Lecture 5 Cytoskeleton Regulation 2023 PDF

Cytoskeleton II: Regulation 1 Accessory Proteins • Filaments (AFs, MTs, IFs) are dynamic and under control of the cell. • Form higher-order structures (e.g. mitotic spindle). • Accessory proteins modify these cytoskeletal dynamics. 2 Nucleation by g-tubulin Protein Complex • • • • MTOC (the c...

Cytoskeleton II: Regulation 1 Accessory Proteins • Filaments (AFs, MTs, IFs) are dynamic and under control of the cell. • Form higher-order structures (e.g. mitotic spindle). • Accessory proteins modify these cytoskeletal dynamics. 2 Nucleation by g-tubulin Protein Complex • • • • MTOC (the centrosome) in animal cells. Spindle pole body in yeast. g-tubulin is highly conserved. gTuRC (ring complex) accelerates MT formation. • Nucleation occurs at the (-) end. gTuRC 3 Nucleation by g-tubulin Complex • MT initiation also occurs in cytosol • e.g. plants. • MTs nucleate daughter MTs. 4 The Centrioles and Centrosome • Centrosome contains 2 cylindrical centrioles. • 9 triplets of MTs. • Centrioles organize PCM (Pericentriolar Material). • Centriole lumen and PCM contain g-tubulin. • PCM initiates MT assembly. 2 pairs of centrioles after duplication during cell cycle. Karp. Cell and Molecular Biology 5 The Centrioles and Centrosome • Centrosome near nucleus. • (-) ends of MTs are anchored. • (+) ends of MTs emanate in astral configuration. 0.1 µm Cooper and Hausman. The Cell: A Molecular Approach 6 Nucleation of AFs • Cell periphery (cortex), where density of AF proteins is highest. • Location related to AF function (cell shape, movement). • Facilitated by ABPs (actin binding proteins) including ARPs (actin related proteins). 7 Initiation of AFs (unbranched) • Initiation by formin, an ABP • Correct alignment for polymerization • e.g. filaments of muscle cells. Initial actin monomer Formin dimer Cooper and Hausman. The Cell. 8 Initiation of AF Branches • Cell periphery • Arp2/3 produces extensive branching. • Complex contains 7 proteins. • Binds at (-) end. • 70º favourable angle. 9 Control of Subunit Pools • Both actin and tubulin are maintained in the cytosol at high concentrations. • Concentration can exceed Cc. • Accessory proteins may sequester unused subunits (sequestering proteins). • Sequestered proteins are not hydrolyzed. • Provide control or regulation of filament elongation. 10 Thymosin Sequesters Actin • Thymosin sequesters, but profilin recruits monomers. • Thymosin makes polymerization less favourable. • Profilin competes with thymosin and promotes assembly. 11 Stathmin Sequesters Tubulin • Stathmin binding prevents polymerization. • Decreases effective [tubulin]. • Promotes dynamic instability (catastrophe). 12 MT-Associated Proteins (MAPs) tau MAP2 • Several binding domains. • Length of projecting domain determines packing of MTs. • MAP2 (neuronal cell bodies); tau (axons). • tau and Alzheimer’s Disease (tau unable to bind to MTs). • Poorly soluble (hyperphosphorylated) tau may induce neurodegeneration. 13 MAP2 overexpression tau overexpression 14 AF Binding Proteins Cofilin destabilizes AFs • binds to side of proteins. • binding induces mechanical stress. • treadmilling, turnover. • cell locomotion. Tropomyosin stabilizes AFs • binds to side of proteins. • muscle contraction. 15 Modifications at AF Ends • “Capping” • Recall elongation slower at (-) end. • e.g. CapZ (+), elongation occurs only at (-) end. • e.g. tropomodulin (-) in muscle contraction. e.g. CapZ 16 Figure 16-43 Molecular Biology of the Cell (© Garland Science 2008) Modifications at MT Ends • Capping in MTs has dramatic effect on dynamic instability. • Important during mitosis. (Kinesins) 17 Cross-Linking Proteins and AFs • Formation of higher-order structures. • Spacing of 2 actin binding domains of crosslinking protein determines type of structure. • 2 major groups: bundling and gel-forming. 18 Cross-Linking Proteins and AFs Cross-linking proteins Bundling Gel-forming (network) Red = actin binding domains 19 Actin Bundling Proteins e.g. Muscle e.g. Filopodia a-actinin Fimbrin 20 Gel-forming Proteins Red blood cell membrane •Forms 2-D network. •Gives RBC membrane flexibility. Cooper and Hausman. The Cell. 21 Gel-forming Proteins Filamin e.g. lamellipodia in locomotion 22 Changes in Cell Shape During Embryonic Development • Vertebrate embryo. • Formation of neural tube during nervous system development. • Cell height – MTs • Folding into tube – AFs. 23 Changes in Cell Shape During Embryonic Development 24 Induction by Extracellular Signals • Example, crawling neutrophil. • Chemical activates WASP... • Polymerizing filaments push membrane. Extension of lamellipodium (+) (+) (-) (-) Neutrophil 25 WASP = Wiskott-Aldrich Syndrome Protein http://astro.temple.edu/~jbs/courses/204lectures/neutrophil-js.html Sequence of steps 1. 2. 3. 4. 5. 6. Extracellular signal Activation of WASP Nucleation and branching by Arp2/3 Promotion of assembly by profilin Elongation reduced by capping proteins Destabilized by cofilin and return of subunits to pool 26 Things to Consider... 1. Think about how each accessory protein affects the stability of AFs and MTs? 2. Many of the dynamic mechanisms that we discussed today do not apply to IFs. Why? 27