BIO320 Cell Bio Semester Review

BIO 320: Exam 1 Study Guide Lecture 0: General background information (self-explanatory)

--- Cell Theory --- Hierarchy of life --- Covalent bonds --- Noncovalent bonds --- Hydrogen bonds --- Ionic bonds --- Van der Waals forces --- Hydrophobic interactions --- Structure of an amino acid --- 4 levels of protein structure --- Biological roles for proteins --- Enzymes

Lecture 1: Methodology and microscopy

--- SDS-PAGE: separates bands of different molecular weight --- Amphiphilic/amphipathic: water-liking or water-hating --- β-mercaptoethanol: reducing agent (breaks disulfide bonds) --- Mass spectrometry: uses trypsin to cleave at C-termini of lysines and arginines; compare M/Z ratios of fragments to database; need entire genome of organism --- Western blotting (immunoblotting): antibody-based protein detection method --- Immunoprecipitation: determine what is linked to what --- Resolution: want lowest R value in order to get better image; light microscope limit at about 200 nm --- Contrast: see outlines of object; can use computational methods or diffractional methods --- Immunofluorescence microscopy: put fluorescence on molecules for easier viewing --- Excitation/emission profiles of fluorescent dyes/proteins: emission wavelengths are always longer than excitation wavelengths --- Confocal microscopy v. conventional light microscopy: confocal microscopy can see objects in thin slices and then put the images together in reconstructed image --- Fluorescent proteins and fusion proteins: put GFP into objects --- How to get things into living cells: electric shock, injection, vesicle transfer, viral transfer --- Transmission electron microscopy: cryo-EM can see atomic structures as well as X-ray crystallography now --- Immuno-EM: use fluorescence along with electron microscopy --- Scanning electron microscopy: coat object with heavy metal atoms; can only see outside of object

Lecture 2: Protein folding, modification, and degradation

--- Protein folding: Protein grows out of the ribosome->folded N-terminal->folded C-terminal->complete folding->cofactor binds + non-covalent modification->covalent modification (acetylation, glycosylation, phosphorylation)->bind to other subunits->mature protein. --- Molecular chaperones: These help unfold misfolded proteins so that they can correctly fold. Two types, molecular chaperones and chaperonin --- HSP70: Monomeric. Binds to stretches of hydrophobic residues, preventing aggregating residue patches. Uses ATP hydrolysis to fold or unfold protein. HSP70 w/ ATP bound to protein->several cycles->protein folds & HSP70 w/ ADP +Pi->releases ADP & takes in ATP-> ATP binds to HSP70. --- HSP60: Multimeric. Misfolded protein goes in HSP60->protein binds to the hydrophobic binding sites in the chaperonin->cap goes on->protein correctly folds with the help of HSP70 + ATP --- Protein aggregation and disease: misfolded proteins aggregate together = bad --- Ubiquitin: 76 AA long, C-terminal glycine --- Ubiquitination (process): E1+SH undergoes ATP hydrolysis where Ub binds to the SH site ->E1+S+ubiquitin-> binds to ubiquitin ligase->E1+S+ubiqitin binds to E2+SH and E3 (conjugating enzyme)->E1+S leaves, leaving E2+ubiquitin+S+E3 --- E2/E3 specificity: Protein with degradation signal (on lysine sidechain) bind to E2+E3 (ubiquitin ligase)->ubiquitin added on to target protein->E1+ubiquitin adds more ubiquitin to target protein to make a polyubiquitin chain. (K48 +K11= proteasomal degradation) ALSO!! For misfolded CYTOPLASMIC PROTEINS: Misfolded proteins associate with HSP70+40-> those bind to the hydrophobic regions->E3 (CHIP) associates with the molecular chaperone ->if enough time have passed for the associated E3 protein to polyubiquitinate->goes the proteasome to become degraded. For misfolded ER LUMEN PROTEINS: Undergoes ERAD where the proteins bind to the chaperone->goes through the ER protein translocator-> N-glycanase is bound to the protein-> protein is polyubiquitinated->degraded in the proteasome in the cytosol --- Proteasome: Made of the Cap and the Core (active site). The opening is too small for properly folded proteins. The protein with polyubiquitin goes into proteasome->binds to the cap and ubiquitin is cleaved and recycled->unfolding protein by ATP hydrolysis and destroyed within the proteasome. --- Ubiquitination and destruction of functional proteins (ex. cyclin): even well-made proteins may be destroyed --- Retro-translocation: go from ER to cytosol --- N-end rule of protein stability: certain amino sequences are more stable --- Phosphorylation/dephosphorylation: Protein Kinases adds phosphate to serine, tyrosine, or threonine residue. Protein Phosphatase removes phosphates. Highly regulated activities. They can either turn on/off proteins.

Lecture 3: Membrane structure

--- Basic functions of biological membranes: Protective Physical Barrier, Selective Chemical Barrier, closes special compartments, support 2d diffusion of molecules, and unique rxn to substrates. --- 3 major classes of lipids (4 major types): All are amphiphilic Phospholipids: Phosphoglyceride (outer leaflet of ER membrane) and Sphingomyelin (Golgi Lumen)

Glycolipid Cholesterol

--- Saturated v. unsaturated lipids: Unsaturated lipids have bent tails w/ double bonds. Saturated lipids have straight tails. --- Fluid mosaic model: current cell model --- Lateral diffusion: Cholesterol can flip/flop by itself. Phosphoglyceride can flip/flop w/ flippase and scramblase. Sphingolipid cannot flip/flop --- Major determinants of membrane fluidity: Fatty acid tails: longer=less fluid b/c tails interact more by hydrophobic interactions. Unsaturated=more fluid b/c bent tails reduces interaction Cis double bond=more fluid b/c tails are more bent/kink. Temperature: Lower Temp=less fluid --- Asymmetric organization of cell membrane: don’t want charged proteins on outside of cytosol; ER membrane has symmetric organization --- Lipid rafts: Highly ordered and tightly packed as organizing centers for signaling complexes --- Types of membrane proteins: Integral mem. proteins: Hydrophobic domains in mem. and Lipid anchored proteins. Peripheral mem. proteins --- How to predict transmembrane domains: find 20-30 amino acid hydrophobic domains for alpha helixes --- How to extract different membrane proteins from the membrane: Integral membrane proteins (Trans)=Detergent Peripheral membrane proteins=Salt Integral membrane proteins (Lipid)= detergent Ionic vs non-ionic detergent: SDS is ionic detergent, beta-octylglucosidae is non-ionic. --- Restriction of membrane protein movement: Self Aggregation, tethering by other molecules, or interaction with proteins on surface of another cell. --- Fluorescence recovery after photobleaching (FRAP): this determines lateral diffusion rates of membrane proteins/lipids. Steep slope on recovery=high lateral diffusion rate.

Lecture 4: Membrane trafficking

--- Secretory pathway: Starts at the ER --- Structure of the endoplasmic reticulum (ER): oxidizing environment; goes from ER → Golgi → membrane (outside) --- ER signal sequence: The protein that enters into the ER lumen is N-terminal --- Signal Recognition Particle (SRP): This binds to ER signal sequence due to hydrophobic pocket with methionines. The pocket can accommodate alpha helix with R-groups w/ different sizes. --- SRP-Receptor: Binding to SRP causes it to dissociate from ribosome-> recycles --- Process of co-translational translocation: Membrane bound ribosomes (in cytosol)->release protein in ER lumen->goes to plasma mem., secretory vesicles, and lysosomes. --- Translocator/Translocon (Sec61): Closed with a short helix and opens to allow translocating polypeptide to the core of the membrane. --- Signal peptidase: cleaves the sequence next to the signal sequence. --- Co-translational translocation v. post-translational translocation Co-translational translocation: Uses SRP and SRP receptor to allow the protein to pass through the protein to pass the ER membrane. Translation gives energy for translocation! Post-translational translocation: Uses ATP hydrolysis through BiP (HSP70) on the Sec 61 complex to move the protein through the ER membrane. Sec 61 is also bound to Sec 62, etc. --- Stop transfer sequence/start transfer sequence: 20-30 A.A. long (hydrophobic) --- Integral membrane protein insertion into ER membrane (single pass, double pass, N-

terminus outside, N-terminus inside): based off of positive inside rule (cytosol) Single Pass w/ N-terminus inside ER lumen: There is a - to + box. The protein goes in + 1st-

>the NH2 sticks out of the membrane to the cytsol->NH2 goes in the ER lumen->translocator opens->signal sequence in the ER membrane + C-term faces cytosol while N-term faces ER lumen. There is a internal non-cleavable signal sequence that acts as a signal sequence to bind SRP and start transfer to insert in membrane.

Single Pass w/ N-terminus outside (cytosol):. There is a + to - box on the protein. The signal sequence goes into the translocator->the C-term goes into the ER lumen while the N-term remains in the cytosol and the signal sequence is in ER membrane.

Double Pass w/ N-terminus outside (cytosol): Contains a stop-transfer/start-transfer sequence. start-transfer enters in first->binds to the hydrophobic binding site->stop-transfer enters->binds to the hydrophobic binding site->protein moves out of translocator-> C-term + N-term facing cytosolic side

--- Positive inside rule (cytoplasm = inside) --- Protein disulfide isomerase: Helps form disulfide bonds on protein within the ER lumen (oxidizing environment). Cytosol is a reducing environment-> The SH groups would still remain on the cysteine. --- N-linked glycosylation: Adds N-acetylglucoamine to the NH2 group on asparagine (in ER lumen). Also carbohydrate added to asparagine by dolichol *Note - this occurs before the protein translocate completely through the ER membrane. --- Oligosaccharyl transferase: Glucose addition on the protein during N-linked glycosylation. --- Topology of secretory proteins: ER Lumen->EC space. Cytosol->Cytosol

--- Protein folding and quality control of protein folding in the ER (cycle of glucose trimming, addition and chaperone binding)

The glycosylated protein goes through glucose trimming (glucosidase) + glucose adding (transferase)->they become associated with calnexin (molecular chaperone) that bind to exposed hydrophobic residues->if protein spends too long->slow-acting mannosidase cleaves off mannose->signals the protein for degradation

--- ER-associated degradation (ERAD): Found in Lecture 2 --- Phosphoglyceride synthesis: They are made in the cytosolic leaflet of the ER membrane. Phospholipids adds to the cytosolic side-> scramblase flips phospholipids For plasma membrane->adds new membrane by exocytosis->flippase flips the phospholipid to cytoplasmic layer. PS and PE are on the inside of plasma membrane because they function as a signal for degradation if they’re on the outside

Lecture 5: Coated vesicles, Rabs, and SNAREs

--- 3 basic players in vesicular trafficking: Donor membrane, target membrane, and transport vesicle --- Properties of COAT proteins: mechanical device that bend and curve a membrane and a mechanism for selecting the cargo proteins to be included in the vesicle. --- Three major vesicle COATs and directionality: Clathrin, COPI, COPII. COP=coatomer protein. Clathrin has light/heavy chains + adaptor protein while COP I &II form COP coats (alpha, beta). --- Trikelion: 3 heavy + 3 light chains. --- PIPs: Phosphoinositides-> they mark specific membrane and organelles to recruit specific proteins. --- Adaptin recruitment and binding to membrane: AP1 is on the trans-Golgi while the AP2 is on the plasma membrane. AP2 recruited to plasma membrane by PIP2->conformational change in AP2->AP2 binds to cargo proteins->causes slight curve on membrane->more binding of AP2 For stable binding of adaptor proteins->PIP2 and cargo proteins have to be bind. --- Formation of clathrin-coated vesicles: Cargo molecule bind to cargo receptor->cargo receptor binds to adaptor protein->the clathrin binds to the adaptor protein->other clathrin molecules interact to make a bend of the membrane-> they form a coated vesicle. --- Dynamin: GTPase that binds and hydrolyzes GTP so that the clathrin coated vesicles can pinch off. This is due to the conformational change and the energy used for pinching. --- Release of clathrin coat from vesicle: PIP2 is dephosphorylated by PIP phosphatase->reduces amount of PIP2->weakens binding of adaptor protein to membrane + molecular chaperone HSP70 acts as a ATPase->uses ATP hydrolysis to remove clathrin coat

BIO 320: Exam 2 Study Guide Lecture 5: Coated vesicles, Rabs, and SNAREs

-- GTPases: GEF converts from GDP to GTP (activates); GAP converts from GTP to GDP (deactivates) -- COP II coat recruitment by Sar1: Sar1-GDP floating in cytosol; amphipathic helix is curled up inside the protein Sar1-GDP + GEF near ER membrane → GDP to GTP → amphipathic helix comes out → Sar1-GTP ER membrane bound Sec23/24 (adaptor proteins)attach to Sar1-GTP COP2 coat on top of Sar1-GTP, Sec23/24 complex If Sar1-GTP is not attached to the membrane, then Sec23/24 complex can’t bind and COPII can’t bind to Sec23/24 complex Want the coat (COPII) to fall off Sar1 hydrolyzes GTP to GDP --> complex leaves the ER Membrane Conformational change --> folds back up the amphipathic helix → adaptor proteins (Sec23/24 complex) and coat (COPII) falls off --‐ SNARE-mediated vesicle fusion: Specificity for vehicle fusion; SNARE + RAB provide specificity; SNARE completes fusion process after Rab brings vesicle close --‐ Rab-mediated vesicle targeting: from ER (donor) to Golgi (target) Rab1-GDP is in the cytosol with lipid covered up by GDI (inhibitor) Rab1-GDP/lipid complex comes into contact with Rab-GEF on ER membrane Rab1-GTP is inserted into the ER membrane Vesicle forms with Rab still inserted in vesicle membrane Rab connects to vesicle with SNAREs, interacts with tethering protein Interaction of tethering protein and Rab -> brings vesicle closer to Golgi SNAREs on vesicle interact with SNAREs on the Golgi membrane Vesicle contact goes into Golgi Rab stuck in Golgi target membrane --> spontaneous hydrolysis of GTP to GDP on Rab --> Rab will leave the membrane --> GDI nearby will bind Rab

Lecture 6/7: Golgi/Lysosome

--‐ Modification of proteins in the ER: Disulfide bond formation (oxidizing environment) N-linked glycosylation/glucose trimming and addition ERAD --‐ 2 models of golgi organization: Neat structure of Golgi in specific area or Golgi everywhere (more of Golgi is everywhere) --‐ N-linked v. O-linked glycosylation N-linked: asparagine + N-acetylglucosamine O-linked: Threonine/serine + N-acetylgalactosamine --‐ Roles of pH: gets more acidic from ER → Golgi → membrane --‐ Lysosome function: hydrolytic enzymes to destroy stuff --‐ M6P tag and traffic of lysosomal enzymes GlcNAC-phosphate added to mannose --> M6P tag M6P: lysosomal enzymes → endosomes → lysosomes; membrane proteins = no M6P --‐ 3 pathways to lysosomal degradation: Phagocytosis, endocytosis to endosome, autophagy to autophagosome --‐ Autophagy: Basal (damaged) & Starvation-mediated (good proteins)

Lecture 7.5: Retrieval from the golgi

--‐ Why retrograde transport is necessary: return vesicles (membrane material) --‐ KDEL sequence: ER resident proteins sequence at C-termini --‐ KDEL receptor and COP I coat: retrograde transport of escaped ER resident proteins with KDEL receptor at Golgi back to ER; KDEL receptor higher affinity at acidic pH (Golgi) and less affinity at less acidic pH (ER) → drops off ER resident proteins at ER --‐ Role of pH in protein retrieval: above

Lecture 8: Endocytosis and Exocytosis

--‐ Types of exocytosis: constitutive secretory or regulated secretory --‐ Regulated secretory pathway: release of stuff triggered by binding of stuff --‐ Phagocytosis: cell-eating (big stuff) --‐ Pinocytosis (2 types): clathirin and caveolin (cell drinking) --‐ LDL receptor endocytosis: Recycling of receptors with clathirin; doesn’t require ligand binding --‐ Roles of ubiquitin in endocytosis: Ubiquitination of receptors sort into MVB’s for degradation

Lecture 10: Cytoskeleton Introduction

--‐ Purpose of the cytoskeleton: Membrane vesicle and organelle organization and movement Chromosome separation (mitosis) Cell division (cytokinesis) Cell shape, motility, and mechanical integrity Muscle contraction Axon formation --‐ 3 types of cytoskeletal elements: microtubules, intermediate filaments, microfilaments --‐ In vitro assembly of actin and microtubules (graph): begins with nucleation and then growth phase and then equilibrium phase

--‐ Nucleation: monomers come together and create nucleus for polymerization --‐ KOn, KOff, Critical concentration: C = koff/kon; if above C, get growth; if below C, get depolymerization --‐ Structure of a tubulin dimer: alpha and beta tubulin dimer; only beta tubulin can hydrolyze GTP to GDP GTP on beta tubulin can be hydrolyzed only after the α/β--‐tubulin dimer has been incorporated into the microtubule lattice --‐ Taxol: stabilizes microtubule structure --‐ Stochasticity: statistical chance of catastrophe/rescue happening --‐ Structure of a microtubule: 13 protofilaments in imperfect helix with seam --‐ Dynamic instability: undergoes catastrophe/rescue with varying polymerization/depolymerization --‐ Catastrophe and rescue: catastrophe when hydrolysis of GTP catches up to end of growth; rescue when hit GTP remnant further down --‐ GTP-remnant model for rescue: stochastically most are GDP; but GTP remnant allows for rescue and changes from depolymerization to polymerization

Lecture 11: Regulators of microtubules and microtubule-base motility

--‐ Microtubule organizing center: Non-free end of microtubules (minus end) =centrosome --‐ Gamma tubulin ring complex: 7 molecules gamma-TuSC + proteins --‐ Tau and MAP2 microtubule spacers: Stabilize microtubules → less catastrophe even when GDP bound because keeps protofilament straight --‐ Plus-end tracking proteins: bind to plus ends of microtubules --‐ Microtubule-based motility assays Faster growth at plus end than minus end; original red tubulin and stabilize with taxol; then add blue labeled tubulin → more growth on plus end because the critical concentration of the plus end is lower than critical concentration on minus end Minus end–leading (microtubule seems to be moving that direction) in old experiments Advanced experiments were allowed to see that the kinesin were actually plus end directed motors --> minus end-leading

--‐ Kinesin structure: plus-end directed motor: two globular heads for movement --‐ Kinesin movement on microtubules Lagging head ATP bound (tightly bound to microtubule) Leading head (ADP-bound): exchanges ADP for ATP → Neck linker of leading head shifts to forward-pointing conformation → pulls lagging head forward after ATP hydrolysis; repeat --‐ Processivity: how many steps before falling off microtubule

Lecture 12: Actin cytoskeleton and cell motility

--‐ Structure of actin and microfilaments: two protofilaments where G-actin makes up F-actin --‐ Similarities and differences between microfilaments and microtubules: Rate-limiting nucleation Assembly rate is concentration dependent --‐ Treadmilling (understand graph) Minus end critical conc. higher than plus end → Plus end grows while minus end shrinks --‐ Nucleation by WASP and ARP2/3 Pi3kinase turns Pip2 into PIP3 → activates GEF (membrane-bound) for Cdc42 and RacGTP --> activate WASP → activate ARP complex; ARP complex grows towards plasma membrane with actin filament growth

--‐ Formin nucleation: Formin binds to plus end of actin filaments but does not cap! formin moves up in seesaw pattern with actin added to plus end --‐ Profilin: faclitates actin subunit addition; whiskers on formin with profilin bind actin and allow for faster actin filament growth --‐ Thymosin: prevents actin filament assembly by binding actin subunits --‐ Gelsolin: severing and capping protein at plus end; high [calcium] activates and high [PIP2] inactivates --‐ Cofilin: ADF that binds ADP-containing filaments; forces filament to twist more tightly so makes filament more prone to severing --‐ Crosslinking proteins: organizes and connects actin filament assemblies --‐ Protrusion of cell membrane by actin network: allows for cell movement with growth at a certain end (polymerization) and shrinkage at other end (depolymerization) --‐ Use of actin by infectious bacteria: for movement

BIO 320: Exam 3 Study Guide Lecture 13: Actin motors and muscle contraction

--‐ Myosin and thick filament assembly: Myosin: two globular heads that can each bind ATP, alpha-helix neck region, dimer tail; Myosin II = thick filaments Phosphorylation of light chains by MLCK active High step size = high step length but low strength --‐ Cycle of myosin head ATPase activity: no nucleotide bind to actin filament, bind ATP so dissociates, hydrolyze ATP so cocked, binds to different place on actin filament, release Pi so go back to original conformation, ADP released so tightly binds, repeat --‐ Structure of skeletal muscles: Consists of myofibrils consisting of sarcomeres --‐ Structure of sarcomere: Nebulin = actin filament length from CapZ Titin: directly binds to myosin from CapZ Tropomodulin = capped minus end Cap Z = capped plus end --‐ How does the action of myosin result in muscle contraction: Troponin recruits calcium and displaces tropomyosin on actin filament & allows myosin to bind actin filaments contraction Calcium leaves, tropomyosin blocks myosin binding site actin filaments slides back into relaxed state --‐ Release of Ca2+ from sarcoplasmic reticulum: Acetylcholine → action potential → T-tubule conformational change and opens DHP calcium channels → calcium released from SER → contraction; when calcium ATPase pumps calcium back into SER & calcium channels close → relaxation

Lecture 14/15: Cell Cycle I/II

--‐ Cyclins and Cdks: CDK complex has both kinase & cyclin; Cdk blocked at first by T-loop cyclin partially activates CAK phosphorylates CDK complex at threonine site for full activation --‐ Cdk-activating kinase (CAK): CAK is always available but amount of cyclin varies (high cyclin = activation of CDK) There are specific CDKs for each cycle, e.g. G1-CDK --‐ Cdk inhibitor (CKI): Rearranges the active site of the CDK complex; can be regulated through E3s --‐ Which cyclins present during which part of cell cycle: M-CDK’s always at same level; M-cyclin reaches max during mitosis, drops, and rises during interphase; CDK-cyclin complex reaches peak during mitosis --‐ Activation of APC (E3) by M-Cdk: regulation of M-CDK levels (ubiquitination) by anaphase-promoting complex activated by Cdc20

--‐ DNA damage and the activation of M-Cdk: ATM/ATR, Chk1/Chk2, Wee1, Cdc25, 14-3-3

--‐ Nuclear lamin and envelope disassembly: Made up of Nuclear lamin A, B, C along with intermediate filaments. Phosphorylation of lamin by M-CDK breaks down nuclear envelope. --‐ Activation of S-Cdk and the G1 to S transition: Rb, E2F, G1-Cdk, G1/S-cyclin, S-cyclin:

1. Active Rb protein is bound to inactivated E2F protein. 2. active G1-Cdk double phosphorylates the Rb protein which inactivates it and activates E2F

protein. 3. E2F (transcription factor) binds to promoter region of large sets of genes which are involved in

DNA replication --> sets up condition suitable for DNA replication and also drives transcription of G1/S cyclin and S-cyclin --> forms S-Cdk complexes

--‐ Mitogens: promote synthesis of MAP kinase with Ras → activates gene regulatory protein → transcribes Myc → transcribes G1 cyclins and E3 that polyubiquitinates CKIs --‐ Assembly of machinery at origin of replication by Cdc6 and S-Cdk activation: Cdc6 & Cdt1 binds to ORC which licenses the origins, thus getting helicase (Mcm) & other replicative proteins → makes pre-RC; S-CDK initiates replication & destroys Cdc6 + exports helicase from nucleus so only obtain one replication of chromosome --‐ DNA damage blocks G1 to S transition

Lecture 17/18: Mitosis I/II

--‐ Why is chromosome missegregation a major cause of cancer: They lead cells to have genomic instability and tumorgenesis. These cancerous cells have “cin” or chromosomal instability that leads the chromosomes to missegregate at high rates --‐ Condensin: They bind to chromosomes which form Condensin complexes which is vital for condensation. M-Cdk phosphorylates chromosomes->actives condensin; SMC forces condensin ring --‐ Centriole duplication: semiconservative replication; Separation of the centrioles need specific E3 and proteasomes. G1/S phase: the G1/S Cdk allows the old centrioles to grow. Centrosome separation allows for recruitment of more gamma-tubulin so can have more nucleated microtubules → more microtubules in M phase. Phosphorylation of proteins associated with microtubule dynamics by M-Cdk → enhanced dynamic instability → increase frequency of catastrophe → increased chance microtubules can meet certain structure (kinetochore) in M phase; Each centrosome consists of two centrioles!!! --‐ Structure of the mitotic spindle What radiates out of the centrosomes is (+) ends. There are 3 sets of microtubules: astral, kinetochore, and interpolar. The astral and interpolar form the nonkinetochore. The astral MT point toward the plasma membrane. Interpolar MT reach each other in the middle where proteins interact and bind them together. Kinetochore MT are connected together in the middle by the kinetochore. --‐ Various kinesins involved in spindle assembly: Dynein is minus-end directed motor that is attached to the plasma membrane. It pulls centriole towards itself plus-end leading positions centrioles to move centrioles away from each other during Anaphase B Kinesin-5 is plus-end directed motor that crosslinks anti-parallel interpolar microtubules after being phosphorylated (activated) by M-Cdk. --‐ Kinetochore and related-motors: Kinetochore is microtubule attachment site (specific proteins that associate with centromere DNA & microtubules) on each chromosome. --‐ Forces involved in moving chromosomes to the metaphase plate

1) Dynamic instability allow microtubules to probe for kinetochore attachments. 2) Rapid transport of sister chromatids to one pole. 3) More microtubules bind to kinetochore → microtubule fiber. 4) Microtubule from opposing pole interacts with kinetochore in bi-orientation . 5) Sister chromatids congress to center. When at center, poleward forces of microtubules

opposed equally by sister chromatid cohesion force → proper tension. Aurora-B kinase causes detachment of microtubule from kinetochores with insufficient tension. When all chromosomes aligned properly → metaphase. MT depolymerase (leading kinetochore) is minus-end directed motor. +TIP (lagging kinetochore) is plus-end directed motor.

--‐ Chromokinesins: Plus-end directed motors bound along chromosome arms. Tells sister chromatids whether they have reached metaphase plate. Attached to sides of chromosomes and push towards the middle where interpolar microtubules overlap equal and opposite forces of kinesins on either side stall at center --‐ Cohesin: Consists of two SMC proteins and two SCC proteins that encircle sister chromatids and keep them together during replication. Need separase to cleave cohesin before anaphase onset. When separase is bound to securin, separase is inactive. APC + Cdc20 = E3 → degrades securin → activates separase → cleaves cohesin.

--‐ How does the cell know when all chromosomes are lined up at the metaphase plate: Spindle assembly checkpoint ensures anaphase doesn’t occur until all kinetochores attached properly to microtubules through lack of tension. Mad2 protein made as long as there are unattached kinetochores → MAD2 + proteins → MCC (mitotic checkpoint complex) → inhibits APC. Mad2 has short half-life so automatically converted to inactivated Mad2 after short time → MCC not made → APC active → degrade securin → active separase → cleaves cohesin → sister chromatids move apart. --‐ Anaphase A and anaphase B Anaphase A: shortening of distance between chromosomes and poles (moving chromatids apart). Force comes from minus-end directed motors Anaphase B: increase in distance between spindle poles (MTOC) → moves centrosomes towards minus-end. Uses plus-end and minus-end directed motors --‐ Cytokinesis: role of actin, myosin, and RhoA GTPase Cytokinesis accomplished by constriction of acto-myosin contractile ring in cleavage furrow. RhoGEF activates RhoA → activates formin → nucleates straight actin filaments RhoGEF activates RhoA → binds Rho-activated kinases (Rock) → phosphorylates and activates myosin II, inhibits myosin phosphatase → local assembly of thick actin bundles → assembly and contraction of actin-myosin ring --‐ 3 models of cytokinesis positioning Astral stimulation model: Astral microtubules radiate out from all directions from two centrosomes → highest concentration is in middle-top and middle-bottom of cell (easily reachable by astral microtubules from both poles). Central spindle stimulation model: Microtubules go from overlapping interpolar microtubules to middle-top and middle-bottom of cell. Astral relaxation model: Astral microtubules go from centrosomes to nearest place on plasma membrane. (Not good model)

Lecture 19/20: Cell signaling I/II

--‐ Major forms of signaling between cells: 1) Contact-dependent: surface of one cell has something that can bind to surface of another cell;

can have reciprocal signaling 2) Synaptic: synapse between two neurons or neuron + muscle cell; one cell will secrete

molecule that will travel short distance and bind to receptor → response 3) Paracrine: ligand travels farther from signaling cell with ligand picked up through diffusion;

gradient of signal conc. → strength to nearby cells is stronger with response dictated by signal strength; has proteases for restriction to local signaling

4) Endocrine: long-distance ligands go through bloodstream to affect far away target cells → slower response time General cell signaling process: ligand binds to receptor → conformational change of receptor → intracellular signaling proteins → effector target proteins -> responses

--‐ 2 types of ligands (bind cell surface receptors, bind intracellular receptors) Intracellular receptors: hydrophobic molecules (nitric oxide) bind to carrier proteins to travel through hydrophilic aqueous solution → go inside hydrophobic target cell; steroids bind intracellularly by binding inactive receptor and exposing NLS sequence to be taken into nucleus → slow gene transcription response Cell-surface receptors: Most ligands are hydrophilic → bind to receptor outside of cell → exert effects through intracellular signaling proteins --‐ Rapid and slow responses of cell to signals Rapid response: altered protein function (muscles) Slow response: altered protein synthesis (steroids) --‐ G-protein coupled receptors (GPCRs): transmembrane receptor: recognizes signal from outside and transfers signal to cytoplasm; trimeric G-protein: transmits signal from receptor to target protein; target (effector) protein examples: adenylyl cyclase → cAMP or Phospholipase C → DAG & IP3 All GPCRs have 7 transmembrane domains Enzymatic cascades allow for amplification of extracellular signals --‐ Structure of trimeric G-proteins and roles of different subunits: Galpha: binds and hydrolyzes GTP; Gbeta; Ggamma Galpha & Ggamma are prenylated (addition of hydrophobic molecules) & membrane-anchored Ligand binding → conformational change of GPCR turns it into GEF for Galpha → activated alpha subunit & Betagamma complex; many inactive proteins can diffuse into activated GPCR and become activated --‐ Cyclic AMP: Ligand binding to Beta-adrenergic serotonin receptor activates adenylyl cyclase and cleaves two phosphates off of ATP to make cAMP → activates protein kinase A (PKA); cAMP is example of second messenger Cholera toxin modifies Gs (stimulatory G protein) → Galpha stays in GTP-bound state → perpetually active → CFTR ion channel perpetually activated → ions pumped out so water molecules go out → lose water → extreme diarrhea --‐ Protein kinase A (including how PKA mediates the fight or flight response): activated by cAMP (cAMP binds to 4 subunits of regulatory subunit so regulatory subunit dissociates → activated PKA slow response: Activated PKA enters nucleus → phosphorylates CREB → CREB binds to CRE (cyclic AMP response element) → transcribes many genes fast response: PKA activates phosphorylase kinase and inhibits glycogen synthase→ activates glycogen phosphorylase → breaks down glycogen into glucose --‐ Signaling termination: trimeric G protein becomes inactive on own but activated proteins downstream need to be phosphatased; can use negative feedback loops (GPCR signaling → synthesis of RGS (regulator of G-protein signaling) which functions as GAPs for Galphas)

--‐ Desensitization of target cell: can involve receptor ubiquitination or inhibitory phosphorylation Rhodopsin: Light photon is signal → Activated rhodopsin stimulates GRK → GRK phosphorylates rhodopsin & Arrestin binds rhodopsin (inhibit ability to activate more G-proteins) → arrestin becomes adaptor for clathirin pits → endocytosis of rhodopsin photoreceptor; RGS also binds Galpha to stimulate GTP-hydrolysis --‐ Free [calcium] inside cell: Free [calcium] kept very low in cytosol through calcium ATPase pumps out of the cell or into ER lumen & mitochondria --‐ Phospholipase C: cleaves PIP2 into DAG & IP3 --‐ Review phosphoinositides (Exam 1): PIP2 is the substrate for Phospholipase C

--‐ IP3 formation and release of intracellular calcium: Activation of GPCR --> G protein --> Phospholipase C --> opens up IP3 gated channels by cleaving PIP2 into IP3 & DAG--> cytosolic Ca conc. rise abruptly --‐ Calmodulin: four binding sites for calcium; activated when two or more binding sites are occupied by calcium → binds to proteins → more conformational changes in calmodulin Example: CaM-kinases require calmodulin binding; partial activation with calmodulin binding to CaM-kinase II → autophosphorylation for full activation (strength of phosphorylation important for learning); phosphatase needed in order to completely inactivate CaM-kinase II even after calmodulin falls off --‐ Protein kinase C: activated by calcium + DAG + PS (phosphatidylserine) --‐ Second messengers: cAMP, cGMP, calcium, lipid-derived messengers (DAG & IP3) --‐ Gap junctions: channels allow small molecules to pass between cells nonselectively; made by connexin proteins; shares signaling information

Lecture 21: Cell signaling III

--‐ Receptor tyrosine kinase (RTK) structure: example of enzyme-coupled receptor --‐ RTK activation and method of signal relay: receptor dimerization of RTK activates RTKs after ligand binding with phosphorylation of kinase domain & outside kinase domains (docking sites for proteins) → assembly of signaling complexes Can concentrate signaling proteins at specific sites on plasma membrane Can recruit proteins for down-signaling (E3s that ubiquitinate RTKs → endocytosis & ubiquitination of receptors → desensitization) --‐ Common signaling protein domains: Phosphotyrosine on receptor recruits proteins with SH2 or PTB domains --> RTK phosphorylates these proteins → more sites for recruiting more proteins WW domain binds phosphorylated serine/threonine PH domain binds phosphoinositides (PIP3) SH3: binds proline-rich sequences --‐ PI 3-kinase (PI3K): activated by GPCRs and RTKs to generate PIP3 from PIP2; PTEN phosphatase counteracts PI3K --‐ Ras GTPase and MAP kinase cascade: Sos acts as Ras-GEF which activates Ras → MAPKKK → MAPKK → MAPK → turns on Myc transcribes G1 cyclins & polyubiquitinates CKIs Docking protein + monomeric activated receptor + adapter protein + Sos can hold scaffold protein which can then get many proteins to be activated --‐ PI3K-Akt signaling pathway (in apoptosis lecture below): Activated receptor tyrosine kinase recruits PI 3-kinase which generates PIP3 from PIP2; phosphorylation and activation of Akt by PDK1 & mTOR only when nutrients are available -->Active Akt phosphorylates Bad (class C) leads to separation of class A and class C → Active apoptosis-inhibitory protein (class A) and inactivated Bad linked to 14-3-3 protein -> apoptosis inhibition PTEN (acting against PI3K) mutations found in many cancers

Lecture 16: Apoptosis

--‐ Three forms of cell death: necrosis, apoptosis, programmed necrosis --‐ Common features of apoptosis: cell shrinks & condenses; cytoskeleton collapses; nuclear envelope disassembles; chromsomes condense and fragmented; cell surface altered (phosphatidylserine so becomes negatively charged) --‐ 2 classes of caspases: Initiator and executioner/effector caspases --‐ Gelsolin activation by caspase and its effects on the cell: Gelsolin cleavage by caspase results in gelsolin’s activation even without high calcium concentrations → collapse of actin cytoskeleton --‐ Extrinsic apoptotic pathway: Signal for apoptosis comes outside of the cell → Fas ligand to Fas receptor → attaches to FADD adaptor protein + death effector domain with procaspase-8 or 10 → formation of DISC (death-inducing signaling complex) → procaspase is cleaved and activated → targets apoptotic cells --‐ Intrinsic apoptotic pathway: Apoptotic stimulus inside cell → Release of cytochrome C by MOMP activates Apaf1 forming CARD domain complex → many Card domain complexes form cartwheel apoptosome → recruits procaspase-9 → cleaved and caspase-9 becomes active → apoptosis --‐ Bcl2 protein family (Class A, B, C): regulate intrinsic pathway of apoptosis Class A: anti-apoptotic (BH1 to 4) Class B: pro-apoptotic (BH1 to 3) Class C: pro-apoptotic (BH3)

Neutralize A with C, then activate B with remaining C; B needs to be activated by some version of class C B FORMS CHANNELS FOR MOMP! A inactivates B so can no longer function in MOMP

--‐ p53 and the Bcl proteins: Transcription of genes in response to p53 increases C and represses A; when repair fails, cells can be destroyed by apoptosis --‐ How to prevent accidental apoptosis: inhibitors of apoptosis (IAPs) bind to caspases; during apoptosis, MOMP releases anti-IAPs which bind IAPs and prevent them from binding to caspases --‐ PI 3-kinase-Akt signaling pathway: Activated receptor tyrosine kinase recruits PI 3-kinase which generates PIP3 from PIP2; phosphorylation and activation of Akt by PDK1 & mTOR only when nutrients are available -->Active Akt phosphorylates Bad (class C) leads to separation of class A and class C → Active apoptosis-inhibitory protein (class A) and inactivated Bad linked to 14-3-3 protein -> apoptosis inhibition

A

C B

Lecture 24/25: Cell Junctions & Cell-Cell Adhesion

--‐ Classic experiment in cell-cell recognition: When early embryo taken and the cell types are dispersed, the cells will self-organize to how they were before (demonstrates self-organizing principle) Cells can migrate and then aggregate together (important in embryo development) High levels of E-cadherin cells will gather together inside low levels of E-cadherin cells --‐ Four major classes of cell junctions Anchoring junctions: anchors cells to each other… Occluding junctions: restriction barriers so materials outside can’t pass through paracellularly to get to other side of cell Channel-forming junctions: involved with 2nd messengers Signal-relaying junctions: relays signals… --‐ Cell junctions found in polarized epithelial/endothelial cells Polarized cells have apical and basolateral sides Tight junctions, cell-cell anchoring junctions, channel-forming junctions found in virtually all epithelial/endothelial cells Adherens junctions: connects actin filaments Desmosomes: connects intermediate filaments --‐ Proteins involved in cell-cell adhesion: Cadherins, selectins immunoglobulin superfamily, integrins); cadherin adhesion is strongest --‐ Cadherins: calcium-dependent adhesion; has transmembrane domains with repeating EC domains on extracellular side; important for embryonic compaction --‐ Homophilic and heterophilic binding: homophilic = same receptor type; heterophilic = different receptor types; most cadherin forms are homophilic-bound --‐ Cadherins and calcium in the extracellular space: EC domain has multiple binding sites for calcium; removing functional calcium results in droopy cadherin domains → cadherin structures can’t meet each other; aggregate adhesion of thousands of cadherins needed for strong adhesion --‐ Cadherins during neural tube closure and epithelial-to-mesenchymal transition Neural tube closure: neural crest cells don’t express cadherin and become migratory; different cadherins for different types of cells Inactivation mutation of cadherins in cancer so can metastasize cancer (migrate) EMT: involved in migratory cells not expressing cadherin Neural tube closure refers to cadherins having neural crest cells coming together into a circle with skin covering by the ectoderm on top (back with spinal bifida); epithelial = together; mesenchymal = migratory (ex: from back to spine, etc.) --‐ Tension at adherens junctions: When no tension, alpha-catenin can bind straight actin filaments Usually attached to neighboring cells Straight actin filaments have myosin motors working on them --> exert pulling force so there will be tension → pulling force ends up changing conformation of alpha-catenin from folded to straightening out → exposes binding site for vinculin on alpha-catenin → recruits additional actin filaments and also generate more force --> adherins junctions act as tension sensors Adherens junctions organized as belts around cells tied together using cadherins → coordination in motility of adjacent cells → transmission of mechanical stress --‐ Desmosomes: linked to intermediate filaments with nonclassical cadherin proteins like desmogloin and desmocollin

--‐ How do epithelial cells serve as selectively permeable barriers? They have tight junctions which can alter permeability barriers between apical and basolateral sides --‐ Tight junction – organization, claudin, occluding, ZO proteins, etc. Tight junction = zonula occludens: seals extracellular space between cells with intermittent contact Claudin: dictates selective permeability to specific ions ZO proteins: scaffold proteins that organize protein complexes that can allow across specific proteins? --‐ Gap junctions and connexin proteins: mediate intercellular communication and allow small molecules to pass through; gap junctions are made of connexin proteins spanning the membrane 6 molecules of connexin → form connexons → connexons associate to form intercellular channels --‐ Selectins: three subtypes (E,L,P); extracellular lectin domain that binds specific oligosaccharides on surface of another cell and intracellular domain that binds actin filaments; function as cell-cell adhesion molecules that are calcium-dependent example: inflammation turns on P-selectin in endothelial cells → binds weakly to oligosaccharides on WBC’s → slows down WBC’s in needed areas -- Immunoglobulin (IG) superfamily [IgSF]: modular repeats of Ig domains that are calcium-independent --‐ Integrins: alpha and beta subunits that are calcium-dependent; mostly binds extracellular matrix instead of other cells; strong adhesion and emigration stronger interaction involving integrin and VCAM endothelial cell (stronger binding) strong enough so WBC’s stops rolling WBC can squeeze between endothelial cells to go to site of inflammation