Photobleaching fluorescent actin in a fibroblast

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Alberts Fig. 16.56 Photobleaching fluorescent actin in a fibroblast The fluorescent mark moves backward with respect to the front cell edge (and with respect to the substratum). Slow moving cell Y-L Wang et al., 1985. JCB 101:597-602 Experiment #1 Conclusion: 1. Actin meshwork is flowing backward 2 New actin is polymerized at the edge

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Photobleaching fluorescent actin in a fibroblast. Experiment #1. The fluorescent mark moves backward with respect to the front cell edge ( and with respect to the substratum). Alberts Fig. 16.56. Slow moving cell. Conclusion: 1. Actin meshwork is flowing backward - PowerPoint PPT Presentation

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  • Photobleaching fluorescent actin in a fibroblastAlberts Fig. 16.56The fluorescent mark moves backward with respect to the front cell edge (and with respect to the substratum).Slow moving cellY-L Wang et al., 1985. JCB 101:597-602Experiment #1Conclusion:1. Actin meshwork is flowing backward2 New actin is polymerized at the edge

  • Models of actin filament growth during protrusion: Treadmilling versus nucleation-releaseVic SmallTim Mitchison

  • Photoactivation of fluorescence in a moving keratocyteThe fluorescent mark moves backward with respect to the front cell edge (but not with respect to the substratum).Fast moving cellTheriot and Mitchison, 1991 Nature 352: 352-131

  • Treadmilling vs. nucleation-releaseThis is figure 4, from Theriot and Mitchison, 1991. It is posted under papers on HuskyCTImage quality is lost when converting to PDF

  • Rate of actin turnover is consistent with nucleation-release model(Small) EM studies show long actin filaments in lamellae of slow moving fibroblasts

    (Others) EM studies in rapidly moving cells show a dense meshwork of actin filaments of different lengthsPhotobleaching experiments show rate of actin filament turnover to be greater than expected if treadmilling of long filaments occurs

    BUT Small criticized interpretation of photobleaching experimentsDepolymerization of actin filaments makes estimate of filament turnover artificially high

  • Current viewDendritic nucleation model (Mullins, 1998)Confirmed by the discovery of Arp2/3 at leading edge of motile cellsVarible lengths of F-actin in lamellae of motile cellsTreadmilling occurs in individual filaments that are nucleated and released from these sites (if uncapped at both ends).Treadmilling occurs in the whole mass of actin filament meshworkQuestion: If the rearward movement of bleached marks is due to treadmilling, then why do we observe faster rearward movement (w.r.t. substratum) in cells with slow rates of actin turnover?

  • When actin polymerization is inhibited the actin meshwork continues to move rearwardMechanical inhibition of retrograde flow(see movie)

  • In fast moving cells contractile forces at the front edge are low compared to the strength of adhesionsNo rearward actin flow so that most newly polymerized actin contributes to protrusion bleached marks are stationary w.r.t. substratum

    In slow moving cells contractile forces at the front are high (but not higher) compared to the strength of adhesions Actin flows rearward because most newly polymerized actin feeds the flow protrusion is limited - bleached marks move back w.r.t. substratumAdhesions allow mechanical coupling between a contractile cytoskeleton and the substratum

  • The tail of Listeria monocytogenes : Lessons learned from a bacterial pathogenFound in soil, on plants & animalsAssociated with eating contaminated dairy products, plantsInfects intestinal cells and spreads from cell to cellIntracellular motility essential for spreadCan cause, meningitis, septicemia, abortions Old, very young, and immunocompromized people at risk

  • MovieListeria rocketing in infected cell

  • Listeria highjacks host cell functionsListeria express surface proteins internalins (e.g. InlA, InlB)InlA binds to E-cadherin A cell-cell adhesion molecule

    Recruitment of adhesion proteins links bacterium to cytoskeleton triggers phagocytosisListeria secrete listeriolysin O (LLO) and escapes into cytoplasmOptimal activity pH 5.5 = same as inside phag. vacuole

    Bacterium recruits host cells actin and ABPs to move intracellularly

    Induce membrane extension phagocytosed by neighboring cell and infects it

  • Advantages of studying Listeria1. Doesnt have the drawbacks of other whole cell systemse.g. Some cytoskeletal mutations are lethal Functions of many ABPs are redundantDifficult to reconstitute cell motility it requires a plasma membrane2. No plasma membrane3. Motility can be reconstituted in vitro4. The tail of Listeria is analogous to a lamellipodium of a moving cell

  • How does actin polymerization drive the movement of Listeria?1. Insertional actin polymerization occurs at back edge of bacteriumPolymerization fluorescently labeled actin shows brighter regions at back edge2. Photobleaching experiments show that the tail remains stationary as bacterium moves forward

    3. Depolymerization occurs at the same rate throughout the tailtail length is usually constanta decreasing gradient of filament density exists from the front to rear of the tailF-actin half life = 30 secDistance um from backFilament densityadditionloss

  • How does actin polymerization become localized at one end of the bacterium?1.Identification of nucleation factors2. Symmetry breaking (later)

    In the early 90s used a genetic screen in mutant Listeria that could not form tails, and normal onesFound a single gene actA - encodes a bacterial surface protein ActACan induce tail formation in:Immotile Listeria, other bacteria, polystyrene beads

  • Act A is required for tail formationDoes not bind directly to actin

    Looked for proteins that localized to the back edge of bacterium that are not seen in the tail.

    1. Found VASP (vasodilator-stimulated phosphoprotein)discovered by immunofluorescence studies Binds to proline rich region of Act-A Known to be associated with F-actin and focal adhesions in lamellipodia2. Profilin binds VASPVASP and profilin accelerate filament elongation but are not nucleatorsEvidence: Actin clouds form in profilin depleted cytoplasmic extractsVASP-actin complexes have no nucleating activity

    How is elongation accelerated?

    Poly proline regions bind multiple VASP moleculesEvidence: Bacterial speed is proportional to number of proline-rich repeats in ActAGFP-profilin concentration at back edge is proportional to speedBact. Memb. anchor sequence

  • ABPs in Listeria tails are the same as in lamellipodiaArp2/3 (Welch et al., 1997)isolated by column chromatography from host cell (platelet) cytoplasm - required for polymerization is activated by ActACapping proteins e.g. gelsolin - found throughout tail Is enriched at bacterial surface but ActA thought to suppress capping hereADF/Cofilin - found throughout tail important for increasing actin filament turnover by 10-100 times compared with in vitroImmunodepletion leads to formation of very long tailsAddition of excess decreases tail length but increases speedCrosslinking proteins -eg. Fimbrin, -actinin - found throughout tail, structural roleintroduction of dom. negative fragment stops bacteria movement

  • Organization of actin filaments in Listeria is similar to that of lamellipodiaY shaped cross-links containing ARP2/3 are present Evidence of other kinds of crosslinking exists