Ac(n cytoskeleton 12/7/11. The Cytoskeleton. Func0ons of the Cytoskeleton. B Visegrady. Elements of the Cytoskeleton

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1 The Cytoskeleton Ac(n cytoskeleton B Visegrady The cytoskeleton is an intricate network of protein filaments that extends throughout the cytoplasm. A skin cell (fibroblast) stained with Coomassie blue. Func0ons of the Cytoskeleton Structural support for the cytoplasm Causes changes in cell shape Facilitates cell division Causes cell movements Causes muscle contrac(on Controls loca(on of organelles Provides transport between organelles Elements of the Cytoskeleton Intermediate filaments Microfilaments (actin) Microtubules Ac0n Structure of ac(n Highly conserved Most abundant intracellular protein (in eukaryotes) Polarity of molecule - and + ends T form and D form G- actin vs F- actin Fig 18-4 Fig

2 Ac0n monomer, depicted above in two display modes, has subdomains 1-4. A simplified cartoon is at right. ATP binds, along with Mg ++, within a deep cle4 between subdomains 2 & 4. Ac(n can hydrolyze its bound ATP ADP + P i, releasing P i. The ac(n monomer can exchange bound ADP for ATP. The conforma0on of ac(n is different, depending on whether ATP or ADP is in the nucleo(de- binding site. G- ac0n (globular ac(n), with bound ATP, can polymerize to form F- ac0n (filamentous). F- ac0n may hydrolyze bound ATP ADP + P i & release P i. ADP release from the filament does not occur because the clet opening is blocked. ADP/ATP exchange: G- ac0n can release ADP & bind ATP, which is usually present at higher concentra(on than ADP in the cytosol. Ac(n filaments have polarity. The ac(n monomers all orient with their cle4 toward the same end of the filament, called the minus end. The diagram above is oversimplified. Ac(n monomers spiral around the axis of the filament, with a structure resembling a double helix. See diagram in Biomachina website The polarity of ac(n filaments may be visualized by decora(on with globular heads (S1) cleaved off of myosin by proteases. Bound myosin heads cause an appearance of arrowheads in electron micrographs. See images in website of the Heuser Lab. In one experiment, short ac(n filaments were decorated with myosin heads. ATer removal of excess unbound myosin, the concentra(on of G- ac(n was increased, to promote further ac(n polymeriza(on. Filament growth at one end, designated plus (+), exceeded growth at the other end, designated minus (-). In electron micrographs, bound myosin heads appear as arrowheads poin0ng toward the nega0ve end of the filament. Barbed ends orient toward the plus end. 2

3 Bundles and Networks Ac(n filaments may undergo treadmilling, in which filament length remains approximately constant, while ac(n monomers add at the (+) end and dissociate from the (-) end. This has been monitored using brief exposure to labeled ac(n monomers (pulse labeling). What is the benefit of these kinds of associations? Stability of ac0n Stability depends on environment ion and g-actin concentration Provides support through DYNAMIC arrangements including both structure and gel-like qualities of cytosol Ac0n filaments dynamics Filaments utilizes 3 steps: lag period, elongation, steady state ATP hydrolysis NOT required for polymerization ATP hydrolysis changes kinetics of polymerization + vs - end? Illustration of treadmilling CBI 25.2 Other cell func(ons for ac(n Ac(n is the most common protein in the cytoplasm. Ac(n polymeriza(on requires MgATP. Ac(n filaments are called microfilaments. There are over 60 ac(n binding proteins. 12/7/11 The Cytoskeleton 18 3

4 Func0ons of Ac0n Filaments Cell Crawling Ac(n filaments are concentrated beneath the plasma membrane (cell cortex) and give the cell mechanical strength. Assembly of ac(n filaments can determine cell shape and cause cell movement. Associa(on of ac(n filaments with myosin can form contrac(le structures. Growth of Filopodia Focal Adhesions Integrins anchor the outside cell surface to the extra cellular matrix by binding to proteins like fibronec(n and collagen. Fibronec(n > molecular flypaper The ac(n cytoskeleton and adhesion sites in a migra(ng cell Figure 1. a A frame selected from a video sequence of a goldfish fibroblast that is expressing green fluorescent protein (GFP)- tagged ac(n (green) and that was microinjected with rhodamine- tagged vinculin (red). Vinculin localizes to adhesion complexes. b An enlargement of the boxed area I (in a), which shows a region of the lamellipodium in the GFP channel (ac(n). c The boxed area I in the rhodamine channel (vinculin), which shows early adhesion complexes (arrow). d An enlargement of the boxed area II in a, which shows a region of the ac(n network that is behind the lamellipodium. Images courtesy of O. Krylyshkina, Austrian Academy of Sciences, Austria. 4

5 In vitro mo(lity assay Specle microscopy This assay represents a simplified model of muscle force and mo(on genera(on (Warshaw et al., 1990; Warshaw, 1996). In this assay, myosin, a molecular motor, is adhered to a nitrocellulose- coated microscope coverslip. The coverslip is one surface of a 20 µl experimental microchamber. Fluorescently labeled ac(n filaments in a solu(on containing MgATP are perfused into the chamber and then the movement of single ac(n filaments over the myosin surface is visualized in an epifluorescence microscope equipped with an image intensified video camera. Ac(n filament veloci(es can be determined by computer (Work and Warshaw, 1992). The movie is a split screen image of ac(n filaments moving over a surface of smooth (let) and skeletal (right) muscle myosin.. FSM images of ac(n (C) and microtubules (B) in the lamellae of living epithelial cells. A comparison of conven0onal image of fluorescent microtubules (A), labeled with X- rho- damine tubulin, Serum- response factor signalling Ac(va(on of SRF gene Cells expressing mutant ac(n show impaired subcellular MAL distribu(on and SRF ac(va(on When SRF signalling was ac(vated by serum s(mula(on, cells showed increased localiza(on of MAL in the nucleus and enhanced transcrip(on of SRF reporter genes 5