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MCB II Exam 2 Notes

by: Raghav Mishra

MCB II Exam 2 Notes Biol-UA 22

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Raghav Mishra
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Notes on MCB Midterm II exam
Molecular Cell Biology II
Claude Desplan
Study Guide
Biology, Molec Cell




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This 46 page Study Guide was uploaded by Raghav Mishra on Friday January 8, 2016. The Study Guide belongs to Biol-UA 22 at New York University taught by Claude Desplan in Spring 2016. Since its upload, it has received 132 views. For similar materials see Molecular Cell Biology II in Biology at New York University.


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Date Created: 01/08/16
Lecture 23: Second messengers, nuclear receptors 05/09/2014 ▯ Endocrine vs. Developmental Signaling  hormones allow communication between different parts of our body in order to maintain homeostasis  Hormonal Signaling: Homeostasis  hormones involved in calcium regulation: o calcitonin- produced by cells which are inside the thyroid o parathyroid hormone  regulation of glucose: o insulin o glucagon  two loops: one goes down and one goes up????  Developmental Signaling – pattern formation is achieved by inductive signals that change the fate of cells or tissues o while you’re developing, cells become specialized, they must talk to one another in order to do this so that they know what they’re job is going to be o a lot of interaction between cells in the formation of tissue and development of an organism ▯ Frog embryo  Famous experiment by Spemann:  took a bit of the tissue of the dorsal lip of one embryo and put it into another embryo on the ventral side  to be able to tell which tissue came from which embryo, used pigmented and non-pigmented embryo  what he got was two embryos that was both pigmented, which meant grafting just a little bit of tissue from the dorsal lip duplicates the axis, makes two embryo instead of one o i.e. a little bit of tissue was enough to be able to form a second embryo  dorsal embryo and ventral embryo o ventral embryo is actually made not from the tissue that was grafted but from the surrounding part of the embryo, i.e. the tissue was recruited to make a new embryo  donor tissue was only a tiny part of the notochord  first notion of induction, b/c the grafted tissue was able to induce the tissue around it to become a new embryo  Key Question: What is the nature of this induction? ▯ The Spemann Organizer  Spemann & Mangold: Dorsal lip functions as an organizer of the embryo.  The organizer induces the formation of a second embryo  What are the signals? ▯ ▯ Extracellular signal molecular binds to receptor protein on the surface of the cell, which will change what the cell is doing/change the state of the cell. It can change the state of the cell in different ways  1.) altered metabolism: o amplification of signal – signal transduction  2.) altered gene expression:  extracellular signal molecule tells cell what its specialty is going to be, changing the fate of the cell o best way to change the fate of the cell is by changing the gene expression of the cell  best way to change gene expression is to induce transcription factors that inhibit and enhance certain genes o ex: you want a nerve cell, induce TFs that activate nerve genes and inhibit blood genes  3.) altered cell shape of movement o change cell shape by acting on the cytoskeleton o ex protein you would affect to change migration of the cell: Rho  Rho is a very typical protein that is altered to change movement of the cell ▯ Distinct responses to developmental signals ▯ Once extracellular signal binds to a receptor, what are the consequences of whatever happens inside of the cell?  1.) Differentiate o ex: differentiate as a nerve cell but not as a blood cell o regulating gene expression  2.) Divide o cell proliferation o induce cells to enter into cell cycle so they can divide and proliferate  3.) Survive o keep cells alive  4.) Die o programmed cell death – apoptosis o macrophages will phagocyte these dead cells by recognizing a signal on the cell surface that signals the phagocytes to come eat it (phosphatidyl serine) ▯ How do cells respond to signals during development? ▯ ▯ Mechanism of Signal Transduction ▯ receptor sees a signal from the outside, and how does it respond to the extracellular signal? ▯ Activation can involve: ▯ - Oligomerization (e.g. receptor tyrosine kinases (RTKs)  receptor can dimerize, and activate some enzyme in the cell that will propagate the signal inside the cell ▯ - Conformational change (e.g. GPCR, G-coupled Receptor)  conformation change of the receptor will activate a molecule inside the cell ▯ - Proteolytic processing (e.g. Notch)  receptor binding to ligand induces a release of the molecule inside of the cell by proteolytic cleavage Oligomerization  receptor tyrosine kinases (RTKs) o ex: insulin receptor, EGFR  inside the cell, you have a tyrosine kinase that can add a phosphate onto a tyrosine  extracellular signal forces dimerization of the receptor, which activates the receptor tyrosine kinase activity o this activation causes a cross-phosphorylation of tyrosine onto those two receptors ▯ Conformational change  ex: GPCR – G-coupled receptor protein  7 transmembrane membrane which spans the membrane like a swinging machine  inside of GPCR is bound to a G protein  ligand binds to receptor, changing its conformation and changing how it interacts with the G protein ▯ Receptor can be: ▯ - Ion channel-linked ▯ - Enzyme-linked, triggering a phosphorylation cascade ▯ - G-Protein-Coupled Receptors (GPCRs) ▯ 7-transmembrane G-coupled receptors (GPCR)  GPCR encoding olfactory receptor, receptors that allow you to smell  b/c every smell you can smell needs specific types of GPCRs, there is so many GPCRs, 1500/24000 proteins are GPCRs, biggest family of proteins!!!!!  7-transmembrane structure with the inside as the C-terminus and the N-terminus on the outside  loops on the inside (C1, C2, C3, and C4) will bind the signal and the inside will interact with the G-protein  G protein itself is first anchored at the membrane  Upon binding of an extracellular signal, conformational change is going to change the way the G protein is interacting with the membrane (i.e. activates the G protein).  Once G protein is activated, it will migrate across the membrane and go to an enzyme and activate an enzyme, and this enzyme will do something to the inside of the cell.  Remember: metabotropic vs ionotropic receptors  Respond to a wide variety of mediators (hormones, neurotransmitters, epinephrine, serotonin) ▯ G-proteins as made of trimeric: ,, and   , and  chains form a tight complex  that anchors G-protein to the plasma membrane   chain can bind and hydrolyze GTP  Trimeric G-proteins disassemble when activated  Inactive G-protein has a bound GDP  When activated: GDP dissociates, new GTP is bound  This causes a to dissociate from    binds to adenylate cyclase, altering its activity ▯ inactive G protein is bound to GDP. When active, trade GDP for GTP, activating them. They hydrolyze GTP and then inactivates again. This makes sense b/c you don’t want to activate a pathway forever. ▯ I.e.  extracellular signal binds to GPCR  this causes recruitment of G protein to GPCR, which allows of exchange of GDP on the inactive G-protein for GTP  activation of GTP allows disassociation of the  subunit, and now you have the activated  subunit and an activated  subunit o activated  subunit has GTP  hydrolyze GTP to inactivate the process ▯ ex of another molecule that goes through this same cycle:  Rho!!!!  Rho is a small G-protein that goes through this same cycle, but it is not a GPCR, it is actually a GEF, so it cannot by itself hydrolyze GTP, needs help, GAP helps Rho hydrolyze GTP ▯ What will the activated G-protein do???  - GPCRs activate trimeric G-protein  - Activated G-protein alters the cellular concentration of a “second messenger” (cyclic AMP or Ca2+) o these molecules diffuse into the cell and activate a bunch of processes  - Gs activation stimulates Adenylate cyclase  - Gi inhibits it  - The second messenger activates a protein kinase  - The protein kinase phosphorylates another enzyme and alters its activity ▯ activated alpha subunit of G-protein will talk to adenylyl cyclase, which is an enzyme that can take ATP and transform it into cAMP ▯ then, hydrolyze ATP to get inactivated, bind back to the beta and gamma subunits and bind back to the membrane ▯ cAMP ▯ adenylyl cyclase takes ATP and cleaves two phosphates and forming two phosphoester bonds, making cAMP  can also do this with GTP, making cGMP ▯ don’t want cAMP to stay forever, want to get rid of cAMP using phosphodiesterase, which transforms cAMP into AMP by breaking the two phosphoester bonds ▯ Activation of PKA by cAMP extracellular signal binds to GPCR, leading to the activation of the G protein  activated GTP-loaded G protein talks to activated adenylyl cyclase, which transforms ATP into cAMP  cAMP can bind to a protein which is an inhibitor of an enzyme called a protein kinase (PKA)  normally, PKA is inhibited by the repressor protein. When this repressor protein binds cAMP, it releases the activated PKA,  what does PKA do? i.e. what is the consequence of having this protein that can now phosphorylate inside the cell? ▯ Control of gene expression by PKA  activated PKA can go inside the nucleus and then talk to the transcription factor CREB  PKA phosphorylates CREB, allowing it to bind DNA and activate gene expression/transcription  PKA is highly specific.. doesn’t phosphorylate anything, they choose which proteins to phosphorylate ▯ Signal amplification  why so many steps? b/c you 1.) produce amplification!  for ex: epinephrine binds to GPCR, activated G- protein binds to adenylyl cyclase o adenylyl cyclase produces a LOT of cAMP o ex: PKA can phosphorylate many transcription enzymes  more better to look at picture….  2.) Also, you can regulate each step: o ex: controlling the production of cAMP  There is also multiple pathways that can feed into these steps ▯ producing cAMP is not the only function of GPCR, there are many many GPCRs that can do a wide variety of things: ▯ GPCR can activate other second messengers  ex: Rather than activating adenylyl cyclase to produce cAMP, it activates an enzyme called PLC  PLC is an enzyme that acts by hydrolyzing phospholipids o upon the function of the PLC, you are going to cut between the lipid and the phospho sugar, producing DAG  DAG can now activate PKC o difference between PKA and PKC?  they both add phosphate onto proteins, but target proteins are different  other than DAG, PKC can also produce IP3, which is a sugar covered w/ 3 phosphate  IP3 is a second messenger which can diffuse into the cell and cause the release of Ca2+ ions from an intracellular store of calcium o IP3 binds to IP3-gated Ca2+ release channels, causing them to open and release calcium from the intracellular storage/ER into the cytoplasm  dramatically increase concentration of Ca2+ inside the cell  Ca2+ is ALSO something that can activate PKC o BOTH DAG and Ca2+ can activate PKC!!!  biochem bullshit….don’t care… ▯ Opsin: a GPCR for Phototransduction  in your photoreceptors, you have cells which have a lot of membranes b/c those cells have a lot of receptors that can detect light  receptor called opsin is a GPCR  if you want to detect light, you are going to need a lot of membranes that contain a lot of opsin ▯ Let’s see what happens if you receive light…. ▯ Opsin  a photon is going to be received by the opsin b/c inside the opsin is something called a chromophore  the chromophore can be either in the cis conformation and the trans conformation, and it can change its conformation depending on the light  With no light, chromophore is in its cis form, light hits it changes it to trans form and it becomes active ▯ Absorption of light by retinal activates opsin and PDE  light changes inactive rhodopsin to active rhodopsin (b/c you have changed the conformation of the chlorophore)  active rhodopsin GPCR trades GDP for GTP, and it’s going to load the  subunit of the G protein with GTP, activating the G protein  activated G-protein binds to PDE (phospho-diesterase)  PDE digests cGMP to make GMP, i.e. destroys cGMP to produce GMP  this lowers the concentration of the cGMP  the more light you have, the lower the level of cGMP o cGMP is able to activate sodium channels o opening sodium channels depolarizes the cell  so, having more light CLOSES sodium channels o in the dark, your photoreceptors are always depolarized o when you see light, sodium channels close and you repolarize the cell  LIGHT causes HYPERpolarization!!!!!!  cave fish – animals that lost their eyes due to spending hundreds of thousands of years in the dark o why is this? o if you spend your life in the dark, your eyes are already depolarized b/c there is no light ever o so the best way to save energy is to just get rid of the eye ▯ De-activation of opsin*  phosphorylation of rhodopsin by a GRK  arrestin now can block the rhodopsin and make sure it does not keep signaling to the G-protein ▯ SUMMARY:  activated GPCR can go 3 ways: it can activate: o adenylyl cyclase ->cAMP->PKA o PLC -> DAG -> PKC  IP3-> Ca2+ -> PKC o PDE -> cGMP -> channel  in all cases, you activate a second messenger which causes a response in the cell  in all cases, you have a phosphorylation enzyme ▯ ▯ Second messengers  biochem bullshit…  ***know structures of shit like glycerol, fatty acids, DAG, ….. fk this ▯ ▯ Surface vs. intracellular signaling  this is going to be DIRECT, rather than having multiple steps  not an example of a receptor binding a ligand at the cell, but a ligand that can get into the cell by itself and do its job directly  this is due to nuclei receptors  ex: steroid hormones ▯ Major pathways that control development  steroid hormones o signal goes directly into the cell and affects transcription in the nucleus  retinoic acid – very hydrophobic molecule with an acid at the end thyroid hormone – made in thyroid, it’s basically a modified tyrosin, at its surface it has an iodine, it’s basically the only molecule in your body that requires iodine  Vit. D – also derived from steroid  all these molecules have the property of being hydrophobic  these hydrophobic molecules can go through membrane with no problem b/c they like lipids (they love to cross membranes) and that they don’t like to be in the blood as they don’t like to be in a hydrophilic area o so, in the blood, these molecules are usually carried by carrier proteins and then released into the cell ▯ Nuclear receptors: e.g. Steroid hormones (as developmental signals)  - Retinoic acid, thyroid hormones & Vitamin D are liposoluble  - They are secreted and transported by carriers proteins  - The hydrophobic signal enters the cell where it binds to a “nuclear receptor”: Transcription factor (e.g. RAR) o this receptor itself is a transcription factor  - The steroid-receptor complex is translocated to the nucleus where it activates transcription of genes containing response element (e.g. RARE) ▯ Nuclear Receptor function  cortisol receptor, estrogen receptor, progesterone receptor, vitamin D rec, thyroid hormone rec, retinoic acid rec, etc.  ALL of these receptors have a DNA-binding domain as well as the domain to bind the steroid hormone  when there is no hormone, everything is folded together and inactive b/c there is a protein that hides the DNA-binding domain by folding over the hormone-binding domain, making the receptor inactive  upon binding of the hormone, you kick out the protein and you unfold the protein, so you expose the DNA-binding domain  molecule can now get into the nucleus and activate transcription ▯ W8, I thought we were talking about developmental signaling?  sex hormones are very important for the functioning of sex organs as well as the making of those sex organs  vitamin D, thyroid hormone, and retinoic acid receptors are also very important for development  ex: thyroid hormone receptor involved in making the cone photoreceptor cells  ex: retinoic acid receptor important for development of body axis….e.g. what is posterior what is anterior etc. ▯ Surface signaling: hydrophilic signaling molecule  Remember: multiple steps b/c  1.) amplification  2.) regulation – the more steps, the more regulation you can have  3.) cross-reactivity – pathways often cross-reacts with different pathways ▯ Intracellular signaling: hydrophobic signaling molecule  steroid hormones carried by carrier proteins ▯ Enzyme-linked receptors  RTK – a receptor binds to a ligand, and the receptor will dimerize  receptor contains enzymatic activity which can phosphorylate tyrosine  the binding and dimerization will lead to activation of the catalytic domain ▯ Cell Surface Receptors ▯ - Signals can be: ▯ ** Direct (JAK/STAT) ▯ ** Relay signal (Cascade of kinases amplifies signal) ▯ - Mechanism of transduction: ▯ ** Conformational change (e.g. GPCR) ▯ ** Oligomerization (e.g. receptor tyrosine kinases (RTKs). ▯ ** Proteolytic processing (e.g. Notch). ▯ - Output of signal can be: ▯ ** Transcriptional (MAPK) ▯ ** Enzymatic ▯ ** Cytoskeleton ▯ Major pathways that control development  these pathways are heavily involved with cancer and disease  ▯ START HERE, SLIDE 6****** ▯ ▯ The Jak/STAT pathway  receptor is bound by ligand, causing dimerization  dimerization causes cross activation of the phospho-tyrosine  this causes activation of the phospho-tyrosine domain, which will phosphorylate the receptor itself  this will recruit the SH2 domain of STAT1  by bringing STAT1 close to the kinase, this is going to lead to phosphorylation of the tyrosine on STAT1  Now you have an SH domain that loves phospho-tyrosine and itself ads its own phospho-tyrosine on its own molecule?????  acidity of the SH2 domain for one phosphotyrosine is lower than the acidity of the SH2 domain for another phosphotyrosine, meaning it loves its own phosphotyrosine better than the other phosphotyrosine o SH2 domain of STAT loves its own phosphotyrosine more than the phosphotyrosine on the receptor b/c its in a better context (the amino acid that flanks the SH2 domain on the receptor is not as good as the amino acid that flanks the SH2 of STAT)  STAT binds to the tail of its neighbor, causing a dimer  *important thing to remember: SH2 domain has different affinity for phosphotyrosine depending on context  OMG REVIEW THIS SHIT******** ▯ Src: the first characterized proto-oncogene  the SH2 domain was found in a protein called Src  Src contains 4 domains that is found in other proteins o SH1 o SH2 o SH3 o SH4  Src homology domain contains a kinase domain, like Jak  we know that Jak is a tyrosine kinase b/c it resembles Src, which is a tyrosine kinase  any protein we know that has an SH2 domain is going to bind to phosphotyrosine  Src was the first tyrosine kinase to be identified  SH3 mediates interaction between other proteins (proline..proline..proline.. pro, prop, pro for ex.)  SH2 of STAT is found in a transcription factor ▯ SH Src-homology regions  SH1 domain: Catalytic kinase domain  SH2: binds Y-PO4 peptides with consensus: (PO4Tyr-Met/Val-X-Met)  SH3: Interacts with Pro-rich targets (mediate protein-protein interactions)  SH4 domain: myristylation (membrane-localization signal, anchors protein to the membrane ▯ The SH2 domain  we know the structure of SH2 domain due to crystallization  why is Src so important?  Src is a chicken virus that causes cancer  first time we could cure cancer!  most other cancers are not caused by viruses, they are caused by mutations in genes  why do u think Src causes cancer? o b/c it contains a tyrosine-kinase, which mimics some regulatory proteins, so it you put too much tyrosine-kinase, you are going to disrupt regulation and cause proliferation of cells, causing cancer  PTB domain – also binds phosphotyrosine  SH3 can bind proline-rich  PH, Pleckstrin homology domain – bind PIP membrane protein to bring proteins to the membrane ▯ The Jak/STAT pathway  Summary: receptor binds ligand, activating tyrosine kinase. Tyrosine kinase phosphorylates the receptor itself, which makes a phosphotyrosine. Jak binds with phosphotyrosine of SH2 domain, but it loves itself so much, that it’s going to form a homodimer by binding to the other phosphotyrosine b/c this phosphotyrosine is in a better context (higher acidity) than other phosphotyrosine, and therefore detaches and instead binds to that one…… fuck u Desplan. Now we have a dimer……  Dimer will go into the nucleus and bind to DNA o STAT is a transcription factor that can activate transcription of target genes once form a dimer! o some of these genes can be against certain viruses, while some genes can be to specify cell fate during development  not as direct as steroids, but pretty direct…  So, in the case of PKA activating a transcription factor or of steroid hormone activating transcription OR of cytokine activating transcription, you are going to change the fate of the cell b/c when you get different signals from the outside you are going to get different activation of different genes, therefore changing the fate of the cell ▯ Receptor Tyrosine Kinase: RTKs  also involves tyrosine kinase, but in the case of the receptor, RTKs, the receptor ITSELF is the tyrosine kinase o while in Jak, the receptor was a receptor that was bound by a tyrosine kinase ▯ Receptor Tyrosine Kinases ▯ • Large family of single-pass transmembrane receptors. ▯ • Often growth factor receptors, e.g. EGF, Insulin. ▯ • Cytoplasmic domain contains protein tyrosine kinase. ▯ • Activation involves trans-phosphorylation of specific tyrosine residues for downstream recruitment and stimulation of kinase activity.  i.e. activation by outside signal is going to lead to activation of the tyrosine kinase inside the cell  EGF controls cell proliferation  insulin is going to bind RTKs  if you don’t have insulin, you get Type I Diabetes o can fix most of the problem with insulin shots  however, some patients don’t have the receptor for insulin, they have a mutation in insulin receptor, and as a consequence, insulin injections won’t work as cells cannot respond – Type II Diabetes  in the case of Type II Diabetes, it is Glucose-transporter 4, GluT-4, cannot go to the membrane and bring in glucose to the cell, resulting in abnormally high levels of glucose in the cell ▯ Most RTKs include a very large variety of proteins. ▯ RTK Family  all receptors have an outside domain which bind to different signals  EGF, insulin, PGF, etc….  different structures of outside domains = different signals they bind to = different specifics in function  inside the cell, they al share the tyrosine-kinase domain, same kind of domain we call SH1 in Src, and the same kind of domain we have in Jak kinase  inside domain will phosphorylate tyrosine on a specific protein  however, not to any phosphotyrosine on any protein, they have specificity  very diverse outside domain, very controlled/reserved inside tyrosine-kinase domain  almost all of these domains are a causing agent for cancer  many cancer drugs seek to control activity of the tyrosine-kinase enzymes  domains on the outside are made of repeats of the same domain over and over again ▯ RTK Activation  when you have binding of outside signal on the outside domain, this causes dimerization  this causes activation of the tyrosine-kinase, which causes addition of a phosphate onto kinase of the tail of the receptor itself  called RTKs b/c they are kinases at the same time they are receptors  interaction of two kinase domains causes activation of each other o how do we tell this?  to test this model, that suggests a trans activation (meaning subunit A and subunit B activate each other) o mutate a single amino acid that prevents the catalytic domain of the tyrosine kinase, making it inactive and it cannot add a phosphate to any other protein o even if you cause dimerization, it never causes activation of RTK o i.e. normal tyrosine kinase domain cannot work if the other tyrosine kinase domain is mutated o this is dominant-negative, meaning one cannot work without the other  this can be useful clinically, b/c an overactive tyrosine kinase domain can cause cancer, so poison one of the tyrosine kinase domains  SH2 domain proteins are what bind to these phosphotyrosines ▯ What is the output of RTK's ?  each phosphotyrosine is going to recruit different proteins that contain the SH2 domain o remember: not all SH2 domains are born equal some like one phosphate while others like another phosphate  this pathway can feed into different pathways depending on which proteins bind to it ▯ Outputs of RTK's  some examples: GEF, Pi3-kinase, GAP, PLC  PLC – a target of GPCR, remember?  GAP and GEF (G-protein exchange factor) –  GEF - these are proteins that can facilitate the exchange of GDP for GTP, meaning that they are going to load G-proteins with GTP and therefore activate G-proteins o So, RTKs may activate Ras GEF, which will activate a G protein called Ras.  may also activate GAP (GTPase activating protein) – inactivates G protein as it hydrolyzes GTP  Pi3k – adds phosphates onto phosphoatidyl phospholipids  these proteins contain the SH2 domain, and these SH2 domains love to bind to the phosphates of the receptor after it has been activated ▯ Signaling Through RTK: The Ras pathway  GEF actually doesn’t really bind directly to the phosphotyrosine  it actually binds to the phosphotyrosine through an adaptor protein o Grb-2 adaptor protein  what GEF does is exchange GDP for GTP, activating G-protein Ras ▯ G-proteins ****THIS WILL BE ON THE EXAM*******  Ras G protein – involved w/ activation of RTKs  GEF activates Ras by exchanging GDP for GTP  GAP hydrolyzes GTP, inactivating Ras  all G proteins go through this cycle of activation and inactivation  Ras is also involved w/ cancer, as it is carried by a virus o it can lead to overactivation of Ras, doing all that shit cancer fucks do by overactivating RTK ▯ Before G protein is inactivated by GAP through hydrolysis of GTP:  active Ras activates the MAP-kinase-kinase-kinase, which is a protein that phosphorylated MAP o it is a serine-kinase like PKA, PKB, and PKC o adds a phosphate on another protein  MAP-kinase-kinase-kinase targets MAP-kinase-kinase, which targets MAP-kinase, and target of MAP-kinase is MAP  MAP is going to activate a bunch proteins which is going to activate transcription/transcription factors  end product is transcription activation, but goes through multiple steps of phosphorylation ▯ Common themes:  cascades – (often cascade of kinase)  at the end phosphorylation of transcription factor o so far the important proteins phosphorylated at the end are TFs, if you want to change shape, these proteins will be cytoskeletal proteins ▯ Outputs of RTK's  some RTKs recruit GEF, some recruit GAP, and some can recruit both  What do you think will be the consequence of mutating the GAP protein? o Ras will remain active all the time, causing cancer  mutation in GEF – lack of activation, no cell proliferation, smaller tissue  PLC can also be recruited to RTKs  Remember PLC in the context of GPCR – o breaks bonds in Pl, converting it into DAG which can talk to PKC o also releases IP3 which releases stores of Ca2+, increasing calcium concentration in cytoplasm and activates PKC  now you see that everything is so well integrated, this is why drug makers have a hard time trying to cure b/c there are SO many pathways that feed into certain mechanisms and shit  remember that Ca2+ levels must be low in cytoplasm, so it is being constantly pumped out of the cell or into the ER storage of calcium ▯ Outputs of RTK's  another pathway that can be recruited: PI3K  PDGF can have thousands phosphates which can recruit PI3-kinase PI3K  Remember:  PI3K takes PIP2 and makes it into PIP3 (adds one more phosphate to PIP2)  this phospholipid plays a very important role inside of the cell  so much biochem bs in this wtf ▯ PI3K now activated by RTK, it is going to activate serine kinase  PI3K activates PKB – a serine kinase  PKB is activated by virtue of the PH domain  PH domain (like SH2 domain) binds to phosphotyrosine  PH domains can bind to PIP3  so basically what these domains do is recruit kinases to the membrane o when you have PIP2, the domain will not be recruited, when you have PIP3, the three phosphates can now recruit the serine kinases to the membrane, and these two proteins will associate w/ each other o what’s going to happen is that PDK1 will activate PKB  a cascade of kinases where one kinase will activate another kinase, and this going to lead to activation of target proteins to change transcription or change the cytoskeleton – GENERAL SUMMARY  what PKB and PDK1 plays a role in apoptosis – programmed cell death  ex. of cell death: forming of hand, kills cells in between fingers, duck do not kill the cells ▯ Signalosome  Preformed signaling complex on scaffold  in every case, it leads to phosphorylation of serine kinase which leads to phosphorylation of transcription factor ▯ ▯ Example of a signaling complex  a whole slew of interactions and pathways that we don’t fully understand, this is why we don’t understand cancer, TOO DAMN COMPLICATED GETTING BOGGED DOWN BY ZE DETAILS sHIT ▯ Wnt signaling  Wnt comes from gene called Wingless – mutation causes a fly with no wings  this gene was cloned  a group of nerds, the same ones who discovered Src, was playing around w/ MMTV virus (which can cause breast cancer/mammary tumor in mice)  trying to clone site of insertion of MMTV virus  found that virus is inserted next to the gene Int-1 (integration 1) o Int-1 site of integration of MMTV  there is int-1, Int-2, Int-3, Int-4, and Int-5  Int-1 in mice was the same Wingless gene in flies  This is why it’s called Wnt  virus causes overactivation of gene, which causes cancer as it tells the cell you should divide you should divide you should divide  What is the relationship between Wingless and Int/MMTV?!?!?!?!?! ▯ Wnt signaling in animal development  gene was found in many other species  How do you make a mutation? o CRISPR  overexpress Wnt in hydra o you get another hydra budding off the other hydra, makes babies all over the place o seems to have some function with buds  Planaria – flatworms, can cut it up and it regenerates o removing the Wnt gene in planaria, you get multiple heads  Flies – wingless is a weak allele of Wnt o when you don’t have wingless, you have a segmentation defect and you fail to separate the different segments of a fly embryo  Frog – o you get duplication of the axis o (remember, review random – Schemann experiment w/ embryos and grafting n shit) ▯ Wnt Ligands and Frizzled Receptors  multiple Wnt genes in each organism  7 Fly Wnt ligands (including Wg)  20 mammalian Wnts  Wnt receptors are called Frizzled receptors (Fz), as the hair on the fly becomes frizzled  4 Fly Fz receptors  10 mammalian Fz  the receptor has several transmembrane domains b/c you want to communicate between the outside and the inside and somehow you must have a domain that binds the Wnt and convey its information inside the cell  the C terminal domain will help convey the information  the CRD domain will help bind the Wnt protein  How do they do this? ▯ Canonical Wnt pathway:  Wg (wingless) binds to a Frizzled (Fz) receptor and recruits a protein called Dishevelled o these names have to do w/ mutations in flies not letting them grow hair very well  Dishevelled will prevent a complex (GSK3/Sgg-Axin-APC) that degrades Arm (ß-catenin), and ß-catenin is a coactivator that goes into the nucleus and activates transcription o SO, disheveled prevents degradation of ß-catenin so ß-catenin can now go into the nucleus and activate transcription.  Remember: -catenin and ß-catenin o adherence junctions, they bind to adherans and help bring cytoskeleton to the membrane  Why same name? B/c it’s the same protein!  Why the same protein at the membrane can be at the nucleus? B/c proteins can be multi-functional!! Woohoo! I don’t care….. ▯ The Wnt pathway  in the membrane you have a transcription factor called TCF, which is attached to a corepressor(coactivator?) and the Wnt-Responsive genes are off  if you have Wnt, you bind to Fz receptor, activate dishevelled, and Dishevelled is going to dislodge the ß-castenin from the GSK complex and this complex will no longer be able to degrade castenin  ß-castenin can go into the nucleus, kick off TCF, the corepressor, and activate Wnt-responsive genes  Questions:  What happens if you have a mutation in APC or GSK or axin? o mimick wing activation in the absence of Wnt o cannot degrade ß-castenin, so it will always be going into the nucleus, and transcription of wnt-responsive genes will always be active!!!! o causes colon cancer!!!!  overactivation of Int causes mammary tumor, overactivation of Wnt causes colon cancer  SO, this is a tumor suppressor gene.. o a gene which prevents tumor from occurring ▯ Remember, losing wingless causes dysfunctional segmentation  find out any other mutation that has the same phenotype  if it has the same phenotype, it is likely in the same pathway  mutation in APC – phenotype you’d expect is the same!  mutation in Fz and dishevelled – same phenotype!  mutation in ß-castenin – signal cannot be conveyed to the nucleus, you also have no adherence junctions  consequence of losing APC – too much signaling, you have opposite phenotype  double mutation in APC and Fz= the same……it doesn’t matter cause signaling pathway is still not working, ß-castenin is wstill always active  double mutation in APC and ß-castenin, APC will lead to no degradation in ß-castenin but mutation in ß-castenin will still result in no transcription activation  SOME OF THIS DOESN’T MAKE FUCKING SENSE REVIEW!!!!!!!  I HATE WHEN HE DOES THAT SHIT WHEN HE’S LIKE THIS AND THAAT I CANT FKN SEE!~!!!!! ▯ Specification of naked cuticle & denticle diversity  in wild type, you have naked cuticle and denticle  in mutant, you fail to make naked cuticle, and you cannot repress the making of the tentacle ▯ b-catenin is stabilized by Wnt signaling  looking at where wg is expressed  engrail (en) is a cell next to the Wg (wingless) cell  Wg will diffuse and by diffusing, it’s going to cause the loss of denticle belt  wg is produced locally, it will diffuse, and prevent formation of denticles  Wg prevents the degradation of ß-castenin ▯ ▯ Canonical vs. non-canonical Wnt pathway: ▯ Canonical Transcription – you should know this shit nigga ▯ Wnt has another type of function: doesn’t have to do w/ ß-castenin or transcription ▯ Non-Canonical PCP - has to do w/ Rho and Rac proteins which are involved in cytoskeleton functions  Wnt goes to Frizzled, which activates Dishevelled, and then goes into the activation of the cytoskeleton proteins Rho and Rac  this leads to the rearrangement of the cytoskeleton ▯ What is the phenotype of losing stuff in the PCP pathway? ▯ ▯ Planar cell polarity (PCP): plane of an epithelium perpendicular to apical-basolateral polarity  the cell is polarized along the apical-basolateral axis, w/ specific proteins on one side and specific additions on one side and tight junctions and adherence junctions  in the case of the epithelium, you have a hair that is formed  notice: your arm hair goes in the same direction o your epithelium is in a polarity in which they all point in the same direction  the hair is on one side of the cell and never on the other side of the cell  PCP – every epithelial cell is polarized into its plane ▯ Non-canonical Wnt pathway: PCP and convergence-extension  in the absence of this pathway, hairs point in random directions o frizzled phenotype  So, Wnt in development is responsible for making the naked cuticle, but after the cuticle is made, it is also responsible for ensuring the PCP and making sure each cell is going in the same direction by knowing where each part of the cell goes in its polar-planar axis ▯ Planar cell polarity (PCP) in epithelial tissues  disrupting any gene involved in the pathway will cause error in PCP ▯ PCP phenotypes in the mouse epidermis  mouse hair also points in the same direction, mutants have loss in hair direction ▯ Planar cell polarity defects in the mouse cochlea  hair cells in cochlea help hear sound  these are also organized in a certain way and point in the same direction  PCP mutant, hair cells point in random orientation ▯ So, not only do you have to arrange cells in basolateral-apical axis, also need to arrange cells in polar-planar axis ▯ Cytoskeletal rearrangements in response to planar polarization ▯ Normal epithelium cell:  accumulate actin on one side of the cell  bundles of actin point in one specific direction ▯ in the case of hair cell:  starts first in the center  but then starts to get polarized in the same direction in an organization that precedes the membrane and perceives sound ▯ Localization of Determinants and PCP in the Wing  accumulation of Fz on the side where Wg signals come from, which makes sense….as Fz wants to go where there is the highest level of Wg  this will lead to the formation of actin filaments which will become the hairs of the cell by accumulating to one side of the cell ▯ The core PCP factors  how do you have a signal come in from one side and activate a receptor on one side of the protein?  you have to realize the problem: o The problem is that you have a bunch of cells and you have a signal coming in… and you want all those 1000s of cell to point in one direction . . . if you think about it, that means they can detect little changes in Wg concentration on one side of the cell compared to the other side of the cell, which is hard to imagine o LOL: the lower is higher than the high here o i.e. the high Wg side of one cell is higher than the high Wg side of the previous cell and so forth o hard to imagine………..very hard for all these cells to have a system in which they detect which side has the most Wg  How does the cell do this???????????  The best way to make this work is dominos!  Wg takes one domino, forces it down, and then they are all going to propogate  each cell reads what it has but also compares w/ its neighbor  so you have a transmembrane protein, frizzled, and Dsh, and Stbm is going to receive the main signal ▯ Logic of interaction among PCP factors  Fz likes Dsh and Dgo.  On the other side of the membrane, it likes Stbm.  This complex (Fz-Dsh-Dgo-Stbm) is going to prevent the formation of Stbm complex on the other side  meaning if you have Fz on one side of the cell, you push the formation of Stbm to the other side of the cell o and vice versa if you put Stbm on one side of the cell, you push Fz to the other side  each cell is going to do this….  although Fz hates Stbm if its on the same side, it loves it if its across the membrane of the neighbor cell o Fz loves to talk to Stbm on the other cell  This means cells will all assemble in the same orientation, this will propagate  self-organized system  this is why when you don’t have Wnt signaling, you still have organization  Wnt comes from direction, and it attaches to Fz, so it tends to attract Fz slightly more on one side than the other  all cells point to Wnt signal  system must not make mistakes! o if system for some reason wanted to invert a cell, it cannot b/c of the interaction between Fz and Stbm will correct it right away o all this came from looking at hair pattern of fly wings  Step 1: Intracellular antagonistic interactions between the Fz/Dsh and Stbm/Pk complexes  Step 2: Positive feedback loop stabilizing the opposite complex across cell membranes  ▯ Non-canonical Wnt pathway is involved in PCP and convergent-extension  reason for short-tailed mutant tadpole in Wnt pathway mutant b/c the path of elongation is disrupted. Each cell is pointing a different direction b/c you have no PCP  Remember: two completely different functions for the same protein! Dishevelled and Fz ▯ ▯ Notch  we have epithelium cells, and sometimes these cells are in the region where we develop nervous system – neural epithelium, the nervous system derives from neural epithelium  some cells will become neural precursor cells  when a cell decides to become a nerve cell it will tell it’s neighbor to stay an epithelial cell, i.e. it will inhibit its neighbor from becoming a nerve cell o lateral inhibition  inhibition signal (which is used in multiple stages of development) is called the Notch signaling pathway  Notch – the receptor on a cell that says don’t become a neuron, remain epithelial cell  membrane-bound inhibitory signal protein – Delta ▯ Proteolytic cleavage  Notch is a large receptor that spans the membrane only once  has a huge domain outside, which is the domain that binds the ligand  What is the ligand, and what happens when the ligand binds to the Notch receptor?  no enzymatic activity here….no kinase activity…  There is an enzyme the cuts the Notch receptor, but the Notch remains together  And then when it binds to the ligand, it’s going to change of conformation, which leads to another cleavage right inside of the membrane, right where the intracellular domain is attached to the membrane  This causes the release of the intracellular peptide – the intracellular Notch fragment  Single used receptor – you protealytically cut your Notch receptor into the outside fragment which will be lost and the intracellular fragment  The intracellular fragment is like ß-catenin, it’s a piece of DNA which is a coactivator  So it will go into the nucleus and help transcription factor to help activate transcription and gene expression  The enzyme number 3 that makes the important cut at the transmembrane domain is called *praseninin?*- the same enzyme that cuts the precursor protein for Alzheimer’s and makes it a configuration that causes Alzheimer plaques ▯ Why do we need this process?  in the neural-epithelium, you have cells which are going to cover the body  from them you are going to delaminate neural precursor cells and they are going to bud off  these cells will become neuroblasts, and again you don’t want too many of these ▯ Stochastic choice: Lateral inhibition  epithelial cell becomes neural stem cell by chance, simply b/c it expresses a little bit more of neural genes  the cell that becomes a neural stem cell tells its neighbors not to become neural stem cells through the Notch pathway  Experiment: o Cell ablation: as soon as you see a cell becoming a neural cell, you kill it o now there is no lateral inhibition, what’s going to happen? o another cell will start becoming a neural cell and start sending the Delta signal to its neighbors o This is called Stochastic choice: choice is random and whatever cell you choose inhibits its neighbors ▯ Proneural genes ▯ Notch and the Neurogenic genes  one cell will have more Notch signaling than the other cell, just by chance, have more Notch receptors  the more Notch you produce, you repress Delta production  so what happens is when you repress Delta you are even more responsive to Delta than you were before  on the other side, you have little Notch signaling and thus more Delta production ▯ ▯ Mutations in Neurogenic genes  `if you don’t have Notch –  if you don’t have Delta – everybody becomes neurons! called the big brain phenotype  if you express only a cleaved form of Notch (just produce intracellular domain of Notch in all the cells) o what you get is no neuroblasts, only epidermal cells! o no neurons can form  reverse genetics – go from gene to the phenotype  knocking out hedgehog gene causes Cyclops phenotype, the two eyes merge together  hedgehog involved in forming the midline between the eyes  Cyclopamin: inhibitor of the hedgehog pathway, (Shh inhibitor)  when sheeps eat cyclopamin, they produce Cyclops babies  cyclopamin used in clinic to take care of overactivation of hedgehog pathway in cancer patients  overactivation of hedgehog pathways usually result in brain tumors  Organizers in the limb bud  in mutant cat, Hemingway cats, you have too many digits o Polydactyl  Wings and legs begin as limb buds  if you take the chick limb, the bud that is going to form the limb, it has a region called the ZPA  if you take the ZPA and graft it to the anterior side (take it from posterior of one chick and put it in the anterior of another chick), you get chicks with two ZPAs, one posterior and one anterior  this is a bit like the dorsal lip duplication in frog embryos, you get duplication of the pattern  ZPA produces an inductive signal that conveys posterior positional information  so you get duplication of 1,2,3 into 1’,2’,3’  ZPA produces sonic hedgehog!  it forms a gradient of sonic hedge hog: high in 3, low in 2, even lower in 1  so, have to put sonic hedge hog producing cells in the dorsal part of the chick bud in order to get patterning of the information  Limb-bud organizer  Zone of polarizing activity (ZPA):Posterior mesoderm  Signals produced by organizer change gene expression in receiving cells, leading to differentiation of particular structures o i.e. the organizer, which is similar to the organizer in frog embryos, is able to organize the tissue around it for forming digits 3 2 and 1  Signal is Shh (hedgehog) produced in ZPA  the same signal exists in the spinal cord: Shh  role of Shh in producing a gradient and producing different fates 3,2, and 1 as its concentration decays is used multiple times in development to pattern other tissue where you may have to specify different fates  ex we will talk about: Shh in the spinal cord  Spinal chord patterning  spinal chord is forming an invagination of the dorsal part of the epidermis  you form a tube which gets closed, and the tube is going to be the spinal cord  spinal cord is made of neurons  what happens if you break the spinal cord? o lose sensitivity and motion o so motor neurons and sensory neurons are contained in the spinal cord  motor neurons drive muscle  sensory neurons get information from environment  when muscle contracts, you need another muscle to relax, need type of signaling pattern to do this o interneurons  there is a plate structure at the bottom of the nerve chord  Below the nerve chord there is a notochord  in the ventral part, you have motor neurons, in the dorsal part, you have sensory neurons  question: How do you specify where the motor neurons, sensory neurons, and interneurons are? Specifically, how do you specify digit 3, digit 2, digit 1?  Sonic hedgehog is produced in the floor plate, and Shh promotes the formation of sensory neurons in place, motor neurons in another, and interneurons in another  depends on concentration of Shh to determine what gets produced where, Red = high concentration yellow= medium conc. green = low conc.  How do we know this?  Five domains in the spinal chord  we can identify different markers that are marking different levels of the nerve chord o each region of the nerve chord is marked by specific genes that mark specific cells, meaning you different cells at different locations o 6 different cell types, some form close to the floor plate, some form far away from the floor plate  Graded Shh activity patterns neural rube  you have different types of motor neurons b/c you have motor neurons that go to one part of the leg and another part of the leg..  and you have interneurons and sensory neurons,….  and at the floor plate, you have a very intense source of Shh  Sonic hh mRNA  in situ hybridization for hedgehog gene  remember, in situ hybridization you are measuring the mRNA for hedgehog  the source of Shh is a diffusing factor  find that diffusion of Shh shows high levels of Shh at the floor plate, medium level, and then lower level  Now we want to know if Shh and the floor plate is responsible for this patterning of motor neurons, interneurons, and sensory neurons  Experiment:  can take tissue in the middle of the invagination,  before the nerve chord is forming, you can put it in vitro in the test tube/plate  For the control, you leave nerve chord as is  For the experimental, you put on top of the tissue a bit of the floor plate or a bit of the notochord, meaning you are going to put on top the source of Shh  see what happens to the fate of the tissue when you put this source of Shh  When you do that, if you put a bit of the notochord, you get motor neurons, if you put a bit of the floor plate, you get motor neurons,  In fact, you can put a filter inbetween notochord and tissue, which allows molecules to go through but not cells, you also get motor neurons  this tells you that there is a diffusible factor that goes from the notochord or the floor plate to the tissue and induce it to produce motor neurons o and this factor is Shh, duh…  great step to try to restore motor neurons if you break your nerve chord  In fact, there is another factor called BMP that comes from the roof plate and diffuse ventrally, so you have two gradients that together pattern precisely all of the six cell types described earlier w/ molecular markers   The Shh pathway  You have a 7 transmembrane receptor called Smoothened o smoothened probably from a mutation in flies which causes no hair  another receptor: Patched  without any Shh, Patched will inhibit Smoothened  b/c Smoothened does nothing, you have a system where you have a transcription factor called Ci protein which is going to be cut in two by a complex of proteins (which includes Costal, suppressor of fused, fused kinase, etc.)  by cutting Ci, it makes it a repressor  if you add Shh signal, you stop the cutting of Ci protein, so that Ci protein is now an activator and it can activate transcription  At the membrane, when Shh binds to Patched receptor, Patched receptor will stop inhibiting Smoothened  Smoothened can now prevent cutting of Ci, and then you get the activator  overactivation of Shh pathway causes medulloblastoma, brain tumors  a mutation that could lead to the formation of brain tumors – kill Patched! so that signal is always active  Patched mutation leads to medulloblastoma  How does Shh create digit 1, digit 2, digit 3? Why is a gradient of Shh important?  Remember: Gradient signaling: high levels produce motor neurons, lower levels produce interneurons, and even lower levels produce sensory neurons   Relay signaling: Another process: Shh induces motor neurons, and motor neurons is going to talk to its neighbor with a different signal to produce interneurons, and interneurons can talk to its neighbor to produce sensory neurons   gradient signaling is present in pretty much every organism you study, can pattern much much more w/ gradient   Morphogens  morphogenic gradient is a gradient of a protein which can pattern a tissue  high concentration you produce blue, medium concentration you produce white, low concentration you produce red  what you have to realize is that there is some sort of threshold, meaning you have a continuous gradient which leads to a discontinuous fate decision  “French Flag Model” – it’s not that blue feeds into white which feeds into red, it is that if you have a certain concentration you will be blue, below that concentration you have white, and below that concentration you will have red o concentration dependent!!!!!!!!!!!!!!!!  Most organisms use molecular gradients  e.g.: bacterial chemotactism   The Bicoid ‘morphogen’ specifies different fates based on its concentration  gradient formed by protein called Bicoid  have an embryo, a huge cell with no membrane  the left side of the embryo, the anterior side, there will be mRNA for bicoid o high level concentration of Bcd at the left side  levels of Bcd fades away as you go through cell and pretty much no Bcd at the posterior end of the embryo  Bcd is a transcription factor that activates gene expression by binding DNA  so, you have genes that are expressed very deep into the gradient and genes that are expressed more and more into the anterior part of the embryo  simple concentration of Bcd can drive the expression of these different genes  What would be the quality of the Bcd binding site in the promoter of hunchback, ems, otd, or btd? o Remember: Bcd acts as a gradient o so high concentration makes otd, the head o lower concentration makes posterior part of the head o and even lower concentration makes hunchback (hb) o so if Bcd binds directly to the promoter of hb, ems, otd, and btd, what would be the nature of the Bcd binding site on the hb promoter as compared to the otd promoter?  i.e. which one will be a better binding site?  The hb will have a better binding site, b/c it is able to bind Bcd at lower concentrations as compared to otd  SO, the way these four genes can be established is simply by choosing the binding site affinity of the different genes for Bcd o if you have very good binding site for Bcd, you are going to bind at very high affinity, even if there’s low concentration of Bcd, such as hb, produces thorax o SO if you want genes to be expressed oNLY in the most anterior part of the embryo, you are going to put genes w/ lousy binding sites for Bcd, meaning only high concentrations of Bcd will be able to activate those genes, such as otd, produces head o BINGO, you have your pattern o otd, ems, btd creates head o hb creates thorax o all depends on affinity of binding site for Bcd and concentration of Bcd  Segmentation cascade  Bcd provided from the mother, right away creates gradient of high at the anterior none at posterior  also makes other genes that work together that makes a striped pattern of genes  you get 14 stripes, which correspond to the 14 segments  So, remember Small, you have patterning starting from a very simple gradient, and just by virtue of the presence of the gradient, you can position certain genes which can then work together to segment the embryo and further segment into 14 stripes  engrail (en): is expressed in each segment, but only in the posterio


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