Cell Bio Test 4 Material-2 PDF

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. -