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 -TuRC)

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 (CapZ): - 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)