1. Lecture 3 Actin and myosin in non-muscle cells; Cell motility Outline:
Actin polymerization in vitro
Regulation of actin dynamics in cells
Actin organization
Cell motility
Paper: Self-polarization and directional motility
of the cytoplasm

2. Actin highly conserved 375 aa, 43 kD protein
the most abundant protein in non-muscle cells  1-5%
roles: cell shape, polarization, locomotion, division; vesicle traffic
monomer = G-actin
polymer = F-actin, microfilaments
inhibitors:
latrunculin, cytochalasin; phalloidin

3. platelet dynamics resting activated retraction
Dramatic morphological changes result from
reorganization of actin cross-linked to plasma membrane

5. Actin Structure
pointed
two-stranded helix
barbed

6. Actin highly conserved, binding proteins are not

7. Actin polymerization dynamics in vitro
Assays to measure:
1) viscometry
2) sedimentation
3) fluorescence spectroscopy-
pyrene actin assembly assay

8. Pyrene actin assembly assay
couple to C-374 of actin
*[free actin] =Cc=
0.1 mM
elongation
*steady state
nucleation
fluorescence 
polymer
time
Mg++, KCl

9. - + steady state - treadmilling of subunits D D D T D D T T D T D
D-Pi T D D
D-Pi T T D T D
Cc(- end) = 0.8 mM > Cc(+ end) = 0.1 mM
filament turnover rate: t1/2 = 30 min
rate limiting step = dissociation of ADP actin from minus end

10. Actin dynamics in vivo Parameters: Model systems:
1. Spatial and temporal control of polymerization/depolymerization
2. Turnover
3. Movement of actin filaments - myosins
Model systems:
1. Fibroblast
2. Keratocyte - epithelial cell
3. Listeria monocytogenes - intracellular bacterial pathogen

11. Listeria
monocytogenes

12. Spatial Control microinject fluorescently-labeled actin
t= 1 min
t= 5 min
polymerization occurs at leading edge of keratocyte,
rear surface of Listeria

13. Filament Turnover microinject “caged” fluorescently-labeled actin
illuminate in specific location with UV light to release caging group
resorufin
caging
group
resorufin

14. Measure rate of fluorescence decay = actin turnover rate t1/2 = 30 sec
actin stays in
same place
as cell moves
forward
t = 30 sec
t = 1 min
Measure rate of fluorescence decay = actin turnover rate
t1/2 = 30 sec

15. in vitro in vivo t1/2 = 30 min Actin Cc = 0.1 mM [Actin]= 500 mM
Actin dynamics in vivo are controlled by actin binding proteins

16. - + 3 3. elongation 4 4. depolymerization D D D T D D D T 2
D-Pi T D D D
D-Pi T 2
2. nucleation D T 1
1. monomer pool

17. Regulation of the monomer pool
Thymosin b4
M.W. 5000
binds 1:1 - enough to buffer all the actin
sequesters actin from polymerizing
localization - diffuse
Profilin
M.W. 14,000
binds 1:1 - can buffer 20% of actin
promotes nucleototide exchange and polymerization
binds PIP2 and proline-rich sequences
localization - diffuse and leading edge, Listeria surface

20. Nucleation Arp2/3 complex
7 subunits, include actin-related proteins 2 and 3
promotes actin polymerization at listeria surface
accelerates actin polymerization in pyrene actin assembly assay
(with activator, eliminates lag phase)
binds (-) ends and filament sides - branching function
localization - lamellipodia
Activators:
Listeria: Act A
cells: WASP family proteins

22. immuno-
EM of
Arp2/3
at actin
branch
points
in leading
edge

23. Elongation Profilin Capping factors: promotes (+) end growth
CapZ (Capping protein) - (+) end
tropomodulin - (-) end
gelsolin - (+) end
can stabilize or destabilize filaments,
prevent elongation

24. Depolymerization gelsolin ADF/cofilin M.W. 87,000
Ca++-dependent severing
ADF/cofilin
M.W. 19,000
binds G- and F-actin
accelerates (-) end
depolymerization 25-fold
+ ADF

25. what controls depolymerization in Listeria tails?
Question:
what controls depolymerization in Listeria tails?
Listeria + cytoplasmic egg extract  motility in vitro
immunodeplete gelsolin or ADF/cofilin and observe effects
control
gelsolin
ADF/Cofilin
Rosenblatt et al., 1997

26. Important Breakthrough: Reconstitution of Listeria motility from
purified components
required:
Actin and ATP
Arp2/3 complex
ADF/cofilin
Capping protein
stimulators:
VASP -binds ActA, actin, profilin
Profilin
a-actinin

27. - + depolymerization: end availability: ADF/cofilin CapZ, gelsolin D T
D-Pi T +
monomer regulation:
thymosin b4, profilin
nucleation:
Arp2/3
complex

28. Organization of actin filaments
Myosins
Cross-linking proteins
Membrane attachments
Assemblies: cell cortex, stress fibers, contractile ring,
cell protrusions, microvilli

29. stationary cell - stress fibers

31. Dividing
cell

32. Dictyostelium amoeba

33. locomoting cell - filopodia and lamellipodia

35. Cell Motility Swimming Crawling purposes:
Microtubule-based – cilia, flagella
Crawling
Actin-based
purposes:
1) wound healing - epidermal cells
2) immune response - leukocytes –migrate to sites of infection
3) development – neural crest cells; neuronal process extension
4) cancer cell metastasis –malignancy determinant

36. 4 processes coordinated:
protrusion
anchorage
forward movement
tail retraction

37. protrusion Proposed mechanisms:
Actin polymerization at leading edge - local force
Proposed mechanisms:
1) “thermal rachet” - actin polymerization pushes
2) myosin I - movement of actin filaments

38. 1) thermal rachet - membrane fluctuations
2) myosin I - dependent
myosin I could also transport assembly factors to membrane

40. anchorage Adhesion plaques: connect cell to substratum
prevent leading lamella from retracting

41. forward movement Proposed mechanisms: Observations:
Actin networks stationary with respect to substratum
Cell body and nucleus rotates
myosin II required
Proposed mechanisms:
1) sarcomere-like contractions in rear
2) transport along actin arrays

42. crawling
Dictyostelium
amoeba

43. tail retraction
passive - cell snaps loose from adhesion plaques

44. How is cell polarity established?
cell loaded with Ca++ -sensitive dye Fura-2