Cytoskeleton

• Cytoskeleton


  The internal framework of eukaryotic cells, composed of three main types of filaments: microfilaments, intermediate filaments, and microtubules
• Microfilaments


  • 5-9nm, Actin polymers
• Intermediate filaments


  • 9-10nm, 6 classes with common structure, show cell & tissue-specific expression except Lamins that form the Nuclear Lamina in all cells
• Microtubules


  • 25nm, Tubulin Polymers
• Cytoskeletal Filaments


  Assembled from Preformed Subunits
• Cells build the large filamentous structures of the cytoskeleton by the repetitive joining of smaller preformed subunits
• Scaffolds of filaments can be quickly removed and reassembled in another location
• Cytosolic pool


  A pool of subunits available at any place the cell requires filaments
• Intermediate filaments


  Made up from small fibrous units
• Microfilaments and microtubules


  Built from yet smaller globular proteins
• Actin microfilaments and microtubules exist in the cytosol as preformed subunits that can be rapidly assembled or disassembled during dynamic shape changes such as those necessary for cell migration
• The static appearance of actin filament bundles in micrographs is an illusion. In common with microtubules, microfilaments are constantly being remodelled
• Depolymerised actin or tubulin subunits re-join the cytosolic pool and are free to diffuse throughout the cell
• Small segments of polymerised filaments act as nucleation centres that rapidly speeds up fibre generation
• Nucleation


  The process where the growth of F-actin from G-actin isn't a linear process as, due to the weakness of their molecular associations, small actin oligomers are unstable and don't become stable until many monomer subunits are in association
• Generation of assembly points (nuclei) of filament elongation is slow, representing the Rate-Limiting-Step in actin elongation
• In cell-free solutions of G-actin the equilibrium between G and F actin may be pushed towards the F-form by "seeding" the solution with small actin filaments
• Actin filament nucleation and assembly sites


  Diverse but often located in the cell cortex, just internal to the plasma membrane
• Actin-related protein complex (ARP)


  Mimics the plus end of an actin filament and permits a stable point for nucleation of a filament, thus bypassing the rate limiting step in the process
• The ARP initiates actin assembly most efficiently when bound to the side of a pre-existing actin filament; the new fibre grows at an angle of 70 degrees to the original and this results in a complex inter-linked mesh
• Formins


  A different family of nucleating proteins that substitute the ARP role when cells require parallel bundles of unbranched filaments as in contractile stress fibres
• Tropomyosin and Cofilin


  Accessory proteins that bind longitudinally to actin assemblies to influence their stability
• Capping proteins


  Accessory proteins that bind to the open ends of actin assemblies to influence their stability
• Polymerization of actin requires ATP
• Polymerization occurs one monomer at a time
• During polymerization ATP becomes hydrolysed and the resulting ADP becomes trapped in the polymer
• Actin polymerization is not driven by ATP hydrolysis, but the hydrolysis weakens the bonds between the monomers making depolymerisation easier (vital for the dynamic nature of the function of the actin cytoskeleton)
• Polymerization can occur at both ends of an actin filament, but is 10 times faster at the plus end
• Gross Actin Format


  Varies according to location and purpose, mediated by different cross-linking proteins
• Focal complexes


  Structures where the actin cytoskeleton interacts with the cells external environment, containing proteins such as integrins, paxillin, vinculin and talin
• Cell migration in mammals


  Accomplished through cell crawling, dependent on a continuous and integrated series of rapid cytoskeletal rearrangements
• Protrusion


  Actin-rich structures (lamellipodia & filopodia) are pushed out at the front of the cell (leading edge), using actin polymerisation to do mechanical work
• Attachment


  The actin cytoskeleton becomes attached to the substratum via intermediate adhesion molecules
• Traction


  The bulk of the trailing cytoplasm is drawn forward, using the myosin II motor protein to do mechanical work
• Protofilament


  The subunit of each microtubule, a heterodimer of α-tubulin and β-tubulin
• A microtubule is a stiff hollow tube formed from 13 protofilaments aligned in parallel
• Nucleation, Elongation, Steady state


  The 3 phases of microtubule formation and maintenance
• In the cell microtubules grow from a specific nucleating site, in most cases the centrosome
• In a typical cell the ratio of microtubules to free tubulin is around 50:50
• The rate of microtubule polymerization is 3 times greater at the plus end than at the minus end