Actin Non muscle Motility Intermediate Filaments I Ameboid Motility Locomotion of Cells a Most cells are capable of locomotion b Normal cell motility outgrowth phagocytosis c Pathological cell motility i Metastasis d Extension of one or more lamellipodia i Embryonic development wound healing tissue remodeling neuron i Assembly of coherent meshwork of actin filaments e Adhesion Some lamellipodia adhere to substratum through focal contacts i Coupling to external substrate ii Clutch may be efficient in rapidly moving cells or slippery iii Rearward flow faster than forward cell movement in slow cells f Translocation cytoplasm flows forward Bulk of i Controlled disassembly of actin meshwork and filaments for recycling testube 1 Actin filament half life in lamellipodia 100x s shorter than g De adhesion Tail detaches and retracts into cell body i Contraction of stress fibers myosin II h Locomoting cells i Actin network enriched at leading edge lamellipodium pushed membrane forward ii Myosin II contractile bundles enriched in tail pull tail forward II Movements driven by actin polymerization assembly Listeria a Listeria also Shigella and vaccinia virus escape phagosome endosome into cytoplasm b Propel themselves through cell cytoplasm by stimulating polymerization of an actin comet tail Exp Listeria moving in the cytoplasm Xenopus egg extract c i Listeria organize a comet tail of actin filaments that push the bacteria through Xenopus egg extract d Bacterial membrane protein Act A recruits cytosolic proteins that nucleate actin filament formation at base of bacterium to push it forward e Exp Act A expressed in E coli or on beads stimulates movement by organizing a comet tail of short actin filaments i Act A is like cell WASP f Force production at cell leading edge is similar to Listeria motility g Cdc42 Rho family G protein activated by GEF h Cdc42 GTP activates WASp intramolecular inhibition until activated by binding RBD i WASp W domain binds actin monomer j WASp A domain binds activates Arp2 3 complex i Arp 2 3 complex contains Arps actin related proteins 2 and 3 and 5 other components ii Activated by Nucleation Promoting Factor e g WASP WAVE VASP k Activated Arp 2 3 complex binds to side of one actin filament and end of second actin filament i Bound actin nucleates actin filament growth at branch point ii 70o angle l Arp 2 3 complex i Actin filament end capping protein ii Nucleates growth of actin filament addition of monomers at end iii Stabilizes dendritic branches of interaction b w short actin filaments at 70o angle m End of actin filaments growing on Arp 2 3 branch become capped by capping protein i Generates short filaments n Continual re initiation by Arp2 3 of new filaments and elongation of filaments pushes bacterium forward o Cofilin binds cooperatively to side of ADP actin filaments i Twists helix ii Breaks filaments more ends and destabilizes ends iii Depolymerization p Actin monomer reactivated by GEF activity of profilin i Diffuses to leading edge ii Adds new filaments III Lamellipodium motility a WASP and profilin stimulate polymerization of actin filaments near specific sites on membrane b Arp2 3 forms branched network of actin filaments c Branched network stabilized by filamin actinin crosslinking IV Elastic Brownian Ratchet Model a Actin filament like a flexible wire b Brownian movement bends flexes filament end enough for actin monomer addition c Flex back to straight position pushes membrane forward d 70o angle angel of Arp2 3 branches is crucial for ensuring flex opens enough space between filament end and membrane V Extracellular signals stimulate formation of different types of actin structures a Communication of extracellular signals to cytoskeleton is mediated by monomeric G proteins Cdc42 Rac and Rho which cross talk b Asymmetric signaling yields directional migration i Cdc42 activation at leading edge stimulates filopodia formation and activation of Rac ii Rac activates Rho activation in trailing region of cell iii Rho inhibits Rac activation in trailing region Microtubule Structure Dynamics and Motility I Cytoskeleton Functions a Structure i Support organize cytoplasm or parts of cell 1 Microvilli cilia flagella b Motility i Cell as a whole 1 Swimming sperm ameboid motility ii Material in cells 1 Mitosis axonal transport II Tubulin a Highly conserved protein across all eukaryotic species i Related protein in bacteria FtZ tubulin GTP permanently b Subunit dimer c The bound i GTP or GDP bound d The subunit GTPase activity III Microtubule Structure Intrinsic polarity a b Microtubule open 25 nm diameter tube c Mictrotubule wall linear protofilaments of dimers i All aligned with same polarity d Protofilament arrangements in walls of MTs cross section i Cytoplasmic MTs 13 protofilaments ii Cilia flagella doublet MTs 13 10 protofilament walls iii Centriole basal body MTOCs triplet MTs 13 10 10 IV Microtubule Polymerization Assembly a Initiation i Tubulin dimers tubulin GTP associate and dissociate at each end ii Tubulin dimer in wall of MT subunit hydrolyzes GTP to GDP Pi b Critical concentration for MT polymerization i MTs like MFs have critical concentration for polymerization 1 Below Cc MTs do not form 2 Above Cc all excess tubulin assembles into MTs c Experiment MT and ends for polymerization i Unpolymerized tubulin added to flagellar axoneme seed which contains uniformly polarized MTs ii Result polymerization greater at end with tubulin subunit exposed end for polymerization d Preferential addition and loss of tubulin from end of MT ii i End is more active for both polymerization and depolymerization In cells ends of MTs are associated with Microtubule Organizing Centers MTOCs e Centriole at center of MTOC in cells i One end of MT associated with MTOC centriole in cell f MT polarity orientation in cells i of MT oriented toward MTOC in cell body except in dendrities 1 Dendrites have mixed polarity g Drugs that affect MT polymerization i Colchicine vinca plants 1 Blocks end dimer addition 2 Depolymerizes MTs in cells 3 Blocks mitosis ii Nocadozole 1 Blocks end of MTs 2 Blocks mitosis of fungi 3 Athletes foot medicine iii Taxol Pacific yew trees 1 Blocks loss of tubulin from MT 2 Polymerizes all tubulin in cell irreversible 3 Blocks mitosis V Microtubule dynamics in cells a Stability of MTs in cells i Dynamic MTs T minutes in cytoplasm and mitotic spindle ii Stable MTs T 100 days in axons of neurons and cilia flagella b Dynamic instability i ii Individual MTs