Cytoskeleton and Cell Motility

Nancy Freitag256-7345nancy.freitag@sbri.org

Objectives

What regulates the shape and assembly of the cell cytoskeleton?

How do pathogens exploit host cell actin assembly?

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)

The actin cytoskeleton and cell motility

Cytoplasmic system of fibers crucial to cell motility

Plays a structural role Undergoes rearrangement which can produce movement

Actin provides framework & determines cell shape

Filaments are organized into bundles & networks held together by

cross-linking proteins

Actin filamentsgive shape tomicrovilli

Cell locomotion

Cell moves forward by extending filipodia & lamellipodia

Focal adhesions are formed

Cell is pulled forward

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

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

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

Actin filaments in solution

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

Elongation: addition of monomerto both ends

Steady state: G-actin monomersexchange with subunits at bothends w/no change in total mass

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

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

Cc

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.

Below Cc (+) end: no filament growth occurs

Between Cc (+) and Cc (-): growth occurs at the (+) end (treadmilling)

Above Cc (-): growth occurs at both ends

Actin filaments grow faster at one end than at the other

Treadmilling

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

Examples of proteins that sequester G-actin

Thymosin 4: sequesters free ATP-G-actin. Acts as a buffer.

Profilin: sequesters actin, and promotes the exchange of ATP for ADP-G-actin.

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.

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

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.

Pathogen-mediated cytoskeletal rearrangements Prevention of uptake: inhibition of phagocytosis or pedestal formation

Invasion: induced uptake Actin-based motility: intracellular motility and intercellular spread

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

From Tilney and Portnoy, J. Cell Biology 1989

L. monocytogenes as a tool for defining actin assembly

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?

Identification of ActA

Search for bacterial mutants unable to spread within cells led to identification of the actA gene product

wild type actA mutant

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

the cytosol

Domains of ActA

SP = signal peptide

WH2 & Arp 2/3 = bind Arp 2/3

AB region = monomeric actin binding

actin,actin + ActA

ActA

Arp 2/3

Arp 2/3 + ActA

ActA + Arp 2/3 function as a highly efficient nucleation site

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

Wiskott-Aldrich protein family (WASP)

Activate Arp 2/3 Contain WH2 domains, acidic domains, and proline rich regions

WASP, N-WASP, Scar

Fascin-mediated propulsion of

Listeria monocytogenes independent of frequent

nucleationby the Arp2/3 comple

J. Cell Biology 165: 233 - 2422004

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Copyright ©2006 by the National Academy of Sciences

Haviv, Lior et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4906-4911

Fig. 5. The transition from asters to stars

Additional references

Actin-based motility of intracelllular microbial pathogens. Micro Mol Biol Rev. (2001) 65: 595 - 626.

Interaction of human Arp 2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science (1998) 281: 105 - 108.