1. Cytoskeleton and Cell Motility
Nancy Freitag

2. Objectives
What regulates the shape and assembly of the cell cytoskeleton?
How do pathogens exploit host cell actin assembly?

3. Overview The cytoskeleton and cell motility
The dynamics of actin assembly
The cell cytoskeleton as a target for pathogens
Actin elongation-based propulsion: bacterial motility and cell movement (PAPER)

4. The actin cytoskeleton and cell motility
Cytoplasmic system of fibers crucial to cell motility
Plays a structural role
Undergoes rearrangement which can produce movement

6. Actin provides framework & determines cell shape

7. Filaments are organized into bundles & networks held together by cross-linking proteins

8. Actin filaments
give shape to
microvilli

9. Cell locomotion
Cell moves forward by extending filipodia & lamellipodia
Focal adhesions are formed
Cell is pulled forward

10. Actin monomers and filaments
Actin is the most abundant intracellular protein. Highly conserved.
G-actin = actin monomer.
F-actin = filamentous polymer.
Each actin monomer contains Mg2+ complexed with either ATP or ADP

11. G-actin has two lobes separated by a deep cleft where ATP binds.

12. G-actin can assemble into F-actin in vitro under the right ionic conditions; no other proteins are required to produce filaments.

13. Actin filaments in solution

15. Dynamics of actin assembly in vitro: a brief overview
Lag phase: G-actin aggregates into short, unstable oligomers.
An oligomer of 3 or 4 subunits acts as a nucleus for further polymerization

16. Elongation: addition of monomer
to both ends
Steady state: G-actin monomers
exchange with subunits at both
ends w/no change in total mass

18. Each actin monomer is bound to a molecule
of ATP. Following addition of monomer,
ATP is hydrolyzed to ADP.

20. Critical concentration (Cc)
The equilibrium concentration of a pool of unassembled actin
The measure of the ability of a solution of G-actin to polymerize
Above Cc a solution of actin will polymerize
Below Cc F-actin will depolymerize

21. Cc

22. Actin filaments grow faster at one end than at the other
The barbed end, or (+) end, elongates
5 to 10 times faster than the
pointed, or (-) end.
The difference in elongation reflects the
difference in Cc values at the two ends.

24. Actin filaments grow faster at one end than at the other
Below Cc (+) end: no filament growth occurs
Between Cc (+) and Cc (-): growth occurs at the (+) end (treadmilling)
Above Cc (-): growth occurs at both ends

25. Treadmilling

26. Actin polymerization is regulated by proteins that bind G-actin
Cc for a cell is ~ 0.2 uM.
Concentration of G-actin is 50 uM to 200 uM.
Pool of G-actin is maintained by proteins that sequester G-actin

27. Examples of proteins that sequester G-actin
Thymosin b4: sequesters free ATP-G-actin. Acts as a buffer.
Profilin: sequesters actin, and promotes the exchange of ATP for ADP-G-actin.

28. Actin filament length is controlled by proteins that cap or sever filaments

29. Actin filament length is controlled by proteins that cap or sever filaments
Gelsolin and cofilin: break actin network into shorter fragments.
Alter conformation of actin subunit, causing breakage, & then remain bound.
Bound protein prevents addition of new monomers, an activity called capping.

32. Assays for actin polymerization
Pyrene actin assays: spectrofluorometric assay. Fluorescently tagged actin gives a wavelength-specific signal when polymerized.
Cytoplasmic extracts: can add or deplete factors.

34. Pathogen-mediated cytoskeletal rearrangements
Prevention of uptake: inhibition of phagocytosis or pedestal formation
Invasion: induced uptake
Actin-based motility: intracellular motility and intercellular spread

36. Pathogens that exploit actin-based intracellular motility
Listeria monocytogenes
Shigella flexneri
Mycobacteria
Burkholderia
Rickettsia
Vaccinia virus

37. L. monocytogenes as a tool for defining actin assembly
From Tilney and Portnoy, J. Cell Biology 1989

39. What is required for actin-based motility within the cytosol?
Immunofluorescence studies indicated the presence of a variety of proteins associated with actin tails…
But which ones are required for movement? Which ones are simply binding actin?

40. Identification of ActA
Search for bacterial mutants unable
to spread within cells led to identification
of the actA gene product
wild type
actA mutant

41. ActA and a host protein complex, Arp 2/3,
were found to co-localize at the base
of L. monocytogenes actin tails within
the cytosol

42. Domains of ActA SP = signal peptide WH2 & Arp 2/3 = bind Arp 2/3
AB region = monomeric actin
binding

43. ActA + Arp 2/3 function as a
highly efficient nucleation site
actin,
actin + ActA
Arp 2/3
Arp 2/3 + ActA
ActA

44. Arp 2/3 complex Complex of 7 polypeptides Present in all eukaryotes
Only known factor that stimulates nucleation of actin at barbed ends
Can bind to the sides of filaments and stimulate polymerization
Requires activation

46. Wiskott-Aldrich protein family (WASP)
Activate Arp 2/3
Contain WH2 domains, acidic domains, and proline rich regions
WASP, N-WASP, Scar

53. Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 comple
J. Cell Biology 165:
2004

54. Figure 1

55. Figure 2

56. Figure 3

57. Figure 4

58. Figure 5

59. Figure 6

60. Figure 7

61. Figure 8

62. Figure 9

63. Figure 10

64. Fig. 5. The transition from asters to stars
Haviv, Lior et al. (2006) Proc. Natl. Acad. Sci. USA 103,
Copyright ©2006 by the National Academy of Sciences

65. Additional references
Actin-based motility of intracelllular microbial pathogens. Micro Mol Biol Rev. (2001) 65:
Interaction of human Arp 2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science (1998) 281: