MCB 2210 001 4 1 2015 Cytoskeletal Functions During Interphase Actin o Membrane protrusion lamellipodia and filopodia Dynamic cell surface extensions containing actin Capable of migration Fibroblast cell of the connective tissue The leading edge regularly extends thin sheet like structures called lamellipodia and thin needle like projections or spikes called filopodia After protruding forward they adhere to the substrate or sweep over the top of a cell as a ruffle Also contain long cables of actin called stress fibers that function in cell adhesion and contraction Actin actively polymerizes near the leading edge The behavior of actin filaments in lamellipodia and filopodia can be traced by microinjecting the cells with fluorescently labeled actin Immediately after injection fluorescent actin is incorporated into filaments at the extreme leading edge o Indicates that actin polymerization takes place here Actin assembly drives membrane protrusion Because actin polymerization at the leading edge is coupled to membrane protrusion the assembly of actin filaments in the network abutting the plasma membrane generates the force that pushes the membrane forward o The energy that drives this process comes from hydrolysis of ATP by actin After actin filaments are nucleated at the leading edge of a fibroblast o The entire network of filaments fluxes backward towards the interior of the cell Called retrograde flow Depolymerization of filaments begins so retrograde flow is coupled to depolymerization Cofilin thought the be major actin depolymerizing activity behind leading edge Actin filaments are oriented with their barbed ends facing the membrane in the direction of protrusion Lamellipodia filaments are organized as a branched network Filiopodia organized into parallel bubbles o Cell Adhesion stress fibers and focal adhesions Stress Fibers long bundles of actin filaments that lie along the lower surface of the cell Ends terminate at integrin containing focal adhesions attach the cell to the underlying substrates Contract in response to phosphorylation of non muscle myosin II light chain by MLCK o Endocytosis and Trafficking clathrin pits and endosomes Both are controlled by the actin cytoskeleton Branched actin assembly facilitates endocytotic internalization o Cytokinesis o Nucleation factors generate actin structures Microtubules o Directs many processes associated with the secretory pathway Organelle positioning ER and Golgi and membrane transport Anterograde Transport ER Golgi Plasma Membrane Retrograde Transport Endosome Golgi o ER membranes are dynamic and connected to MTs o Golgi Positioning depends on MT s and their motor proteins MT s help position the Golgi and the ER The Golgi is positioned near the centrosome in most cells and when MTs are disrupted by drugs or disassembled during mitosis the Golgi is fragmented The ER consists of a network of tubules that are organized along MTs and branched throughout the cytoplasm When MTs are disrupted the ER collapses When MTs repolymerize ER reforms normal structure MTs also control localization of mitochondria o Mitosis Rho family GTPase signaling o Rho family GTPases Rho Rac Cdc42 etc members of a family of GTP binding and hydrolyzing proteins called the Ras superfamily o Active bound to GTP Can interact with molecules that carry out downstream cellular functions o Inactive bound to GDP o GEFs mediate conversion of inactive GDP bound to active GTP bound forms Catalyze exchange of bound GDP for free GTP o GAPs catalyze the conversion of the active GTP bound form to the inactive GDP bound form o GDIs stabilize the inactive GDP bound form WASP proteins and Formins integrate signals from Rho GTPases to generate lamellipodia filopodia and stress fibers o WASP proteins activated by small GTPases Cdc42 or Rac Assemble actin in membrane protrusions o WASP inactive autoinhibited form in the absence of activated Cdc42 GTP o When Cdc42 GTP binds to the GBD of WASP relieves autoinhibition The WASP then interacts with the Arp2 3 complex and actin causes nucleation of a new filaments Similar with formins o Rho Rac and Cdc42 are crucial for signaling cascades that activate the WASPs and or Formins to drive assembly They regulate the formation of different F actin structures in cells Microinjection experiments using activated forms of Rho Rac and Cdc42 that are trapped in the GTP bound state have shown that each of these proteins controls the formation of a different actin containing structure in cells Microinjection of o Rac GTP causes formation of lamellipodia and membrane ruffles o Cdc42 GTP causes formation of filopodia o Rho GTP causes formation of stress fibers
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