MCB130.Midterm2.review - Midterm2 Review MCB130 Spring'08...

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Midterm2 Review MCB130 Spring ’08 Drubin 1 1. Visualizing the cytoskeleton: a. Cytoskeletal filaments are dynamic structures that continuously undergo rapid assembly/disassembly cycles. b. Imaging techniques have always been important for studies of the cytoskeleton because its organization is intimately tied to function. c. Contrast and resolution must be optimized for image analysis. d. Electron microscopy provides spatial resolution of ~2nm but can only be used on fixed (dead) cells. e. Resolution for light microscopy is limited to ~200nm. Higher numerical aperture lenses provide better resolution, as does shorter wavelength light. f. Contrast is the main limiting factor for visualization of biological specimens by light microscopy. Dyes, fluorescent probes, DIC and Nomarski optics all help to create/enhance contrast in biological samples. g. For fluorescence microscopy, the emission wavelength is always longer than the excitation wavelength since energy is lost when a flurophor is excited by incident light and then emits a photon. h. Immunofluorescence provides a powerful approach for visualization of specific molecules. i. GFP fusions allow real-time analysis of protein localization in living cells. 2. Actin filaments – Structural and Dynamic Properties a. Actin filaments are involved in cell motility, cell adhesion, cell contraction and intracellular transport. b. Actin is 42 kDa protein that binds and hydrolyzes ATP. c. Salt will promote actin assembly into polarized filaments (+ and – ends), stimulating ATP hydrolysis. d. + (barbed) ends tend to be oriented toward membranes in cells e. Assembly normally involves a lag phase because formation of an assembly nucleus or ‘seed’ composed of three actin monomers is rate limiting. Addition of a nucleus (short actin filament or nucleating protein(s)) eliminates the lag. f. Cytochalasin, latrunculin and phalloidin affect filament assembly in different ways g. Thanks to ATP hydrolysis, the + and – filament ends have different assembly properties (in contrast to an “equilibrium filament”. This is because the assembly and disassembly reactions are not the same. ATP actin assembles and ADP actin disassembles. Plus ends (0.1 μ M) and – ends (0.8 μ M) have different critical concentrations. The net critical concentration for the filament is ~0.2 μ M actin. Unassembled actin will reach this concentration at steady state and subunits will treadmill through filaments.
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Midterm2 Review MCB130 Spring ’08 Drubin 2 h. The Arp2/3 complex nucleates assembly of new actin filaments. This complex contains two actin-related proteins (Arp2 and Arp3). The complex remains bound at the filament – end with a free + end growing away from the complex. The Arp2/3 complex also binds to sides of actin filaments, resulting in assembly of branched filament arrays. Importantly, the Arp2/3 complex requires activation. Both WASP and the sides of pre- existing actin filaments help to activate the Arp2/3 complex. WASP in turn is activated by Cdc42 GTPase.
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