Cell Biology - Exam 3 (Chapter 15 to 18, 20)

(Chap. 18) Describe the composition, structure, organization and cellular functions of microtubules

*Actin filaments use ATP; Microtubules use GTP* Composition: alpha-beta-tubulin dimers assemble in polarized manner GTP bound to alpha-tubulin is non-exchangeable GTP bound to beta-tubulin is exchangeable and is necessary for proper assembly Once assembled, beta-tubulin hydrolyzes GTP to GDP A microtubule fiber is comprised of 13 protofilaments and can be found in singlets, doublets & triplets *know the structures on slide 51* MTOC = microtubule organizing center

(Chap. 20) Describe signaling pathways associated with CAMs (e.g. integrins)

20-38 Model for integrin activation. - Form heterodimer •(a) Integrin activation - conformational changes: •Movements near the propeller and βA domains - increase affinity for ligands •(top) Inactive, low-affinity, "bent" conformation •(bottom) Active, high-affinity, "extended" conformation •Greater transmembrane and cytoplasmic domain separation - induced by ligand binding or resulting from altered interactions with the talin and kindlin adapter proteins •(b) Integrin αIIbβ3 molecules: •(top panel) Inactive (bent) conformation - no bound ligand or talin •(bottom panel) Activate (straightened) conformation - activated by bound talin head domain Integrin adhesion receptor-mediated signaling pathways control diverse cell functions. •Integrins: •Interact via adapter proteins and signaling molecules with a broad array of intracellular signaling pathways •Influence cell survival, gene transcription, cytoskeletal organization, cell motility, and cell proliferation •Outside-in signaling - extracellular ligand binding - •Changes integrin cytoplasmic domain conformation - •Alters interactions with cytoplasmic proteins including •Adapter proteins (e.g., talins, kindlins, paxillin, vinculin) •Signaling kinases that transmit signals via diverse signaling pathways (Src-family kinases, focal adhesion kinase [FAK], integrin-linked kinase [ILK]) •Influences cell proliferation, cell survival, cytoskeletal organization, cell migration, and gene transcription •Components of several signaling pathways: •Some - associated directly with the plasma membrane •Many of the pathways - shared with other cell-surface activated signaling pathways (e.g., receptor tyrosine kinases) •Inside-out signaling - intracellular signaling pathways cause adapter proteins to modify integrin ability to bind extracellular ligands

(Chap. 20) Describe the major components of the basal lamina and their functions

A basal lamina separates epithelial cells and some other cells from connective tissue. •Basal lamina: A meshwork of filamentous proteins that associates with the plasma membrane and connective tissue collagen fibers. Play most important roles in epithelial tissue, also present around other tissue. •Functions in organizing cells into tissues and distinct compartments, protecting cells, repairing tissues, forming permeability barriers, and guiding migrating cells during development •One side - linked to cells by adhesion receptors including hemidesmosome and integrins that bind to laminin in the basal lamina •Other side - anchored to adjacent connective tissue by a layer of collagen fibers embedded in a proteoglycan-rich matrix Laminin is a heterotrimeric multi-adhesive matrix protein found in all basal laminae. •(a) Laminin molecule: •16 vertebrate laminin isoforms - assembled from 5 α, 3 β, and 3 γ chains •Coiled-coil region - three peptides covalently linked by several disulfide bonds •Globular domains - bind to/crosslink adhesion receptors and various matrix components •(b) Structure (EM): •(left) Characteristic cross shape •(right) Carbohydrate-binding LG domains near the C-terminus Structure and assembly of type IV collagen. •Sheet-forming type IV collagen is a major structural component of the basal lamina. •Type IV collagen: •400-nm-long molecule •N-terminus - small globular domain •C-terminus - large globular domain •Collagenous triple helix - interrupted by nonhelical segments that introduce flexible kinks into the molecule •Formation of dimers, tetramers, and higher-order sheet-like networks: •Lateral interactions between triple-helical segments •Head-to-head and tail-to-tail interactions between the globular domains •Multiple, unusual sulfilimine (-S=N-) or thioether bonds between hydroxylysine (or lysine) and methionine residues covalently cross-link some adjacent C-terminal domains and contribute to the stability of the network.

(Chap. 18) Name different classes of intermediate filaments and describe their locations and functions

Acidic keratins: where? epithelial cells; functions? tissue strength and integrity Basic keratins: where? epithelial cells; functions? tissue strength and integrity Lamins: where? nucleus; functions? nuclear structure and organization

(Chap. 18) Describe the structures and functions of microtubules in cilia, flagella, and the mitotic spindle apparatus

Basal body: nine linked triplet Axoneme: the membrane-bound core of the cilium or flagellum Cilia and flagella bending mediated by axonemal dyenin; involves doublet tubules Mitotic spindle: involves singlet tubules -Astral microtubules: from the spindle poles to cell cortex -Kinetochore microtubules: link the spindle poles to the kinetochores on sister chromatid pairs -Polar microtubules: each spindle pole body toward the opposite one and interact together

(Chap. 20) Identify the 4 major types of cell adhesion molecules (CAMs) and describe what they bind

Cadherin (E-cadherin): form Ca2+-dependent extracellular HOMOPHILIC interactions (repeat domains) with E-cadherins on adjacent cells Immunoglobulin (Ig) superfamily: form HOMOPHILIC linkages (as shown for NCAM) and some can form heterophilic linkages Integrins: HETEROPHILIC; heterodimeric (alpha and beta chains) and bind to very large, multi-adhesive matrix proteins such as fibronectin Selectin: contain a carbohydrate-binding lectin domain that recognizes specialized sugar structures on adjacent cell glycoproteins/glycolipids, often form higher-order oligomers within the plane or the plasma membrane, HETEROPHILIC (selectins bind to sugar chains of glycoproteins) Adherens junctions - adhesion type: cell-cell - principal CAMs or adhesion receptors: cadherins - cytoskeleton attachment: actin filaments - intracellular adapters: catenins Desmosomes - adhesion type: cell-cell - principal CAMs or adhesion receptors: desmosome cadherins - cytoskeleton attachment: intermediate filaments Tight Junctions - adhesion type: cell-cell - principal CAMs or adhesion receptors: occludin, claudins, JAMs - cytoskeleton attachment: actin filaments Gap junctions - adhesion type: cell-cell - principal CAMs or adhesion receptors: connexins, innexins, pannexins - cytoskeleton attachment: via adapters to other junctions

(Chap. 17) Describe the role of Rho protein in cell movement and signal transduction

Cell Movement 1. Lamellipodium extends from leading edge 2. Lamellopodia adhere to surface via focal adhesions 3. Cell cytosol is pushed forward as the rear contracts 4. Trailing edge remains attached to surface until tail eventually detaches and retracts into cell body. Membrane and integrins internalized to make new focal adhesions Signal Transduction -Detection of signals lead to activation of small GTPases (Cdc42, Rac, and Rho), which interact with effectors to induce cytoskeleton changes -Back: Rho activation -> leading to myosin II activation (contraction) -Front: Rac activation -> leading to Arp2/3 activation (branched) Cdc42 activation at the front *Cdc42 -> Rac -> Rho High Rho concentration at trailing edge, High Cdc42 concentration at leading edge (creating polarity)

(Chap. 20) Describe different types of cell-cell junctions and their protein components

E-cadherin: •Exoplasmic domains - •Clustered by cis and trans interactions at adherens junctions on adjacent cells •Form Ca2+-dependent homophilic interactions (black dots are where calcium ions bind) •Cytosolic domains - •Bind directly/indirectly through multiple adapter proteins (e.g., β-catenin) to actin filaments (F-actin) of the cytoskeleton •Participate in intracellular signaling pathways Desmosomes. - Mediated by integrins •(a) Desmosomes contain two specialized cadherins - desmoglein and desmocollin: •Cytosolic domains - •Distinct from those in classical cadherins •Bind adapter proteins - including plakoglobin, desmoplakins, and plakophilins - attach to sides of intermediate filaments •(b) Desmosome connecting two cultured differentiated human keratinocytes (EM): •Bundles of intermediate filament bundles radiate from the two darkly staining cytoplasmic plaques that line the inner surface of the adjacent plasma membranes. •(inset) EM tomograph of desmosome linking two human epidermal cells (plasma membranes, pink; desmosomal cadherins, blue) Tight junctions. - Tightly link cells together via proteins that "stitch" them together •Seal off body cavities • Restrict diffusion of membrane components between apical and basolateral membrane regions •(a) TJ between two intestinal epithelial cells: Honeycomb-like network of ridges and grooves •(b) Structure - Linkage of rows of protein particles in adjacent cells Proteins that compose tight junctions. •(a) Occludin (green) and tricellulin (red) localization in mouse intestinal epithelial cell tight junctions: •Occludin - throughout TJ •Tricellulin - predominantly concentrated in tricellular junctions •(b) Protein structures and functions: •Occludin and claudin: •Principal integral membrane proteins of tight junctions •Four transmembrane domains •Occludin C-terminal cytoplasmic domain binds PDZ-containing scaffold proteins (ZO-1-3 proteins). •Claudin larger extracellular loop - contributes to paracellular ion transport selectivity •Cytoplasmic domain of TJ proteins bind to actin •Exoplasmic domain determine what molecules can pass through Gap junctions. - More important in signaling •Composed of connexins - 21 different human connexin genes - different sets of connexins are expressed in different cell types •Allow small molecules to pass directly between cytosols of adjacent cells •(a) Gap junction (connecting two mouse liver cells): •Adjacent cell plasma membranes - separated by a "gap" of 2-3 nm for a distance of several hundred nanometers •(b) Numerous hexagonal connexon structures on the cytosolic face of a gap junction: •Each aligns with a similar structure on an adjacent cell to form tunnel connecting cytoplasms. •(c) Gap junction connecting two plasma membranes: •Both membranes contain connexon hemichannels - cylinders of six dumbbell-shaped connexin molecules •Two connexons join in the gap between the cells to form a gap-junction channel. •(d) Human Cx26 gap junction (x-ray crystallography; 3.5-A resolution): •(left) Two attached connexons (space-filling model): •Each connexin - four transmembrane helices •(Structures of the loops connecting the transmembrane helices are not well defined and not shown.) •(right) Connexon central pore channel: •Diameter ∼14 A •Lined by polar/charged amino acids •cAMP in G-coupled pathway Principal types of cell junctions connecting the columnar epithelial cells lining the small intestine. •(a) Intestinal epithelial cells: •Basal surface - adhesion to basal lamina •Apical surface - fingerlike microvilli project into the intestinal lumen •Tight junction - •Primarily in epithelial cells •Surrounds the cell below the microvilli - connects to all neighboring cells •Regulates paracellular transport of substances between the intestinal lumen and internal body fluids (blood) via the extracellular space between cells •Boundary between apical and basolateral regions of the plasma membrane •Transmembrane proteins cannot pass through / move - Restricts some proteins to apical vs. basal •Gap junctions - allow movement of small molecules and ions between cytosols of adjacent cells •Adhesion junctions - •Adherens junction - •Continuous junction with all neighboring cells •Circumferential belt of actin and myosin filaments associated with the adherens junction - functions as a tension cable that can internally brace and control cell shape (only found in epithelial cells) •Desmosomes - spot cell-cell junctions •Hemidesmosomes - spot cell-ECM junctions •(b) Junctions between rat intestinal epithelial cells (EM)

(Chap. 20) Describe the major proteins of connective tissue and their structures and functions

Elastic and collagen fibers in connective tissue. •Elastic fibers permit many tissues to undergo repeated stretching and recoiling. •(a) Lung loose connective tissue (nuclei, stained blue): •Elastic fibers - thin fibers (purple) •Collagen fibers (bundles of collagen fibrils; pink) •(b,c) Elastic fibers and collagen fibrils (coll) (mouse skin - longitudinal and cross-sectional EM images): •Elastic fibers - solid core of elastin fibers (e) integrated into a bundle of fibrillin-fibulin-LTBP microfibrils (mf) •Elastin fibers - •Aggregates of monomeric tropoelastin molecules that are covalently cross-linked via a lysyl oxidase-mediated process similar to that for collagen •Repetitive proline- and glycine-enriched hydrophobic sequence motifs contribute to ability of tropoelastins to self-associate, extend under stress, and recoil efficiently. Interactions of fibrillar collagens with fibril-associated collagens. •Type I and II collagens - associate with nonfibrillar collagens to form diverse structures •(a) Tendons: •Type I fibrils - all oriented in the direction of the stress applied to the tendon •Proteoglycans and type VI collagen - bind noncovalently to coat the type I fibril surface •Type VI collagen microfibril globular and triple-helical segments - link type I fibrils together into thicker fibers •(b) Cartilage: •Type IX collagen molecules - covalently bound at regular intervals along type II fibrils •Chondroitin sulfate chain covalently linked to the α2(IX) chain at the flexible kink - projects outward from the fibril •Globular N-terminal region - projects outward from the fibril

(Chap. 15) Distinguish 4 types of cell-cell signaling: endocrine, paracrine, autocrine, contact

Endocrine: signaling molecules synthesized and secreted by signaling cells, transported through the circulatory system, act on target distant from site of synthesis Paracrine: signaling molecules released by signaling cell only target cells that are close by Autocrine: cell responds to signaling molecules that itself released Contact: membrane signaling proteins of one cell bind to receptors on adjacent cell

(Chap. 17) Describe generally that different actin-binding proteins organize different actin structure

FIMBRIN: microvilli, filopodia, focal adhesions ALPHA-ACTININ: stress fibers, filopodia, muscle Z line SPECTRIN: cell cortex FILAMIN: leading edge, stress fibers, filopodia DYSTROPHIN: linking membrane proteins to actin cortex in muscle

(Chap. 17) Describe the composition of microfilaments (actin) and the cellular structures they form

G-actin: globular, free actin monomer F-actin: filamentous actin Mesh-like networks (cortical actin network): LAMELLIPODIUM; strength, semi-rigid, shapes cell surfaces Elongated "cables": STRESS FIBERS (stretches cell); bundles of F-actin filaments; contractility comes from sliding of filaments Slender needle-like extensions: FILOPODIA ("feelers"); rapid extension and disassembly

(Chap. 15) Describe how heterotrimeric G proteins mediate signals from GPCRs and effectors and the sequential steps from inactive, to active, and back to inactive

G-protein coupled receptor components: •Receptor with 7 transmembrane alpha helices •Heterotrimeric G-protein (G-alpha and G-beta-gamma) •Membrane-bound effector protein (Adenylyl cyclase) •Proteins that participate in desensitization of signaling pathway •Ligand binds to receptor, inducing conformational change •Activated receptor binds to G-alpha subunit of G-protein •Conformational change in G-alpha subunit induces dissociation of GDP from G-protein •GTP binds to G-alpha subunit, induces conformational change that results in G-alpha subunit dissociating from receptor and G-beta-gamma subunits •Ligand dissociates from receptor, free G-alpha subunit binds to membrane-bound effector protein to activate it •Hydrolysis from GTP on G-alpha subunit to GDP, induces G-alpha subunit dissociating from effector protein and reassociation with G-beta-gamma subunits •This diagram shows an effector protein being activated by G-protein complex, but different G-protein complexes can inhibit effector proteins as well

(Chap. 20) Describe the essential roles of cell-ECM interactions in cell differentiation and development

Integrins mediate linkage between fibronectin in the ECM and the cytoskeleton. •Stress fibers - long contractile bundles of actin microfilaments •Stress fiber actin microfilaments terminate at cell membrane where extracellular fibronectin fibrils connect to integrins in the cell membrane. •Binding of cell-surface integrins to fibronectin in the ECM induces the actin cytoskeleton-dependent clustering of integrin molecules in the focal contact. •Integrin-based adhesion shape, distribution, and composition varies depending on cell environment. Extracellular Matrix Proteins (~ 1000 genes in mammals code ECM proteins) •Proteoglycans - a unique type of glycoprotein: cushioning: hyaluronic acid used to treat knee injury. •Collagens - form fibers: integrity and strength. •Multi-adhesive matrix proteins - Important organizers of the extracellular matrix: Fibronectin and laminin: long, flexible, bind collagen, sugar, signaling molecules, and integrins; regulate cell-matrix adhesion and cell shape and behavior Organization of fibronectin and its binding to integrin. •(a) Fibronectin: •Homodimer linked by disulfide bonds near C-termini (one chain shown) •Each chain contains 2446 amino acids in three types of repeating sequences/domains (type I, II, or III). •ECM binding - specific binding sites for heparan sulfate, fibrin (a major constituent of blood clots), collagen •Cell-surface integrin binding - two type III "cell-binding" domains - bound by integrin extracellular domains. •(b) "Cell binding" / integrin-binding domain structure: •RGD motif - binds to integrin The repeating disaccharides of glycosaminoglycans (GAGs). •Proteoglycans and constituent GAGs play diverse roles in the ECM. •Four classes of GAGs: contain some carboxylate or sulfate group - negative charges •Formed by polymerization of monomeric units into repeats of a particular disaccharide and subsequent modifications •Modifications - can have different modification at each repeat •O-linked oligosaccharide - three-sugar "linker" attached to hydroxyl side chains of Ser (or Thr in other cases) •N-linked: sugar linker attach to N of Asn (learned in Ch13) •Hyaluronan: •Made by HA synthase (plasma-membrane-bound enzyme) - secreted directly into extracellular space as it is synthesized •Resists compression, facilitates cell migration, and provides cartilage its gel-like properties •Surrounds migrating and proliferating cells, particularly in embryonic tissues •Forms the backbone of complex proteoglycan aggregates found in many ECMs including cartilage •Aggrecan - predominant proteoglycan in cartilage, not type of GAG •4 types of GAGs: Heparin, chondroitin sulfate, keratin sulfate, hyaluronic acid •Proteoglycans cushion cells, bind extracellular molecules

(Chap. 15) Describe three major effector pathways downstream of GPCRs: ion channel activation, cyclic nucleotides, and intracelluar Ca²⁺

Ion channel activation: •G-beta-gamma opens K+ channel (Effector protein) •GTP hydrolysis causes G-alpha to bind with G-beta-gamma •Slows down rate of muscle contraction Cyclic nucleotides: •GPCR activate/inhibit Adenylyl cyclase (Effector protein), which may then increase/decrease cAMP levels •Increase cAMP •Activates PKA -> Phosphorylate glycogen -> Inhibit glycogen synthesis •PKA ->Glycogen degradation •Inhibits Phosphoprotein phosphatase (PP) -> No dephosphorylation of enzymes in kinase cascade or of inactive glycogen synthase •Decrease cAMP •Inactivates PKA, no inhibition of PP -> Glycogen synthesis and inhibition of glycogen degradation Intracellular Ca²⁺: •Ligand binds to GPCR, leads to dissociation of G-protein subunits, and one subunit (in this case, G-beta-gamma) binds to Phospholipase C (Effector protein) •Phospholipase C cleaves PI(4,5)P2 to produce IP3 and DAG •IP3 diffuses through cytosol and opens IP3-gated Ca2+ channels in ER membrane •Ca2+ ions released into cytosol •Protein kinase C (PKC) recruited to plasma membrane •DAG activates PKC PKC can then phosphorylate various cellular enzymes and receptors

(Chap. 16) Identify signals, receptors, transcription factors for major signaling pathways: Wnt signaling pathway Identify its mechanism of signal transduction (e.g. protein phosphorylation, subunit dissociation/degradation, ion channel activation, 2nd messenger) and whether it is reversible or irreversible.

Irreversible (a) Absence of Wnt: •The TCF transcription factor is bound to promoters or enhancers of target genes, but its association with transcriptional repressors such as Groucho (Gro) inhibits gene activation. •β-catenin is bound in a complex with Axin (a scaffold protein), APC, and the kinases CK1 and GSK3, which sequentially phosphorylate β-catenin at multiple serine and threonine residues. •The E3 TrCP ubiquitin ligase binds to two phosphorylated β-catenin residues, leading to β-catenin ubiquitinylation and degradation in proteasomes. (b) Binding of Wnt to its receptor Frizzled (Fz) and to the LRP co-receptor: •Triggers GSK3 and CK1 phosphorylation of LRP •Phosphorylated LRP binds disheveled scaffold protein and Axin. •Axin binding to phosphorylated LRP disrupts the Axin-APC-CK1-GSK3-β-catenin complex, preventing phosphorylation of β-catenin by CK1 and GSK3 and leading to accumulation of β-catenin in the cell. •Free β-catenin translocates into the nucleus and binds to TCF, displacing the Gro repressor. •Recruitment of co-activator proteins including Pygo, LGS, and others activate gene expression. •Many targets of Wnt pathways also control Wnt signaling (feedback loop) •Involve degradation/dissociation

(Chap. 18) Distinguish the two types of motor proteins, kinesins and dyneins, with respect to their structures and functions

KINESINS -analogous to myosins -kinesin-1 and kinesin-2 --have head and tail --involved in ANTEROGRADE organelle transport --move toward + end -kinesin-5 --have two heads, 4 heavy chains --move toward + end --microtubule sliding at mitotic spindle -kinesin-13 --have only head --microtubule disassembly DYNEINS --RETROGRADE organelle transport --move toward - end --involved in microtubules sliding for flagella

(Chap. 15) Explain how signal transduction pathways and second messengers amplify and integrate signals.

Kinase - adds phosphate group Phosphatase - removes phosphate group Guanine nucleotide exchange factor (GEF) - induce release of GDP from G protein and promotes addition of GTP -> accelerates G protein signaling GTPase-activating protein (GAP) - Induces GTPase activity (hydrolysis) of G protein -> Terminates G protein signaling •cAMP - generated from ATP by adenylyl cyclase; activates PKA •cGMP - generated by guanylyl cyclase; activates PKG and specific cation channels •IP3 and DAG - made from PIP2 by phospholipase C; IP3 opens channels to release Ca2+ from the ER; DAG activates PKC with Ca2+ •Ca2+ - released from intracellular stores or transported into the cell; activates calmodulin, specific kinases (PKC), and other regulatory proteins •Epinephrine = 1st messenger •cAMP = 2ND messenger -> No amplification DURING the process of 2nd messenger binding to receptor (in this case, no amplification occurs DURING cAMP binding to Protein kinase A) •Amplification steps are why a relatively low percentage of occupied receptors can elicit a near-maximal cellular response

(Chap. 18) Describe the process of MT disassembly and assembly

Microtubule with GTP-beta-tubulin on the + end of each protofilament will grow; after binding, GTP gets hydrolyzed to GDP Microbutubles with GDP-beta-tubulin on the + end of each protofilament will form curved structure and undergo rapid dissambly Microtubule associated proteins (MAPs) regulate microtubules assembly Dynamic disassembly via Kinesin-13 and Stathmin Proteins bind to alpha-beta-tubulin and promote disassembly Hydrolysis of ATP on Kinesin-13 enhances its activity Stathmin binds to curved GDP-beta-tubulin; inactivated by phosphorylation

(Chap. 18) Describe the role of MTOCs in nucleating MT polymerization

Microtubules are nucleated at and extend from MTOC. Associated proteins (Gamma-TuRC including Gamma-tubulin) Promote assembly of Mts MTOC in animal cells has centrioles; special microtubules 'seeds'; perpendicular orientation *GAMMA-TUBULIN IS ONLY FOUND IN MTOCs*

(Chap. 17) Describe myosins and how they function in skeletal muscle contraction

Myosin: actin-based motor proteins that move along actin filaments Monomer (I) or Dimer (II and V) form Head is an actin-activated ATPase Motor activity is driven by ATP binding/hydrolysis Class I have single head domain, tail bound to plasma membrane (FUNCTION: membrane association, endocytosis) Class II have two head domains and two tail, can assemble into bipolar filaments (FUNCTION: contraction) Class V have two head domains and six tails, walk along actin filament (FUNCTION: organelle transport) ALL THREE CLASSES OF MYOSINS MOVE TOWARDS + END OF ACTIN FILAMENTS Skeletal Muscle Contraction 1. ATP binds to myosin head, and it releases from actin filament 2. Myosin head hydrolyzes ATP to ADP and Pi, and it rotates to cocked state 3. Cocked myosin head binds to actin filament 4. Myosin head release Pi and elastic energy to move actin filament (power stroke) 5. Myosin head is tightly bound to actin filament until ADP is replaced with ATP *Myosin heads walk along actin microfilaments toward plus end and Z bands get closer together -> muscle contraction (contraction limited by length, strength) *Ca2+ regulates the ability of myosin to bind to actin (troponin [Ca2+] and tropomyosin)

(Chap. 17) Describe the polarity of actin filaments and their growth at actin concentrations below at and above critical concentration; regulation by cofilin, profilin, and thymosin

Nucleation is slowest step Elongation occurs immediately after nucleation and quickly builds actin filament Steady state assembly and disassembly occur, but there is no net growth Critical concentration: concentration of free G-actin at which the assembly onto a filament end is balanced by loss from that end; defined at steady state + end critical concentration is lower than that of - end. Filament formation occurs at critical concentration Intrinsic ATP hydrolysis -> ATP is slowly hydrolyzed to ADP, release of Pi At [G-actin] < Cc+, there is no filament growth Treadmilling condition: at [G-actin] > Cc+ but <Cc-, there is growth from the (+) end only At [G-actin] > Cc+ and Cc-, there is growth from both ends, but the (+) end grows faster Thymosin Beta4: bind to ATP G-actin, inhibit actin polymerization, conserves G-actin reservoir Profilin: bind to ADP-G-actin, promotes actin polymerization Cofilin: promotes actin depolymerization by cleaving fragments with ADP-G-actin CapZ: stabilizes actin filaments and prevents growth at + end Tropomodulin: stabilizes actin filaments and prevents disassembly at - end

(Chap. 17) Identify key proteins that nucleate branched vs unbranched actin filament assembly: formins and Arp2/3

Nucleation step: Formins (unbranched) dimerize and form seeds Rho-binding domain of inactive formin binds to membrane-bound active Rho-GTP, which reveals the FH2 domain recuits Profilin-ATP-actin complexes and feed into + end of growing filament Arp2/3 (branched) actin filaments actin subunit bind to nucleation promoting factor (NPF). two NPF-actin complexes bind to Arp2/3 complex. together, Arp2/3 bind to side of actin filament and actin subunits on NPFs bind to Arp2/3. assembly of branch at + end at 70-degree angle

(Chap. 15) Use ligand binding plots and Kd = [R][L]/[RL] to infer Kd, receptor concentration RT.

R=(free) receptor L=(free) ligand/hormone RL=occupied receptor (bound ligand and receptor) Plots of physiological response and ligand binding affinity are different Maximal cell response is induced when less than 100% of receptors are occupied (blue curve) Practice Problem: F19 Exam 3 SA1 Part C

(Chap. 16) Identify signals, receptors, transcription factors for major signaling pathways: Receptor Tyrosine Kinase pathway (RTK) Identify its mechanism of signal transduction (e.g. protein phosphorylation, subunit dissociation/degradation, ion channel activation, 2nd messenger) and whether it is reversible or irreversible.

Reversible 1. Without ligand, RTKs in monomeric form with poorly active kinases 2. When ligands bind to extracellular domains of two RTKs, they dimerize. The kinases on cytosolic domains phosphorylate each other at tyrosine residue in activation loop 3. Activation loop moves out of kinase catalytic site, increasing ability of ATP and/or protein substrate to bind; Can serve as docking site for SH2 and other binding domains on downstream signal-transducing proteins •Major mechanism of signal termination is internalization and endocytosis of ligand-bound receptors ---HER/Erb family of RTKs and their ligands ---Erb-B2 (HER2) - Forms heterodimer with ligand-binding Erb-B1, 3, or 4 ---Erb-B3 (HER3) - Signals only when complexed with Erb-B2

(Chap. 16) Identify signals, receptors, transcription factors for major signaling pathways: Cytokine signaling via JAK-STAT Identify its mechanism of signal transduction (e.g. protein phosphorylation, subunit dissociation/degradation, ion channel activation, 2nd messenger) and whether it is reversible or irreversible.

Reversible 1.In absence of ligand, two receptors form homodimer, while JAK kinases are not active 2.When ligand binds to receptors, JAK kinase domains are brought closer together and phosphorylate each other at tyrosine residue in activation loop, ultimately activating JAK kinases 3.Active JAK kinases phosphorylate multiple tyrosine residues in receptor cytosolic domain -Can function as docking sites for STAT proteins Activation and structure of STAT proteins Epo = Erythropoietin cytokine that triggers erythrocyte/RBC production 1. After ligand binding, SH2 domain of inactive monomeric STAT transcription factor binds to phosphotyrosine in the receptor, which brings the STAT close to active JAK associated with receptor 2. JAK phosphorylates STAT and phosphorylated STATS dissociate from receptor and spontaneously dimerize 3. STAT homodimers are stabilized by two phosphotyrosine-SH2 domain interactions, so will not bind to receptor 4. STAT homodimer translocates to nucleus, bind to promoter sequence, activating transcription of target gene Signal termination SHP1 - Short term regulation -SHP1 - Phosphotyrosine phosphatase -SH2 domain of SHP1 binds to phosphotyrosinein activated receptor, revealing SHP1's catalytic site -SHP1 dephosphorylates tyrosine in activation loop of JAK kinase, inactivating it SOCS proteins - Long term regulation -SH2 domain of SOCS binds to phosphotyrosine on receptor or on JAK to block binding of other signaling proteins -SOCS box targets receptor and JAK for degradation by ubiquitin tagging

(Chap. 16) Identify signals, receptors, transcription factors for major signaling pathways: Ras/MAP kinase pathway (MAPK) Identify its mechanism of signal transduction (e.g. protein phosphorylation, subunit dissociation/degradation, ion channel activation, 2nd messenger) and whether it is reversible or irreversible.

Reversible •Receptor tyrosine kinases activate Ras via adapter proteins 1. A specific phosphotyrosine on an activated, ligand-bound receptor provides a specific site for binding the cytosolic adapter protein GRB2 SH2 domain. 2. GRB2 SH3 domains bind to SOS (proline-rich motifs) and recruit SOS to the membrane to interact with Ras∙GDP. 3. The SOS guanine nucleotide exchange factor (GEF) activity promotes release of GDP and formation of active Ras∙GTP. •Ras activates highly conserved cascade of three protein kinases (Raf → MEK → MAP kinase) •14-3-3 protein dimer bound to Raf phosphoserine-259 and phosphoserine-621 and stabilizes Raf in an inactive conformation •In stimulated cell: 1. Active RTK receptor activates formation of active Ras·GTP. 2. Ras·GTP interacts with the Raf N-terminal regulatory domain, which results in dephosphorylation of one of the serines, release of 14-3-3, and activation of the Raf kinase activity. 3. Ras GTP hydrolysis to Ras·GDP releases active Raf. 4. Raf phosphorylates and activates MEK. 5. MEK, a dual-specificity protein kinase, phosphorylates MAP kinase on both tyrosine and serine/threonine residues 6. 6: MAP kinase phosphorylates many different proteins in different cells, including nuclear transcription factors, that mediate cellular responses

(Chap. 16) Identify signals, receptors, transcription factors for major signaling pathways: TGT-beta via Smads Identify its mechanism of signal transduction (e.g. protein phosphorylation, subunit dissociation/degradation, ion channel activation, 2nd messenger) and whether it is reversible or irreversible.

Reversible Signals: Smads Receptors: RIII, RII, and RI (receptors have serine/threonine kinase domain) Transcription factors: Mechanism: •TGF-Beta binds to RIII, which brings TGF-Beta to RII OR TGF-Beta binds to RII directly •TGF-Beta bound RII recruits and phosphorylates juxtamembrane segment of RI •Activated RI phosphorylates Smad2 or Smad3 -> Conformational change that reveals its nuclear localization signal (NLS) •Two phosphorylated molecules of Smad2/3 bind to co-Smad (Smad4) and to importin to form complex •Entire complex translocates to nucleus •Ran-GTP causes importin dissociation •Nuclear transcription factor associates with Smad2/3-Smad4 complex, which binds to regulatory sequences of target gene •Complex recruits transcriptional co-activators and induces gene transcription •Nuclear phosphatase dephosphorylates Smad2/3 •Smad2/3 recycled through nuclear pore to cytosol To turn off TGT-beta: dephosphorylation of Smad2/3, inhibition of Smad3-Smad4 interaction (Ski), activating histone deacetylase (Ski/SnoN)

(Chap. 18) Describe the coordination and cooperation of cytoskeletal elements during cell migration

slide 61 Cdc42 regulates microfilaments and microtubules independently to polarize a migrating cell

(Chap. 16) Identify signals, receptors, transcription factors for major signaling pathways: Phosphoinositide signaling pathway (PI3K) Identify its mechanism of signal transduction (e.g. protein phosphorylation, subunit dissociation/degradation, ion channel activation, 2nd messenger) and whether it is reversible or irreversible.

•In unstimulated cells, PKB (Akt) is in the cytosol with its PH domain bound to and inhibiting its catalytic kinase domain •In simulated cells: 1. Hormone stimulation leads to activation of PI-3 kinase, which forms PI 3-phosphates (Inositol-3-phosphates, IP3) 2. The 3-phosphate group binds the PH domains of PKB (Akt) and PDK1, docking both to the membrane and partially activating PKB activity. 3. PDK1 phosphorylates the PKB activation loop serine and PDK2 phosphorylates a PKB C-terminus serine fully activates PKB activity, which induces many cellular responses. •The PI-3 kinase pathway is negatively regulated by PTEN phosphatase, which dephosphorylates the 3-phosphate •Cdc42 can be activated by PI3K (cell migration)

(Chap. 16) Describe how different external signals can integrate on common internal signaling pathways or molecules.

•Insulin, Wnt, and TGFbeta leads to activation or inhibition of adipose gene expression. Integrated responses control adipocyte differentiation. •Many signaling proteins, such as Wnt and TGF-β, oppose the action of insulin and prevent preadipocyte differentiation into adipocytes by directly or indirectly regulating PPARγ and C/EBPα genes expression. •PPARγ and C/EBPα are induced early in adipogenesis and each enhances transcription of the gene encoding the other, leading to a rapid increase in expression of both proteins during the first two days of differentiation. (a) Insulin activates by several PKB pathways, leading to activation of PPARγ expression by inhibiting two repressors of the PPARγ gene. (b) Wnt signaling release β-catenin that activates the transcription factor TCF, which blocks expression of the PPARγ and C/EBPα genes, thus inhibits adipogenesis. (c) TGF-β pathway lead to Smad3 phosphorylation. Phosphorylated Smad3 binds to C/EBPα protein and prevents it from activating expression of the PPARγ gene.

(Chap. 16) Identify signals, receptors, transcription factors for major signaling pathways: Notch-Delta signaling pathway Identify its mechanism of signal transduction (e.g. protein phosphorylation, subunit dissociation/degradation, ion channel activation, 2nd messenger) and whether it is reversible or irreversible.

•Irreversible process •Pathway determines the fates of many types of cells during development. •Without Delta: •The Notch transmembrane subunit is noncovalently associated with its extracellular subunit, which is folded so that it cannot be cleaved by the ADAM 10 cell-surface protease •In presence of Delta •Delta binds to extracellular domain of Notch on an adjacent cell •ADAM10 cleaves Notch •Delta+Notch (extracellular domain) is endocytosed •Notch (cytoplasmic domain) is further cleaved by γ-secretase complex •Notch fragment translocate to nucleus and act as transcription factor Mediates lateral inhibition