1. CYTOSKELETON
intermediate filaments:
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

2. Intermediate filaments are staggered arrays of coiled coil proteins

3. Distinct IFs in different cell types (Table 16-1)

4. Tubulin: monomers bind GTP; GTP-GDP in polymer (b monomer only)
13 protofilaments

5. Actin: monomer binds ATP; ATP-ADP in polymer

6. Polymerization in vitro:
- critical concentration
- time course of polymerization
- plus, minus ends
(Panel 16-2)

7. - MTs growing from a stable MT
bundle (core of a cilium): plus end
grows faster

8. Hydrolysis of GTP or ATP in polymer:
- results in treadmilling (actin filaments) or dynamic instability (MTs)

9. Treadmilling of a microtubule in vivo

10. Dynamic instability of microtubules:
catastrophe and rescue

11. Dynamic instability of MTs:

12. Dynamic instability of MTs in vivo

13. Drugs that affect actin and MTs (Table 16-2)

14. MT polymerization is nucleated by
g-tubulin ring complexes (g -TuRC)

15. MTOCs:
- animal cells: centrosome with centrioles
- plants, fungi: no centrioles, MTOCs in nuclear envelope

16. EM of centrosome

17. 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)

18. ARP 2/3 complex: nucleates actin filaments from the minus end

20. 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

21. Actin binding proteins in yeast
(genetics or biochemistry)

22. - bind G-actin

24. Bind sides of actin filament:
- tropomyosin (stabilizes)
- cofilin (destabilizes ADP-bound filaments)

25. + end capping (CapZ):
- filament grows more slowly above critical conc.
- filament shrinks more slowly below critical conc.

26. Actin cross-linking proteins - conserved actin-binding sites:
- bundle (a-actinin, fimbrin)
- cross-link at right angles (filamin)
- web-forming (spectrin)

29. Actin-severing:
- sever only: accelerates assembly of new filaments
- sever and cap + end (gelsolin): slows filament growth

30. (extracellular matrix receptor)
Attach actin filaments to plasma membrane:
- mediate cell adhesion

31. - microinjection of constitutively active forms
Actin cytoskeleton: global responses to extracellular signals depend on Rho family proteins (Ras superfamily - small monomeric G-proteins)