Dec 17, 2015
Cytoskeleton and Cell Motility
Nancy [email protected]
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.