1. (and intermediate filaments)
Microtubules, Actin
(and intermediate filaments)
Macrophage microtubulin green
Intermediate filaments red
DNA blue

2. Mitochondria, actin

7. Cytoplasmic microtubules
Cytoplasmic mtubules
brkdwn
Mitotic spindle
Differentiated cell types have
stable mtubule structures
Cilia, flagella form from
basal bodies

8. So what are Microtubules??
… hollow, rigid tubes composed of a/b heterodimers
POLAR.. w/o polarity, it can not function.

9. Each subunit binds GTP
… but the beta
subunit has intrinsic
GTPase activity.

11. MicroTubule Organizing Centers
Microtubule networks assemble and disassemble
Assembly is initiated at MTOCs
MicroTubule Organizing Centers
(aka centrosomes)

12. MTOC 100’s of g subunits serve to nucleate mtubules, Centriole pair
(sometimes)
Similar to
Basal bodies

13. Anti a tubulin, anti g tubulin

14. Growth vs. shrinking
is related to the amt.
of free tubulin (Cc)
Growth when balanced
tipped toward >[Cc]

16. CATASTROPHIC!! Microtubule assembly and disassemble is
Occurs preferentially at the plus ends
Critical Concentration, Cc, above grows/below shrinks
Does not require GTP hydrolysis for assembly
The rapid nature of disassembly is due to its constrained
internal structure (GDP tubulin)

17. Colchicine (from crocus) binds free tubulin
Autumn Crocus
Toolbox:
Colchicine (from crocus) binds free tubulin
Taxol (from yew) stabilizes microtubules
Both cancer therapy drugs, both block mitosis.
How? If opposite modes of action?
Yew

18. Microtubules in the mitotic spindle of a mouse fibroblast in culture.  
Image by Steve Rogers, UNC Dept. Biology.  (Confocal Microscope)

20. Organelles, vesicles with
cargo move around the cell.
Why’s that?

21. How do we know?
Pulse-chase with radioactive amino acids injected into rat ganglion
Assay, slice up axon over time, SDS PAGE autoradiography

22. Microtubules are needed for trafficking vesicles (10 cm/day)
but this is dramatically faster than diffusion in the axon.
…in a differentiated cell- dynamic instability is suppressed… by capping proteins.

23. In vitro,
Extruded
Exoplasm
ATP-dependent

24. Centrosomes …… away from

25. Video microscopy
increases resolution and
visualizes movement.
This movement requires ATP.
But AMP-PNP (analog)
Competitively inhibits

26. Michael Sheetz, Columbia
Ron Vale, UCSF
… and AMP-PNP
Michael Sheetz, Columbia
17.5 kinesin.mov
Movie from Jeff Gelles lab

27. Sliding microtubule assay- used to purify kinesin.

28. Video microscopy can be used to track the movement of a
single kinesin molecule

29. One example of microtubules in action…

33. Attached ciliary dynein

34. Filaments (F actin) are another important
means by which the cytoskeleton is used for trafficking.
It is important for cell shape, location and contraction

35. Abundant: 5% of total protein, half as filaments Actin monomers
in 4 colors

36. F actin grows at BOTH ends but the plus ends grows faster.
ATP hydrolysis is not required for assembly.

37. Phalloidin binds F actin, from Death Angel
Cytochalasins and lantrunculin
bind G actin

38. Actin filaments are rarely single …
Nets and bundles.
Profilin
thymosin
filamin
gelsolin
myosin

40. Filopodium (spikes) are similarly formed

41. Actin-related proteins
promote branching
More distally

42. Racs (Cdc42 is a member) are small GTPases related to the Ras members
discussed before.
These can be activated by recptors and in turn activate actin assembly

43. Myosin is a motor that runs along (or tugs at) actin.
Actin-based movement of vesicles
Myosin-driven cell shape changes
Muscle contraction
Myosin I and myosin I

44. One head (ATPase) and one tail, tail varies

45. Dimer
300 heads here, bind actin

47. Best understood example … contraction in muscle cells.
Multinucleate (formed by cell fusions
50 mm diameter and centimeters long)

48. … just one sarcomere.

49. Plus ends

50. Each filament has ~300 heads.
Each head binds 5X/s
32 mm in 0.1 s
Attached, No ATP, “rigor”
Released, binds ATP
Cocked, hydrolysis
Loose Pi
Power stroke, loose ADP
Attached again,

51. T-tubules bring PM up close to the SR …