Cytoskeleton and Cell Motility Nancy Freitag 256-7345 nancy.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 filaments give shape to microvilli

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 monomer to both ends Steady state: G-actin monomers exchange with subunits at both ends w/no change in total mass

Each actin monomer is bound to a molecule of 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.

Actin filaments grow faster at one end than at the other Below Cc (+) end: no filament growth occurs Between Cc (+) and Cc (-): growth occurs at the (+) end (treadmilling) Above Cc (-): growth occurs at both ends

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 b4: 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

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.

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

L. monocytogenes as a tool for defining actin assembly From Tilney and Portnoy, J. Cell Biology 1989

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 base of 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

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

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 nucleation by the Arp2/3 comple J. Cell Biology 165: 233 - 242 2004

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Fig. 5. The transition from asters to stars Haviv, Lior et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4906-4911 Copyright ©2006 by the National Academy of Sciences

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.