CYTOSKELETON intermediate filaments 8 12 nm diameter fibers
CYTOSKELETON intermediate filaments: - 8 -12 nm diameter fibers - rope-like assemblies of IF proteins - provide mechanical strength microtubules (axonemal, cytoplasmic): - 25 nm diameter cylinders - polymers of a, b-tubulin - organize organelles, direct intracellular transport actin filaments (microfilaments): - 5 -9 nm diameter filaments (F-actin) - polymers of actin (G-actin) - concentrated in cell cortex - determine shape of cell surface, whole cell motility accessory proteins methods
Intermediate filaments are staggered arrays of coiled coil proteins
Distinct IFs in different cell types (Table 16 -1)
Tubulin: monomers bind GTP; GTP-GDP in polymer (b monomer only) 13 protofilaments
Actin: monomer binds ATP; ATP-ADP in polymer
Polymerization in vitro: - critical concentration - time course of polymerization - plus, minus ends (Panel 16 -2)
- MTs growing from a stable MT bundle (core of a cilium): plus end grows faster
Hydrolysis of GTP or ATP in polymer: - results in treadmilling (actin filaments) or dynamic instability (MTs)
Treadmilling of a microtubule in vivo
Dynamic instability of microtubules: catastrophe and rescue
Dynamic instability of MTs:
Dynamic instability of MTs in vivo
Drugs that affect actin and MTs (Table 16 -2)
MT polymerization is nucleated by g-tubulin ring complexes (g -Tu. RC)
MTOCs: - animal cells: centrosome with centrioles - plants, fungi: no centrioles, MTOCs in nuclear envelope
EM of centrosome
Actin filaments assemble at the cortex (nucleated at the plasma membrane): a) all actin filaments - most formed before cells were permeabilized (fluor-phalloidin) b) newly formed filaments - 5 min. after labeling (rhod-actin monomers)
ARP 2/3 complex: nucleates actin filaments from the minus end
Actin-binding proteins organize actin filaments in cells: - nucleate - bind G-actin (sequester or promote monomer addition) - bind filament sides (stabilize or destabilize) - cap (+ or - ends) - sever - bundle (loose or tight; parallel or anti-parallel) - crosslink - branch - link to membranes (direct or via membrane proteins - regulate MT-binding proteins
Actin binding proteins in yeast (genetics or biochemistry)
- bind G-actin
Bind sides of actin filament: - tropomyosin (stabilizes) - cofilin (destabilizes ADP-bound filaments)
+ end capping (Cap. Z): - filament grows more slowly above critical conc. - filament shrinks more slowly below critical conc.
Actin cross-linking proteins - conserved actin-binding sites: - bundle (a-actinin, fimbrin) - cross-link at right angles (filamin) - web-forming (spectrin)
Actin-severing: - sever only: accelerates assembly of new filaments - sever and cap + end (gelsolin): slows filament growth
(extracellular matrix receptor) Attach actin filaments to plasma membrane: - mediate cell adhesion
- microinjection of constitutively active forms Actin cytoskeleton: global responses to extracellular signals depend on Rho family proteins (Ras superfamily - small monomeric G-proteins)
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