellular Matrix Focal Adhesions and Hemidesmosomes Gap Junctions Allow Small Molecules to Pass Directly from Cell to Cell A Gap Junction Connexon Is Made Up of Six Transmembrane Connexin Subunits 39 Connexins form a 6 member connexon channel between two adjacent cells Fig 9 15 39 Gap junctions are used for communication Via small molecule second messengers Gap Junctions Have Diverse Functions The Permeability of Gap Junctions Is Regulated In Plants Plasmodesmata Perform Many of the Same Functions as Gap Junctions S ummary 39 See Fig 1919 39 Multicellular animals use a variety of junctions as permeability barriers structural support cellcell communication CELL CELL ADHESION MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 47 Cadherins Mediate Cazidependent Cell Cell Adhesion 39 Any cell adhesion molecule is generically referred to a CAM 39 There are different kinds of cadherins 39 Virtually all cells in a multicellular organism express some sort of cadherin 39 Most cadherins are single pass glycproteins 39 Extracellular domains are often found in tandem repeats Fig 19 24 39 These cadherin repeats are structurally related to antibodies immunoglobulins 39 They bind calcium Cadherins Mediate Cell Cell Adhesion by a Homophilic Mechanism 39 Three different possibilities shown in Fig 19 26 39 Cells expressing one type of cadherin tend to seek out similar cells Fig 19 27 Cadherins Are Linked to the Actin Cytoskeleton by Catenins Fig 1929 Selectins Mediate Transient Cell Cell Adhesions in the Bloodstream 39 Blood cells need to move about the body 39 Selectins are cell surface proteins on blood cells that bind to carbohydrates lectins Fig 19 30 39 White blood cells also express their own lectins 39 When a cell is damaged it flags down a white blood cells by expressing lectins on its surface 0 This interaction is weak allowing the white blood cell to move around 0 But this induces the expression of integrins which allow stronger binding and penetration of the white blood cell into the tissue Members of the lmmunoglobulin Superfamily of Proteins Mediate Ca2 MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 48 independent Cell Cell Adhesion The most notable one is N CAM o Homophilic interactions 39 Another one is I CAM o Heterophilicinteractions 39 Modification of N CAM with sialic acid prevent cell adhesion 39 N CAM directed cell cell interaction are not as strong as that generated by cadherins 39 N CAMs might be important for nerve cell cell interactions Multiple Types of Cell Surface Molecules Act in Parallel to Mediate Selective Cell Cell Adhesion Nonjunctional Contacts May Initiate Cell Cell Adhesions That Junctional Contacts Then Orient and Stabilize 39 This allows cells to move past one another 39 If permanent residence is taken up then they need to set up cell junctions Summary see Fig 1932 39 Calcium dependent cell cell adhesion is mediate by cadherins via homophilic interactions 39 N CAMs play a role in neural development MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 49 THE EXTRACELLULAR MATRIX OF ANIMALS 39 A vast network of proteins and polysaccharides upon which cells move and attach 39 It is made by the cells that occupy and traverse the matrix Fig 19 35 39 The matrix can be diverse in function 0 Calcification of the matrix gives rise to bones and teeth 0 It can become transparent to give rise to the eye comea o Ropelike organization gives rise to tendons The Extracellular Matrix Is Made and Oriented by the Cells Within It 39 Fibroblast cells secrete the matrix over much of the body 39 Chondroblasts make cartilage 39 Osteoblasts make bone 39 The extracellular matrix is made up of proteoglycans and fibrous proteins 39 Glycosaminoglycans GAGs are the polysaccharides on the proteoglycans 39 The GAGs create a gel like matrix which provides a spongy protection and a milieu for cells to move around in o The matrix in the knee joint provides a cushion during running 39 Fibrous proteins include o collagen provides mechanical strength 0 elastin provides stretchabilityelasticity o fibronectin o laminin Glycosaminoglycan GAG Chains Occupy Large Amounts of Space and Form Hydrated Gels Fig 19 37 Fig 1938 Hyaluronan ls Thought to Facilitate Cell Migration During Tissue Morphogenesis and Repair 39 Hyaluronan is not attached to protein but is a highly hydrated polysaccharide Proteoglycans Are Composed of GAG Chains Covalently Linked to a Core Protein Proteoglycans Can Regulate the Activities of Secreted Proteins 39 Some signaling proteins bind to the extracellular matrix MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 50 GAG Chains May Be Highly Organized in the Extracellular Matrix Fig 19 41 Cell Surface Proteoglycans Act as Co Receptors 39 Some cell surface receptors require co association with proteoglycans embedded in the cell membrane Collagens Are the Major Proteins of the Extracellular Matrix 39 Collagen forms an extend triple alpha helix Fig 19 43 39 Collagen 9 collagen fibrils 9 collagen fibers Fig 19 44 39 Crosslinking of collagen helps provide mechanical strength Elastin Gives Tissues Their Elasticity Fig 1952 39 Skin blood vessels lungs bladder lining and the birth canal are examples of tissue that need to expand Yet they need to be strong 39 Elastin is a protein that provides elasticity much more than rubber 39 When mixed with collagen you get both strength and elasticity 39 Crosslinking and the folding properties of elastin provide the molecular basis for their stretchability MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 51 The Controlled Degradation of Matrix Components Helps Cells Migrate 39 Cells need to move through the matrix and not get caught up in it 39 They do this by secreting proteases that locally degrade the matrix 39 Collagenase is a protease that degrades collagen 39 When cancer cells metastasize migrate to different parts of the body they need to secrete proteases 39 Some cells secrete protease inhibitors which block cell movement in that area 0 A class of inhibitors called serpins inhibit serine proteases 39 The extracellular matrix is composed of polysaccharides and glycoproteins 39 The polysaccharides are highly hydrated providing a gel for movement of cells and a cushion against physical pressure 39 Some matrix proteins provide mechanical strength like collagen 39 Others like elastin provide elasticity 39 Cells move through the matrix by secreting proteases MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 52 INTEGRINS Integrins are cell surface receptors of the matrix 39 Integrins connect the matrix to the cytoskeleton 39 They trigger intracellular signaling events in response to the type of matrix that they are touching 39 Unlike typical signaling receptors integrins are much more abundant and have very low affinity for the matrix Why 0 A large number of low affinity contacts allow the cell to move rapidly over the matrix 0 Otherwise its like trying to run in thick mud up to your ankles lntegrins Are Transmembrane Heterodimers Fig 1964 39 They depend on divalent cations for matrix binding 0 The divalent cation helps integrins fold properly lntegrins Must Interact with the Cytoskeleton to Bind Cells to the Extracellular Matrix 39 Most interact with actin filaments via oc actinin filamin and other adaptor proteins 39 Clustering of integrins as a result of matrix binding results in re organization of actin filament and thus altered cell movement and shape 39 Clustering of the actin filament also re enforces the clustering of integrins allowing them to bind the matrix more efficiently Cells Can Regulate the Activity of Their Integrins 39 In some cells like white blood cells the integrins are on the cell surface but many are not in a conformation that binds to the matrix 0 This allows cells to move around unimpeded until a signal triggers them to stick around 39 Signals emanating from damaged tissue bind to cell surface receptors and elicit an intracellular signal transduction cascade that results in activation of the integrins O The white blood cell then sticks around the damaged tissue longer 39 This also happens with blood platelets allowing them to form a blood clot at the wound site MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 53 lntegrins Activate Intracellular Signaling Pathways 39 Integrins bound to the matrix may induce localized cytoplasmic changes 39 This is particularly true in axon guidance in developing nerves where local interactions of transmembrane adhesion proteins affect the placement of actin fibers and the plasma membrane S ammary 39 Integrins are the principle transmembrane receptor used to bind the matrix 39 They link the matrix to the cytoskeleton 39 They can direct an intracellular signal transduction cascade that directs cell movement localized growth and survival MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 54 CHAPTER 17 THE CELL CYCLE AND PROGRAMMED CELL DEATH 39 The cell cycle is the process by which a cell duplicates all of its components resulting in two cells 0 In unicellular organisms like bacteria yeast protozoans each round of the cell cycle generates a new organism o In multicellular animals each cell division just adds more cells to the organism 39 An ordered series of events constitute the cell cycle 39 A control system monitors these events making sure that all the event occur in the proper order 39 Each cell also monitors things happening outside of the cell so it know when to proliferate and when to remain quiescent MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 55 AN OVERVIEW OF THE CELL CYCLE The Cell There are four phase of the cell cycle Fig 17 3 1 G1 a G stands for gap b Cells have a standard set of chromosomes i Diploid 7 2n Haploid 7 1n c Cells accumulate mass grow in G1 This is the phase cells normally step off the cell cycle when they are not proliferating d The cells step out of the cell cycle and into G0 resting phase but they are hardly resting e Extracellular growth factors are required for cells to move through G1 2 S a S stands for synthesis b DNA replication occurs here c Chromosomes are duplicated d So a normally 2n diploid becomes transiently 4n 3 G2 a G2 stands for 2 1d gap phase b Chromosomes have duplicated and now the cell is preparing to divide 4 M a M stands for mitosis b The nuclear envelope breaks down c Chromosomes condense d Chromosomes align e Chromosomes separate to opposite sides of the cell f Cytokinesis takes place physical division of the cell into two After mitosis we re back to G1 Interphase represents those phases in which the chromosomes are not condensed 0 That includes everything but mitosis As cells move through G1 they get to a point of no return in late G1 call start in yeast or the restriction point mammalian cells Cycle Control System Is Similar in All Eukaryotes The molecules that control the cell cycle are essentially the same in all eukaryotes So you can study cell cycle control in yeast to learn how it is done in humans Cell cycle control is so important because loss of control leads to cancer MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 56 The Cell Cycle Control System Can Be Dissected Genetically in Yeasts I Fungi represent a diverse phylogenetic kingdom I Two fungi in particular have been studied intensively as an easy way to study the cell cycle 0 Schizosaccharomycespombe I Fission yeast I Used to brew African beer 0 Saccharomyces cerevisiae I Budding yeast I Baker s and brewers yeast I Cerveza means beer in Spanish 0 These two yeast species are about as far apart from each other evolutionarily as they are from humans I Advantages of using yeast as an experimental organism 0 They grow fast rapid cell cycle I Yeast A 90 min I Bacteria 7 20 min I Human cells 7 24 hours or longer 0 Genome size is 01 the size of mammals 39 Very little junk DNA 39 Few introns and little alternative splicing 39 Less genes 39 Short intergenic regions control regions 0 Many yeast species have had their genome sequenced and thus are amenable to comparative genomics 0 Powerful genetic system 39 Easy to delete genes 39 Easy to mutate genes 39 Easy to set up screens 0 Cell proliferation occurs in the haploid 1n state 39 Problem with diploids is that you have to knock out both genes to see an effect 39 With yeast knock out a single gene I Isolation of yeast mutants that have defects in the cell cycle 0 Why If a mutant fails to move through the cell cycle than the wild type counterpart must be required to move through the cell cycle 0 If a mutant fails to move through the cell cycle how can you propagate it 0 Need to isolate conditional mutants that fail to move through the cell cycle only at a certain temperature 39 For example at 30 C they grow and divide normally but at 37 C they stop in one phase of the cell cycle Fig 17 5 0 How do you isolate such mutants 39 Mutagenize cells with ultraviolet light or a chemical mutagen 39 Plate out cells on agar media so that isolated colonies arise at 30 C MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 57 39 Transfer a sample of each colony to two new plates 39 One is incubated 30 C control 39 The other at 37 C Those that grow at 30 C but not at 37 C are temperatureisensitive mutants ts I For all ts mutants look under the microscope for cells that look like they are stuck in one phase of the cell cycle 39 Grow cells at 30 C 39 Shift to 37 C for a few hours 39 Look under the microscope for entire populations of cells stuck in one phase of the cell cycle 39 Often various phases of the cell cycle can be visually identified as phase specific phenoytpes 17 6 39 Normally a population of wild type cells will have cells in all phases of the cell cycle 0 Such mutants have been called cdc mutants cell division cycle and their genes cdc genes 39 Normal wild type cells are asynchronous in their cell cycle 0 Wild type refers to the non mutant form 0 Asynchronous means that in a population different cells will be in different phases of the cell cycle The Cell Cycle Control System Can Be Analyzed Biochemically in Animal Embryos 39 Yeast genetics provides a powerful means of identifying genes involved in the cell cycle 0 But what to those genes do 0 Need to turn to biochemistry o Now a days yeast biochemistry is quite easy but back in my day they were considered to be a sack of proteases and thus were unsuitable for protein isolation 39 Animal eggs are ideal for biochemical cell cycle studies 0 They are huge in comparison to normal cells 0 About 100000 times bigger and thus with 100000 times more cell cycle proteins 0 Eggs are just poised to go into a flurry of cell division 39 G1 and G2 phases are largely eliminated 39 Thus at the early stages of cell division there is no growth 39 Just 11 rounds of S amp M phases yielding 212 4096 cells But no growth 0 Xenopus frog eggs have been the favorite eggs to use Fig 17 7 MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 58 Cell Cycle Control in Multicellular Organisms Can Be Studied in Cultured Mammalian Cells 39 Its easiest to watch individual cells go through the cell cycle rather than watching cells that are part of a multi cellular organism Cell Cycle Progression Can Be Studied in Various Ways 39 The most popular method uses uorescence activated cell sorting FACS using a ow cytometer O O O S ummary A dye is added to a population of cells that stains the DNA The dye binds DNA and uoresces Cells with twice the normal DNA content will uoresce twice as bright FACS rapidly looks at thousands of cells and reports the amount of uorescence in each A histogram report gives you sense of what portion of the cell population are in different phases of the cell cycle Fig 17 12 39 There are four phases of the cell cycle know them understand them 39 Yeast genetics have been used to identify cell cycle regulatory genes 39 Egg cells are used to isolate and study cell cycle regulatory molecules MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 59 COMPONENTS OF THE CELLCYCLE CONTROL SYSTEM The Cell Cycle Control System Triggers the Major Processes of the Cell Cycle 39 Essentials of cell cycle control A timing device Maintaining an order of events Once and only once per cell cycle Finish what you start Back up systems Work under a variety of conditions 099 NB The Control System Can Arrest the Cell Cycle at Specific Checkpoints 39 The cell cycle cannot proceed through checkpoints in the cell cycle unless the checklist of events since the previous checkpoint has been completed The cell cycle will not proceed through the G1 checkpoint unless appropriate signals are received from other cells At the G2 checkpoint cells cannot enter mitosis unless the cell affirms that all the DNA is replicated 39 There are also other kinds of checkpoints that only come up under certain situations DNA damage checkpoint occurs if the DNA has been damaged The cell cycle is aborted and put on hold until the DNA is repaired and the checklist of repairs is complete Checkpoints Generally Operate Through Negative Intracellular Signals 39 Signals emanate from incomplete jobs 39 Only when the job is complete does the negative signal cease MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 60 The Cell Cycle Control System Is Based on Cyclically Activated Protein Kinases I These protein kinases are call Cdks cyclindependent kinases I Their activity rises and falls with the cell cycle I Cdks are regulated by cyclins Fig 17 16 I Cyclins bind to Cdk and activate their protein kinase activity I Cyclins undergo cycles of synthesis and degradation during the cell cycle I Cdks do not I There are four classes of cyclins o GlS cyclins I Bind Cdks at the end of G1 I Commits the cell to DNA replication 0 S cyclins I Bind Cdks during Siphase I Required for the initiation of DNA replication 0 M cyclins I Promote mitosis o Gl cyclins I Promote passage through Start I Names of cyclins o Vertebrates cyclin A cyclin B etc 0 Yeast Cln3 Cle etc I What do cyclins do 0 Activate Cdks by altering the conformation of Cdks 0 Direct Cdks to their target proteins I Cdks also require activation by phosphorylation o Cdkactivating kinase CAK does this but only after cyclins bind Fig 17 17 Cdk Activity Can Be Controlled Both by Inhibitory Phosphorylation and by Inhibitory Proteins I The inhibitory phosphate is put on by the Weel kinase I It is removed by the Cdc25 phosphatase Fig 17 18 I Regulation of Cdks is a lot like an integration device on a computer Fig 3 66 0 Read p 179 of the Alberts teXt MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 61 The Cell Cycle Control System Depends on Cyclical Proteolysis 39 Cyclins are degraded by a ubiquitin system 39 The proteosome recognizes ubiquitinated cyclins and degrades them 39 During mitosis this is carried out by the anaphase promoting complex APC Fig 17 20b Cell Cycle Control Also Depends on Transcriptional Regulation S ummury 39 Phosphorylation no surprise via Cdks controls the cell cycle 39 Cyclins phosphorylation andphosphatases control the Cdks 39 Cyclin levels are controlled by proteolysis MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 62 INTRACELLULAR CONTROL OF CELLCYCLE EVENTS S Phase Cyclin Cdk Complexes S Cdks Initiate DNA Replication Once Per Cycle I Passage through M phase is required for a cell to regain the ability to go through S phase I Steps in DNA replication Fig 17 22 0 The ORC origin recognition complex binds to replication origins on the DNA I They remain bound throughout the cell cycle I Unlike in bacteria 0 Cch binds ORC early in G1 I Cdc6 prevent premature firing of the origin 0 Mcm proteins then bind the DNA to form a pre replicative complex pre RC I This Mcm complexes are helicases that unwind the DNA in preparation for DNA replication 0 S Cdks S phase cyclins Cdk triggers DNA replication I Cdc6 is phosphorylated dissociates from the pre RC and is degraded I ORC is phosphorylated I S Cdk activity remains high after DNA replication to prevent re assembly of the pre RC 0 After mitosis Cdk activity is reduced to zero allowing replication to begin anew MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 63 The Activation of M Phase Cyclin Cdk Complexes M Cdks Triggers Entry into Mitosis I At the end of S phase the DNA is duplicated 0 So a diploid 2n now has 4n amount of DNA 0 The duplicated chromosomes are still attached to each other at their centromere not to be confused with centrosome or centriole cytoskeleton stuff I We re now in G2 I During G2 the M Cdk complex accumulates but it is kept inactive Via the Weel kinase that puts an inhibitory phosphate on Cdk I DNA replication signals are sent to the CchS phosphatase keeping it inactive until replication is complete I Entry into mitosis is triggered by a large cooperative transition involving the following positive feedback loop Fig 17 23 0 Inhibition of the Weel kinase 0 Activationphosphorylation of the Cdc25 phosphatase Entry into Mitosis Is Blocked by Incomplete DNA Replication The DNA Replication Checkpoint I DNA replication checkpoint ensures no entry into mitosis until every nucleotide is replicated I Some unknown molecular sensor that emanates from unreplicated DNA shuts down M Cdk Via inactivation of cdk25 until replication is complete I Any guesses as to what this could be M Cdk Prepares the Duplicated Chromosomes for Separation I M Cdk Via phosphorylation does the following o Induce assembly of the mitotic spindle I What is the mitotic spindle made of o Chromosome condensation I Phosphorylation of the condensin complex 0 Nuclear envelope breakdown I Via phosphorylation and dissociation of nuclear larnins intermediate filaments o Actin cytoskeleton rearrangement o Reorganization of the ER and golgi MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh Sister Chromatid Separation ls Triggered by Proteolysis During DNA replication in what phase a protein called cohesin is laid down along the sister chromosomes or chromatids as their called before separation o Cohesin is related to but is different from condensin I One holds sister chromatids together I The other packages chromosomes in preparation for mitosis Cohesin keeps the sister chromatid aligned When the replicated chromosome is attached to the mitotic spindle the sister chromatids are pulled in opposite direction But cohesin prevents this APC anaphase promoting complex comes along and promotes cohesin destruction Fig 17 26 0 Cdc20 activates APC 0 APC ubiquitinates a protein called securin o Securin inhibits a protease called separase o Separase proteolyzes cohesin Sister chromatids are then free to be pulled apart by the mitotic spindle M Cdk 9 9 Cdc20 9 APC securin separase cohesin sister chromatid separation Unattached Chromosomes Block Sister Chromatid Separation The Spindle Attachment Checkpoint The sensor monitors whether the kinetichore is attached to spindle The kinetichore is a specialized region of the chromosome attaching the centromere to the microtubule spindle A negative signal is sent out unless the kinetichore is properly attached Exit from Mitosis Requires the Inactivation of M Cdk Cdc20APC complex 9 proteolysis of M Cdk 9 loss of Cdc20APC Negative feedback loop in late mitosis MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh The G1 65 Phase Is a State of Stable Cdk Inactivity MCdk 9 Hctl AFC Cdk inhibitor are also active M cyclin genes are turned down Accumulation of G1 cyclins sets Cdk on down another cell cycle path The Rb Protein Acts as a Brake in Mammalian G1 Cells G1 is where cells generally hang out when not dividing Therefore in cancer cells something is screwed up in G1 Transcriptional activator E2F turns on genes involved in S phase including S Cdk Fig 17 30 The Retinoblastoma protein Rb binds to and inhibits EZF thus preventing exit from G1 Gl Cdk phosphorylates Rb causing it to dissociate from EZF leading to progression into S Loss of Rb causes cancer in the eye Cell Cycle Progression ls Somehow Coordinated With Cell Growth MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 66 Cell Cycle Progression is Blocked by DNA Damage and p53 DNA Damage Checkpoints 39 Prior to S phase if there is DNA damage you don t want to start replicating your chromosomes 0 Replication machinery might stall at damaged DNA 0 Want to wait till damage is repaired 39 DNA damage causes activation of p53 a transcriptional activator of DNA damage response genes Fig 17 33 39 One of the genes expressed by p53 is p21 39 p21 binds GlS Cdk and S Cdk and inhibits them 39 p53 is normally down regulated by proteolysis by Mdm2 ubiquitin ligase 39 In almost half of all cancers p53 is mutated 0 Without p53 cells accumulate DNA damage and continue on through DNA replication causing the accumulation of stably inherited mutations 0 Cells that accumulate mutations also accumulate mutations in checkpoint control genes leading to their inactivation and ultimately to cancer DNA damage 9 protein kinases 9 phospho p53 9 p21 GlS Cdk and S Cdk Normal state of the cell 9 Mdm2 p53 S ummury 39 Know Table 17 2 39 Review Fig 17 34 39 Cyclins Cdks control events throughout the cell cycle MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 67 PROGRAMMED CELL DEATH APOPTOSIS Cells die in your body all the time 39 Billions every hour 39 What s more is that these cells commit suicide 39 They initiate a genetic program to destroy their cellular constituents 39 Why 0 Cells that are no longer needed for development are destroyed Fig 1736 0 To maintain a constant size cell proliferation must be counter balanced with cell death apoptosis Apoptosis Is Mediated by an Intracellular Proteolytic Cascade 39 Apoptosis is not the same thing as cell necrosis due to trauma Fig 17 37 0 Busted open cells cause an in ammation response 39 In apoptosis cells neatly package things up 0 Chromosomes are cut up 0 Nuclear envelope breaks down via destruction of nuclear lamins o A white ag signal is placed on the cell surface so that the undertaker phagocytic cells can take them away 39 Caspases are the proteases responsible for carrying out the apoptotic pathway 0 They act in a cascade of protease activation which serves to amplify and re enforce their fateful decision Procaspases Are Activated by Binding to Adaptor Proteins 39 Cells have death receptors on their cell surface Fig 17 39 0 Fas is a death receptor 0 Binding of the Fas ligand presented by lymphocytes leads to death 0 Other cells might present both the ligand and receptor leading to suicide 39 Activation of the death receptors leads to caspase activation 39 There are also other pathways that lead to caspase activation Bcl Z Family Proteins and IAP Proteins Are the Main Intracellular Regulators of the Cell Death Program MICRBBMB 252 lecture 1 22 notes prepared by B F Pugh 68 S ummury 39 Programmed cell death is a normal developmental fate of a cell 39 Apoptosis is mediated in part by cell surface death receptors or by intracellular signaling events 39 Caspase proteases are responsible for executing cell death ENTRXCEILI FLA R CONTROL OF CEL 1 DHquot ISIUN CELL EROW IT HAND APOPTOSIS Mitogens Stimulate Cell Division Cells Can Delay Division by Entering a Specialized Nondividing State l v togens Stimulate Gl Cdk and GlS Cdk Activities Abnormal Proliferation Si Cause CellCycle Arrest or Cell Death Human Cells Have a Built in Limitation on the Number of Times They Can Divide Extracellular Growth Factors Stimulate Cell Growth Extracellular Survival Factors Suppress Apoptosis Neighboring Cells Compete for Extracellular Signal Proteins Many Types of Normal Animal Cells Need Anchorage to Grow and Proliferate Some Extracellular Signal Proteins Inhibit Cell Growth Cell Division and Survival Intr icately Regulated Patterns ol Cell Division Generate and Maintain Bod y Form S ummary
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