Cell Bio Test 4 Material-2 PDF
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This document provides a comprehensive overview of cell signaling mechanisms, including endocrine, paracrine, and other types. It highlights different signaling pathways, explains signal transduction, and covers regulatory mechanisms like GTPase switch regulation. The document also explores important aspects of hormones and G-protein coupled receptors.
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Cell Signaling - Endocrine Signaling - Epinephrine, insulin - Signaling molecules synthesized and secreted by signaling cells - Transported through circulatory system - Affect distant target cells expressing receptor - Paracrine Signaling - Neurot...
Cell Signaling - Endocrine Signaling - Epinephrine, insulin - Signaling molecules synthesized and secreted by signaling cells - Transported through circulatory system - Affect distant target cells expressing receptor - Paracrine Signaling - Neurotransmitters, growth factors - Signaling molecules secreted by cell - Some bind to EMC and are released when EMC is degraded - Affect nearby target cells expressing receptor - Neuronal Signaling - Signal delivered to individual cells over long distances - Electrical impulse sent along axon to synapse - Neurotransmitter released - Diffuses across synapse to target cell - Autocrine Signaling - Growth factors - Cells respond to signals they secrete - Ex: tumor cells may overproduce and respond to growth factors - Juxtacrine Signaling - Membrane protein signals - Signaling neighboring cells by direct contact with surface receptors - Or the signal can be transmitted through gap junctions directly from cytoplasm of one cell to cytoplasm of another - Signal Transduction Pathway - Each protein alters the next protein, usually by phosphorylation - Steps/Path - 1st messenger (ligand) ⇨ receptor ⇨ G-protein ⇨ effector ⇨ 2nd messenger ⇨ target protein - 2 types of internal switch activate other proteins in cascade - Phosphorylation state - G proteins - Kinase/Phosphate Switch Regulation - Kinase phosphorylation and phosphatase dephosphorylation to regulate target protein activity - Protein kinase transfers terminal phosphate from ATP to specific Ser/Thr or Tyr–OH - Protein phosphatase hydrolyzes P off protein restoring Ser/Thr or Tyr–OH - Protein kinases and phosphatases regulated by signaling processes and modify specific protein targets containing target motifs - GTPase Switch Regulation - Alternate between on and off conformations - G-proteins (GTP-binding proteins) are on/off switches - ON/ACTIVE: Bound to GTP - promoted by GEFs - GEFs catalyze dissociation of bound GDP and replacement it with GTP (not phosphorylation of GDP) - OFF/INACTIVE: Bound to GDP - GTPase activity - GTP → GDP + Pi (ON to OFF) - Accelerated by GAPs and RGSs - STAY OFF - Bind to GDIs - Prevent release of GDP all together - GTPase switch proteins - Heterotrimeric - activated by direct interaction with surface receptors (GEFs) - direct - Monomeric - activated by GEFs that are activated by surface receptors or other proteins - Indirect - Intracellular 2nd Messengers - Transmit signals through cytosol - 4 main types - cAMP: - Generated from ATP by adenylyl cyclase - Activates PKA - cGMP: - Generated by guanylyl cyclase - Activates PKG and specific cation channels - IP3 and DAG: - Both made from PIP2 by phospholipase C - IP3 opens channels to release Ca2+ from the ER - DAG - with Ca2+ activates PKC - Calcium ions (Ca2+): Intracellular second messenger - Released from intracellular stores or transported into the cell - Activates calmodulin, specific kinases (PKC), and other regulatory proteins - Nitric Oxide (NO): Intracellular/Extracellular second messenger - Formation catalyzed by the enzyme nitric oxide synthase Hormones - Synthetic Analogs - Widely used in research and in drugs - Agonist - Mimics natural hormones - Binds to and activates receptor - Induces normal cellular response - Antagonist - Inhibits function of natural hormone - Binds to binding site but induces no response - Reduces natural hormone binding, reduces activity G-Protein Coupled Receptors - Structure of a G-Protein Coupled Receptor (GPCR) - Interact with cytoplasmic G proteins. - Seven α-helical transmembrane domains - Four extracellular segments and four cytosolic segments - Many G proteins are heterotrimeric - Composed of 3 subunits α, β and γ - Bound to membrane by covalently attached lipid chains - Bound to GDP the three proteins are tightly associated as a complex - Activation by receptor induces exchange of GDP for GTP - Alters conformation and releases the Gα and Gβγ subunits which interact with effector proteins - Mechanism of Activation - Step 1: - Ligand binding induces receptor activation conformational change - Step 2: - Activated receptor binds to trimeric G-protein - Step 3: - Activated receptor GEF activity stimulates Gα subunit release of GDP. - Step 4: - GTP binding changes Gα conformation - Dissociates Gβγ - Activates Gα - Step 5: - Gα GTP activates effector enzyme - Step 6: - Gα intrinsic GTPase activity hydrolyzes GTP to GDP - dissociates Gα and turns off effector enzymes - G protein active for minutes or less - Generalized Signal Transduction Pathway involving G-protein - Step 1: - Hormone binding to its cell-surface receptor - Step 2: - Activated receptor (GEF) activates trimeric G protein - Step 3: - G protein alpha subunit binds to and activates a second messenger-generating enzyme. - Step 4: - Activated enzyme generates multiple second messenger molecules. - Step 5: - Second messenger activates a protein kinase. - Step 6: - Kinase phosphorylates and changes activity of one or more target proteins. - 1st: Cytosolic target proteins induce changes in cellular function, metabolism, or movement. - 2nd: Target transcription factors induce changes in gene expression. - Step 7: - Signal termination - Deactivation of a G-protein - GAP stimulates rapid GαGTP hydrolysis to GDP. - GαGDP dissociates from the effector protein - Effector becomes deactivated - Alteration of 2nd Messenger - Second messengers are either degraded or sequestered - cAMP phosphodiesterase (PDE) catalyzes hydrolysis of cyclic bond - AMP, not second messenger - Deactivation of GPCR - Binding of Arestin - GPCR phosphorylated and allosterically inhibited - Synthesis of 2nd Messengers - Phospointosides- phosphorylated forms of phosphatidylinositol produced by the activities of specific kinases - An enzyme called phospholipase C (PLC) is activated by some G proteins - PLC cleaves PIP2 into phosphatidylinositol-inositol triphosphate (IP3) and diacylglycerol (DAG) - IP/DAG Pathway - The IP3/DAG pathway causes a rise in intracellular Ca2+ concentration from ER and external sources - ER is main intracellular storage for Ca - Ca ATPases pump Ca2+ into the ER - Phospholipase 3, IP3, DAG are second messenger to know - Nitric Oxide in Signaling - NO is both intracellular and extracellular messenger - A signal transduction pathway activated by NO and cyclic GMP leads to the relaxation of smooth muscle - NO produced by nitric oxide synthase - Acetylcholine activation of its GPCR - G-protein activation of PLC - PLC generation of IP3 (+ DAG) - Cytosolic Ca2+ increase activates calmodulin - Calmodulin activates NO synthase - NO makes cGMP - cGMP decreases cytosolic calcium and relaxes smooth muscle - Tyrosine Kinases - Activated by two types of receptors - Receptor Tyrosine Kinases (RTKs) - Tyrosine kinase enzyme is part of receptors’s polypeptide chain - Cytokine Receptors - Receptor activates a second kinase encoded by another gene - Both activate multiple signal transduction pathways that regulate gene transcription - Autophosphorylation - Kinase receptors autophosphorylate tyrosine residues on the cytoplasmic region of the receptors - Signaling proteins associate with activated kinase receptors via domains that bind phosphorylated tyrosine residues. - SH2 Domain - Recognize a 4 amino acid motif starting with phosphorylated tyrosine - Mediated phosphorylation-dependent protein-protein interactions - PTB Domain - Can bind to phosphorylated tyrosine (pTYR) - Some can bind to phosphorylated tyrosine - Components of Tyrosine phosphorylation - Adaptor Proteins - Linkers: enable 2 or more proteins to join together - Contain SH2 and one other protein-protein domain - Docking Proteins - Supply receptors with additional Tyr phosphorylation sites - Dock protein = phosphorylation site - PTB or SH2 domain plus phosphorylation sites - Transcription Factors - Enter the nucleus to affect transcription - SH2 domains and phosphorylation sites - Signaling Enzymes - Protein kinases, protein phosphatases, lipid kinases, phospholipases, GTPase activating proteins - SH2 domains associate with activated RTKs and enzymatic activity is turned on - directly or indirectly - Activation of Cytokine Receptors - Cytokine receptors lack intrinsic kinase activity, they activate a separate kinase - Absence of Ligand - Cytokine receptor cytosolic domain binds tightly and irreversibly to JAK protein tyrosine kinase - Receptors form homodimer - JAK kinases poorly active - Ligand Binding - Causes receptor conformational change that brings together JAK kinase domains - JAK kinase domains trans-autophosphorylate each other on tyrosine residue (activation loop) and activate kinases - Active JAK kinases phosphorylate multiple tyrosine residues in the receptor cytosolic domain - Function as docking sites for signal-transducing proteins, including STAT proteins - Activation of STAT proteins - JAK kinases activate STAT transcription factors - STAT proteins (like Smads) bind to open chromatin DNA sites - Phosphorylation and dimerization of STAT proteins exposes the NLS (nuclear-localization signal) - STAT dimer translocates into the nucleus, where it binds to promoter sequences and activates transcription of target genes. - Termination of Cytokine Signal Transduction - Short Term Regulation: phosphotyrosine phosphatase - Phosphotyrosine phosphatase SHP1 is present in an inactive form in the cytosol of unstimulated cells. - SHP1’s SH2 domain binds phosphotyrosine in activated receptor - Activates phosphatase catalytic activity - Allows activate phosphatase to dephosphorylate the JAK2 activation loop - This inactivates JAK kinase activity - Long Term Regulation: SOCS Protein Negative Feedback - STAT5 induces SOCS protein expression in erythropoietin-stimulated erythroid progenitor cells. - SOCS binds to specific phosphotyrosine residues on EpoR or JAK2 - blocks binding of other signaling proteins. - SOCS targets the receptor and JAK2 for degradation - recruits ubiquitin enzymes to target the proteins for proteasomal degradation - Activation of RTKs - Generally only single TM domain - Not effective at inducing conformational change - Ligand has two binding domains - Two separate ligands interact with two receptors - Results in logan dimerization - Brings cytoplasmic tails together to activate kinases - Receptors trans-autophosphorylate each other - Phospho-residues act as docking sites - Can bind proteins with SH2 and PTB domains - Autophosphorylation sites on RTKs - Regulate kinase activity - Act as binding sites for cytoplasmic signaling molecules - How RTKs activate enzymes - translocation to the membrane - places them in close proximity to their substrates - allosteric mechanism - binding to pTyr results in conformational change in the SH2 domain - regulated directly by phosphorylation - Termination of RTK signaling - Short Term - Regulation of distinct phosphatases inactivates the receptor - Long term: - Terminated by internalization of the receptor and lysosomal degradation - RAS Transduction - Activated Ras-GTP binds and activates a serine/threonine kinase - initiates a phosphorylation cascade of ST kinases - Receptor tyrosine kinases activate RAS via adaptor proteins - Phosphorylated RTK recruits the GRB2 adaptor protein. - GRB2 recruits the Son of Sevenless (SOS) protein. - SOS is a guanine nucleotide exchange factor (GEF) protein that activates the Ras G-protein. - RAS/MAP Kinase Pathways - Ras·GTP triggers the downstream kinase cascade by interacting with the Raf - results in dephosphorylation of one of the serines, release of 14-3-3, and activation of the Raf (MAP 3K) kinase activity. - Ras GTP hydrolysis to Ras·GDP - releases active Raf. - Raf phosphorylates and activates MEK (MAP 2K). - MEK phosphorylates MAP kinase (MAP 1K) on both tyrosine and serine/threonine residues. - MAP kinase phosphorylates many different proteins in different cells - PI3K Pathway - Phosphoinositide 3-Kinase - A family of Serine/Threonine kinases Promote cell growth and survival - Promote cell growth and survival, - Insulin-like growth factors are major ligands - PI-3 Kinase recruited to membrane by activated tyrosine receptors - Adds 3-phosphate to PI(4)P to yield PI(3,4)P2 and to PI(4,5)P2 to yield PI(3,4,5)P3 - The phosphorylated inositol phospholipids serve as docking sites for specific intracellular signaling proteins - Protein Kinase B (PKB) - Most important phosphorylated inositol phospholipid - Active kinase, targets wide range of cytosolic proteins - Regulates their activity through phosphorylation - This promotes cell survival - In unstimulated cells PKB is in cytosol with PH domain bound to catalytic kinase domain (inhibits it) - Hormone stimulated cells: - 1: Hormone stimulation leads to activation of PI-3 kinase - 2: The 3-phosphate group binds the PH domains of PKB (Akt) and PDK2, docking to the membrane and partially activating PKB. - 3: PDK1 phosphorylation of the PKB activation loop serine and PDK2 phosphorylation of a PKB C-terminus serine fully activates PKB activity, which induces many cellular responses. - Pathway negatively regulated by PTEN phosphatase - Dephosphorylates 3-phosphate - In many types of cancer PTEN is deleted - Leads to uncontrolled cell growth - SMAD Pathways - TGF-𝜷 - Transforming growth factor 𝜷 signaling molecules - Inhibit cell proliferation and regulate development - SMADS interact with regulatory DNA sequences at sites adjacent to those occupied by cell-specific master transcription factors to induce target genes cell-specifically. - General Mechanism - TGF-𝜷 signaling molecules secreted by most styles of cells - Ligands to activate large group of TGF-𝜷 receptors with S/T Kinase activity - S/T Kinases phosphorylate-activate SMAD transcription factors - Regulate growth and differentiation pathways - TGF-𝜷 Dimer - Three monomer intrachain disulfide linkages form a cysteine knot domain and another disulfide bond links the two monomers. - secreted as a latent TGF-β complex that binds to ECM until released as a free signaling molecule by extracellular proteases. - 3 TGF- 𝜷 receptors activate signaling pathways - Activation requires ligand binding and dimerization - Active receptor phosphorylates the SMAD transcription factors which alters their conformation - dimerization - relocation to the nucleus. - Pathway deactivated by nuclear phosphatases - dephosphorylate SMAD proteins. - Ubiquitination and Protein Degradation Regulated Pathways - Signaling pathways involving ubiquitination and proteolysis of target proteins are irreversible - Protein degradation regulates life spans of intracellular proteins - vary from as short as a few minutes for mitotic cyclins to as long as the age of an organism for proteins in the lens of the eye - Polyubiquitination targets proteins for proteasomal degradation - 3 main types of pathways - WNT - Hedgehog - NF-𝛋B - WNT (Canonical) Pathway - Targeted protein degradation inhibits activation of specific biological process - Activation of signal transduction pathway prevents protein degradation - Hedgehog (Hh) Pathway - Hedgehog proteins act as morphogens to signal nearby cells - Morphogen- signaling molecule that acts directly on cells to produce a response dependent on concentration, diffuse through cells and create concentration gradient - Gradients drive cellular differentiation of stem cells into different cell types - Cells synthesize Hh precursor - Precursor undergoes autoproteolysis (in ER) - Hh precursor undergoes a nucleophilic attack by the thiol side chain of cysteine 258 on the carbonyl carbon of the adjacent glycine 257 - forms a high-energy thioester intermediate. - C-terminal domain enzyme activity catalyzes formation of an ester bond between glycine 257 and a cholesterol hydroxyl group - cleaving the precursor into two fragments. - The N-terminal signaling fragment retains the cholesterol group and is modified by the addition of a palmitoyl group to the N-terminus - tether the Hh protein to the plasma membrane. - NF-𝛋B - master transcriptional regulator of the mammalian immune system - dimeric transcription factor composed of p50 and p65 subunits - bound to its inhibitor I-κBα in resting cells. - Polyubiquitination of I-κBα for degradation by proteasomes leads to removal of I-κBα - Removal revelas NLSs in both subunits of NF-κB, - They translocate into the nucleus where they can activate target gene expression. - Notch/Delta Pathway - Controlled by protein cleavage - Important growth factors/signaling proteins are released by matrix metalloprotease cleavage of transmembrane proteins. - Notch receptor interacting with Delta on an adjacent cell undergoes proteolytic cleavages that release a Notch cytosolic segment - modulates transcription of cell fate-determining genes. - If notch is not associated with delta it cannot be cleaved - If notch is bound to delta it can be cleaved - Initiation of Delta endocytosis by the signaling cell stretches the Notch extracellular domain open for ADAM 10 cleavage - regulated intramembrane proteolysis (RIP). - The released Notch extracellular domain remains bound to Delta - endocytosed by the signaling cell. - The four-protein γ-secretase complex nicastrin subunit binds to the Notch stump generated by ADAM 10. - The four-protein γ-secretase complex presenilin 1 protease catalyzes an intramembrane cleavage that releases the Notch cytosolic segment. - The Notch segment translocates into the nucleus - interacts with several transcription factors to affect expression of genes that determine cell fate during development - Cell Signal Responses - Signal DIvergence - One signal - Can lead to multiple responses in one cell or different responses in different cells - Signal Convergence - Multiple signals - Activation of same pathway or effector - Crosstalk - Multiple signals - Signal from one pathway effects another pathway - Signal Combinations - Multiple Signals - Combination determines response Cell Organization and Movement - The Cytoskeleton - Network of filamentous structures - Intermediate filaments, microtubules, and microfilaments - Reversibly and dynamically assemble each type of filament from specific subunits - Maintains and changes cell shape, cell motility, intracellular transport - Cell signaling regulates cytoskeleton function - Cell-surface receptors transmit external signals from the extracellular matrix, other cells, or soluble factors across the plasma membrane - Activates specific cytosolic signaling pathways - regulate cytoskeleton organization and function. - Microfilaments composed of Actin monomers - Actin - G-actin - ATPase activity - ATP/ADP binds at the bottom of the left cleft and contacts both lobes - F-actin - 2 helically wound strands with repeating 28 subunits - the ATP-binding cleft of every actin subunit is oriented toward the same end of the filament - The filament end with an exposed binding cleft is the (−) end; the opposite end is the (+) end. - This causes structural and functional polarity and makes the ends distinguishable - In Vitro G-actin Polymerization - Polymerization of G-actin monomers to rom F-actin filaments - 3 stages of polymerization - 1: Nucleation (lag) phase - Inefficient formation of three ATP–G-actin “nucleus/seed” initiates formation of a filament. - 2: Elongation phase - Actin subunits rapidly assemble onto each end of a filament. - 3: Steady State phase - G-actin monomers exchange with subunits at the filament ends, but there is no net change in the total length of filaments. - If short actin filament seeds are added then the nucleation phase is skipped - The nucleation phase is very slow - Critical Concentration - Concentration below which filaments cannot assemble and above which filaments assemble and form steady state mixture - Actin Filaments - Each monomer binds an ATP molecule that is hydrolyzed upon polymerization - Once ATP is hydrolyzed the actin filaments associated less - Hydrolysis promotes depolymerization - ATP-G actin assembly is faster at + end than - end and disassembly is roughly the same - Actin Treadmilling - Addition of actin filaments at + end and dissociation at - end - Cell migration, endocytosis, exocytosis - Filament Regulation by Actin Binding Proteins - Actin-binding proteins regulate the rate of assembly and disassembly of actin filaments as well as the availability of G-actin for polymerization - Profilin binds to ADP–G-actin opposite the nucleotide-binding cleft - opening the cleft and catalyzing the exchange of ADP for ATP. - Profilin binding sterically blocks ATP–G-actin assembly on the filament – end but allows the unblocked G-actin monomer end to assemble onto the filament + end. - ATP–G-actin–profilin complex assembly on the + end dissociates profilin to interact with another ADP–G-actin. - Cofilin fragments ADP-actin filament regions, - enhancing overall depolymerization by making more – filament ends. - Thymosin-β4 provides a buffered reservoir of ATP–G-actin for polymerization - sequesters G-actin at high concentration; releases G-actin at low concentration to polymerize - Filament Capping Proteins - Block assembly and disassembly at filament ends - + end capping proteins - CapZ - Limits actin assembly and disassembly to - end - Gelsolin - Severs actin filaments by inserting itself between actin subunits - Blocks + end - Some are activated by rise in Ca2+ concentration - - end capping proteins - Tropomodulin - Blocks end where filament disassembly occurs - Stabilizes filament - Actin Nucleation - Nucleation is 1st step in polymerization - 2 major classes of proteins regulated by signal pathways nucleate actin - FH2 Regulation - Form a dimer (from two formins) that binds two actin subunits to nucleate formation of a new filament - Protects the + end from being immediately capped - Arp2/3 - Nucleates branched filament - Binding of two NPF-actin complexes induces conformational change that actives the Arp2/3 complex - The activated Arp2/3 complex binds to the side of an existing actin filament and binds the - ends of actin subunits. - Additional G-actions assemble onto the + end of new actin filament. - Myosins - Myosin 1 - One heavy chain with head and neck domain - Only single-headed myosin - Variable number of light chains associated with neck domain - Can associate directly with membrane through tail-lipid interaction - Myosin 2 - Two heavy chains each with head and neck domain that bind 2 light domains - Long helical tail homodimerizer into coiled-coil formation - Dimers bind one another through tails forming bipolar myosin filaments with the heads at either end - contractile force of muscles, contractile rings of dividing cells and other contractile bundles in non-muscle cells - - Myosin 5 - Two head domains, 6 light chains per neck - Heavy chain helical tail homodimerizer through a coiled-coli interaction - End of tails interact with specific receptors on organelles and transports them along actin filament tracks - Myosin 1,2,5 move toward + end (+ end directed motors) - Myosin 6 is the only - end directed motor - Powerstroke mechanism say step size and neck chain length should be proportional - Skeletal Muscles - Composed mostly of myofibrils - Myofibrils: groups of myosin 2, actin and accessory proteins grouped into sarcomeres - Actin filaments slide past myosin filaments during muscle contractions - Each sarcomere contains thick filaments of myosin II in its center and parallel arrays of thin filaments of actin extending in from either end of the sarcomere - Stimulation promotes ATP hydrolysis, myosin movement and the sliding of actin bundles toward the center of the sarcomere, resulting in muscle contraction - Accessory Proteins in Skeletal Muscles - Actin Filaments - CapZ and Tropomodulin cap + and - ends - Nebulin binds actin subunits and determines filament length - Titin - Long elastic molecules - One end attached to Z disk, one end attached to M band - Centers the thick myosin filaments by interacting with both ends - Muscle Contraction Triggered by Ca2+ - Neuronal signaling stimulates release of Ca2+ from the sarcoplasmic reticulum via voltage gated ion channels - This Ca2+ regulates a molecular switch that allows contraction to occur - Switch controlled by troponin and tropomyosin - Tropomyosin binds the groove of actin filaments and prevents myosin heads from interacting with the filament - Ca2+ binding to troponin induces conformational change in troponin complex that shifts tropomyosin, allowing myosin heads to bind the actin filaments - Locomotion - Cell Migration/Movement - Wound healing, immune function, development, cancer metastasis - Step 1: An Arp2/3-dependent mechanism extends one or more lamellipodia at the cell leading edge. - Step 2: Lamellipodia adhere to the substratum by formation of focal adhesions in which integrin mediates a connection between the actin cytoskeleton and extracellular matrix proteins such as fibronectin and collagen. - Step 3: Actin-myosin II-dependent contraction at the rear of the cell propels the bulk of the cytoplasm forward. - Step 4: Deadhesion and endocytic recycling at the back of the cell: - Microtubules - Hollow , cylindrical structure, globular proteins arranged in longitudinal rows called protofilaments - Contain 13 protofilaments - Tubulin Dimer - Composed of stably associated, highly conserved and structurally similar α-tubulin and β-tubulin monomers - α-tubulin – GTP is never hydrolyzed and nonexchangeable - Β-tubulin- GDP exchangeable with GTP which can be hydrolyzed in the site - Structurally polarized tube - Aligned end to end with same orientation - A and B slightly offset so a binds to a except at seam where a binds to b - Subunits added preferentially at + end where b tubulin is exposed - Singlet- 13 protofilaments - cytoplasm - Doublet- additional wall of 10 protofilaments forms second tubule - Cilia and flagella - Triplet- two additional 10 protofilament walls - Basal bodies and centrioles - Assembled from MTOCs - Centrosomes initiate microtubules in animal cells - It contains barrel-shaped centrioles surrounded by pericentriolar material (PCM). - Microtubules terminate in the PCM. - Dynamic Instability - Rapid microtubule polymerization alternate with periods of shrinkage - Depends on the presence or absence of a GTP-β-tubulin cap - MT with GTP-β-tubulin on the end of each protofilament - Lateral protofilament-protofilament interactions in the GTP-β-tubulin cap are sufficiently strong to prevent protofilament unpeeling at the MT end. - Catastrophe: assembly to disassembly - Rate of GTP hydrolysis is greater than gtp-tubulin addition - Rescue: disassembly to assembly - Rate of gtp-tubulin addition is greater than GTP hydrolysis - MAP Proteins - attach to the surface of microtubules to increase their stability and promote their assembly. - MAP/tau phosphorylation can regulate MT interactions - Side associations with several monomers along protofilaments stabilize MTs and dampen dynamic instability. - Kinesin Family - Conserved motor domain fused to a variety of class specific nonmotor proteins - Kinesin-2 - Two closely related but non identical heavy chains and a third cargo-binding subunit - Kinesin-5 - Four heavy chain assembled in bi-polarconfiguration - Can slide antiparallel microtubules past each other - Kinesin-13 - “Motor” domain in middle of chain has no motor activity - Destabilize microtubules ends for disassembly - Kinesin-1 - Organelle transport - Sends vesicles down axons toward + end of microtubules - Homodimer of 2 identical heavy chains - Head motor domain: MT and ATP/ADP binding sites - Flexible linker domain: connects head to coiled-coil stalk - Required for motor activity - Regulated by head-to-tail interaction - When head folds back and makes contact with tail it is inactive and no ATPase activity - When motor binds to vesicle head breaks contact with tail and ATPase activity returns so cargo can be transported to + end - Cytoplasmic Dynein - Large protein with globular,force generating head - Minus end directed microtubule motor - Requires an adapter to interact with membrane-bound cargo - Dynactin (adapter) links dynein to cargo and regulates activity - Power Stroke - Force generation mechanism - ATP-dependant change in the position of linker causes movement of microtubule-binding stalk - Cilia and Flagella - Axoneme (central core) with microtubules in a 9+2 configuration - Axoneme: central sheath, connected to the A tubules of peripheral doublets by radial spokes that prevent structural collapse during bending - Interdoublet bridge connects doublets to each other - Composed of nexin - Cilia tend to occur in large numbers on cell structure - Flagella can move differently depending on cell type (sperm vs alga) - Mechanism of Locomotion - –Swinging cross-bridges generate forces for ciliary or flagellar movement. - –The Dynein arm of an A tubule binds to a B tubule and undergoes a conformational change that slides tubules past each other. - –Sliding alternates from one side of the axoneme to another leading to bending - Intermediate Filaments - Heterogenous group of proteins divided into 5 classes - Assembly - Basic building block is a rod-like tetramer formed by two antiparallel dimers. - Both the tetramer and the IF lack polarity - IFs are less sensitive to chemical agents than other types of cytoskeletal elements - Plakin proteins can also cross-link intermediate filaments to actin filaments. -