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7/25/2019 Bilder.2 Actin
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Today:
Cytoskeleton filament systemsActin polymerization dynamicsActin polymerization and force generation
Myosin motors
Signals that regulate actin
ECB Chap. 17
Last time:Cellular scales and microscopy techniquesLipid bilayers (membranes) as organizers of
cellular compartments
how molecular properties of a single protein, along with its family of regulators,
enable the formation of elaborate and dynamic structures that underlie many of
the cells functions.
Cells have three major cytoskeletal systems:
1. actin microfilaments2. microtubules
3. intermediate filaments
}
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cell shape, movement, adhesion,division, intracellular transport
shape, adhesion
From Lodish, Molecular Cell Biology 6e
Dynamic More static
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What does
actin do?
Cell shapeand
structure,mechanical
strength
Cell migration:Converts ATP into motion
Cell adhesion,cortical tension
Actin: an abundant (1-5% of total protein)and evolutionarily conserved filament-
forming protein present in all eukaryotes
The actin cytoskeleton:Assembly of filaments in different times and places enable different shapesand functions
Epithelialmicrovilli
Cytokinetic furrow individing cells
Stress fibers instatic cells
Filopodia andlamellopodia in
motile cells
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Movie: actin-GFP in a moving cell
From Lodish, Molecular Cell Biology 6e
Properties of actin 1: exists in two forms
From Lodish, Molecular Cell Biology 6e
Monomer (G-actin) Polymer (F-actin: filament)Chain of monomers, non-
covalently bound
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Properties of actin 2: filaments are polar with different ends
seed
Lodish, Molecular Cell Biology 6e
Subunits all face the same direction: ATP-binding cleft faces (-) end
Growth can happen at both ends of a filament: are they different?
Expt: take short seed filament -> add G-actin to polymerize
Slowergrowing
Fastergrowing
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Actin polymerization: nucleation, elongation and steady state
From Lodish, Molecular Cell Biology 6e
Dynamics!In absence of seed 3 stages of polymerization
1). Nucleation: formation of stable nucleus (slow)
2). Elongation: monomer addition at ends (fast)
3). Steady state: rate of total addition=rate of total lossthis happens at C
c
: critical concentration
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Actin polymerization: kinetics in vitro
From Lodish, Molecular Cell Biology 6e
Lagphase
No change inmass over time
Fast!
Actin polymerization dynamics in vitro: treadmilling
From Lodish, Molecular Cell Biology 6e
At steady state: [g-actin]=Cc=0.2 uM
If [g-actin]>Cc..polymerizeIf [g-actin]
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From Lodish, Molecular Cell Biology 6e
Seeds ornucleating
protein eliminate
log phase
Actin polymerization: kinetics in vitro vs in vivo
Figure 17-31 Essential Cell Biology ( Garland Science 2010)
Actin function in cells: controlled byactin-binding proteins
[G-actin] in non-muscle cells: ~200 uM. 50% of total actin.This is well above critical concentration.
Why dont unorganized filaments spontaneously form?
Thymosin-B4,Profilin
Arp2/3
Cofilin
Myosin
CP
Fascin
ERM
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Actin polymerization dynamics drive cell migration
de-adhesion
Stages in the actin-
based cycle
Cells pull on substrate using actin-generated forces
extension/protrusion
adhesion
de-adhesion,
translocation
A model system for protrusion:intracellular actin-based movement of the bacterial pathogen Listeria monocytogenes
bacteria actin
From Lodish, Molecular Cell Biology 6e
Hijacks actin cytoskeleton!
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Reconstitution of motility with pure proteins:actin polymerization is sufficient for force generation
From Lodish, Molecular Cell Biology 6e
FORCE
Which proteins are sufficient?(with bacteria, ATP, G-actin)
1). Arp2/3 complex: NucleatorOrganizes Y-branches (70 angle)
optimal for force generation at
bacterial surface
2). Capping protein:
Caps (+) ends of old filaments
so that newer ends (closer tobacteria) polymerize
3). Cofilin: severs ADP filaments->disassembly, monomer recycling(maintain high local [ ] of monomer)
How do cells move?A mechanism similar to Listeria movement drives lamellipodia protrusion
during cell migration.A branched actin network with outward-pointing Plus-ends pushes PM forward.
Lodish, Molecular Cell Biology 6e
FORCE
Cell: an Image Library
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Myosin motors: mechanochemical enzymes thatharness ATPhydrolysis to drive conformational changes and movement along actin
From Kodera et al. Nature Nov 4;468(7320):72-6. Epub 2010 Oct 10.http://www.nature.com/nature/journal/v468/n7320/full/nature09450.html
http://www.youtube.com/watch?v=vJ9ffKeUCvE
Myosin II:Muscle contraction, cell migration
Myosin V:Vesicle movement
From Lodish, Molecular Cell Biology 6e
Most myosin motors move towards the + end of actin filaments
If you attach the myosin motor domain (head) to glass and add actin filamentsand ATP as depicted below, the actin filaments will move along the glass.Which end of the actin filament will be leading, and which will be lagging?
A.
- end leading, + end laggingB.
+ end leading, - end lagging
By pulling actin filaments relative to the membrane or to eachother, myosins can generate contractile forces that are essential
for vesicle transport, locomotion, cortical tension, muscle contraction etc.
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Signaling from Rho GTPases to actin:
regulate cell migration
From Lodish, Molecular Cell Biology 6e