Lecture 3
description
Transcript of Lecture 3
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Lecture 3
Actin and myosin in non-muscle cells; Cell motility
Outline:Actin polymerization in vitroRegulation of actin dynamics in cellsActin organizationCell motility
Paper: Self-polarization and directional motilityof the cytoplasm
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Actin
roles: cell shape, polarization, locomotion, division; vesicle traffic
highly conserved 375 aa, 43 kD protein
the most abundant protein in non-muscle cells 1-5%
monomer = G-actin
polymer = F-actin, microfilaments
inhibitors:latrunculin, cytochalasin; phalloidin
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platelet dynamics
resting activated retraction
Dramatic morphological changes result from reorganization of actin cross-linked to plasma membrane
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Actin Structure
barbed
pointed
two-stranded helix
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Actin highly conserved, binding proteins are not
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Assays to measure:
1) viscometry
2) sedimentation
3) fluorescence spectroscopy-pyrene actin assembly assay
Actin polymerization dynamics in vitro
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pyrene couple to C-374 of actin
Mg++, KCl
elongation
*steady state
nucleation
fluorescence
polymer
time
Pyrene actin assembly assay
*[free actin] =Cc=0.1 M
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-
Cc(- end) = 0.8 M > Cc(+ end) = 0.1 M
D D D D-Pi
D D D-Pi T
T
+
steady state - treadmilling of subunits
T D
D
T
filament turnover rate: t1/2 = 30 min
rate limiting step = dissociation of ADP actin from minus end
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Actin dynamics in vivo
2. Keratocyte - epithelial cell
3. Listeria monocytogenes - intracellular bacterial pathogen
1. Spatial and temporal control of polymerization/depolymerization
2. Turnover
3. Movement of actin filaments - myosins
Parameters:
Model systems:1. Fibroblast
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Listeriamonocytogenes
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Spatial Controlmicroinject fluorescently-labeled actin
t= 5 min
t= 1 min
polymerization occurs at leading edge of keratocyte, rear surface of Listeria
t= 0
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Filament Turnover
microinject “caged” fluorescently-labeled actin
illuminate in specific location with UV light to release caging group
caginggroup resorufin resorufin
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Measure rate of fluorescence decay = actin turnover ratet1/2 = 30 sec
t = 30 sec
t = 1 min
t = 0
actin stays insame placeas cell movesforward
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[Actin]= 500 M
in vivo
t1/2 = 0.5 min
in vitro
t1/2 = 30 min
Actin Cc = 0.1 M
Actin dynamics in vivo are controlled by actin binding proteins
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- D D D D-Pi
D D D-Pi T
T
+
T D
D
1
1. monomer pool
2
2. nucleation
3
3. elongation
4
4. depolymerization
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Regulation of the monomer pool
Thymosin 4M.W. 5000binds 1:1 - enough to buffer all the actinsequesters actin from polymerizinglocalization - diffuse
ProfilinM.W. 14,000binds 1:1 - can buffer 20% of actinpromotes nucleototide exchange and polymerizationbinds PIP2 and proline-rich sequences localization - diffuse and leading edge, Listeria surface
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Nucleation
Arp2/3 complex7 subunits, include actin-related proteins 2 and 3promotes actin polymerization at listeria surfaceaccelerates actin polymerization in pyrene actin assembly assay
(with activator, eliminates lag phase)binds (-) ends and filament sides - branching functionlocalization - lamellipodia
Activators:Listeria: Act Acells: WASP family proteins
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immuno-EM ofArp2/3at actinbranchpoints in leadingedge
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Elongation
Capping factors:CapZ (Capping protein) - (+) endtropomodulin - (-) endgelsolin - (+) end
• can stabilize or destabilize filaments,• prevent elongation
Profilinpromotes (+) end growth
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Depolymerization
gelsolinM.W. 87,000Ca++-dependent severing
ADF/cofilinM.W. 19,000binds G- and F-actinaccelerates (-) end depolymerization 25-fold
+ ADF
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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
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Important Breakthrough:• Reconstitution of Listeria motility frompurified components
required:• Actin and ATP• Arp2/3 complex• ADF/cofilin• Capping protein
stimulators:• VASP -binds ActA, actin, profilin• Profilin• -actinin
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- D D D D-Pi
D D D-Pi T
T
+
T D
D
end availability:CapZ, gelsolin
nucleation:Arp2/3complex
monomer regulation:thymosin 4, profilin
depolymerization:ADF/cofilin
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Organization of actin filaments
Cross-linking proteins
Membrane attachments
Myosins
Assemblies: cell cortex, stress fibers, contractile ring,cell protrusions, microvilli
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stationary cell - stress fibers
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Dividingcell
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Dictyostelium amoeba
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locomoting cell - filopodia and lamellipodia
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Cell Motility
SwimmingMicrotubule-based – cilia, flagella
CrawlingActin-based
purposes: 1) wound healing - epidermal cells2) immune response - leukocytes –migrate to sites of infection3) development – neural crest cells; neuronal process extension4) cancer cell metastasis –malignancy determinant
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protrusion
anchorage
forward movement
tail retraction
4 processes coordinated:
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protrusion
Actin polymerization at leading edge - local force
Proposed mechanisms:
1) “thermal rachet” - actin polymerization pushes
2) myosin I - movement of actin filaments
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2) myosin I - dependent
myosin I could also transport assembly factors to membrane
1) thermal rachet - membrane fluctuations
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anchorage
Adhesion plaques:
connect cell to substratumprevent leading lamella from retracting
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forward movement
Observations:Actin networks stationary with respect to substratumCell body and nucleus rotatesmyosin II required
Proposed mechanisms:
1) sarcomere-like contractions in rear2) transport along actin arrays
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crawlingDictyosteliumamoeba
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tail retraction
passive - cell snaps loose from adhesion plaques
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How is cell polarity established?
cell loaded with Ca++ -sensitive dye Fura-2