Bilder.2 Actin

7/25/2019 Bilder.2 Actin

1/12

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

}

}

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


Properties of actin 1: exists in two forms


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


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


Lagphase

No change inmass over time

Fast!

Actin polymerization dynamics in vitro: treadmilling


At steady state: [g-actin]=Cc=0.2 uM

If [g-actin]>Cc..polymerizeIf [g-actin]


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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


Hijacks actin cytoskeleton!


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Reconstitution of motility with pure proteins:actin polymerization is sufficient for force generation


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


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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12/12

Signaling from Rho GTPases to actin:

regulate cell migration


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