Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex - PubMed (original) (raw)
Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex
William M Brieher et al. J Cell Biol. 2004.
Abstract
Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.
Figures
Figure 1.
Listeria motility continues in the absence of Arp2/3-mediated nucleation. Bacteria are labeled blue and actin is green or red. (A) Listeria moving in brain cytosol. (B) Listeria in cytosol containing the Arp2/3 inhibitor CA. (C) Listeria moving initially in cytosol and Alexa® 488–labeled actin (green), then switched to cytosol containing CA and TMR-labeled actin (red). (D) As per C, but then switched to CA and TMR-labeled actin alone. Bar, 10 μm (applies to all panels).
Figure 2.
Actin comet tails consist of branched filaments and parallel bundles. Thin-section EM of L. monocytogenes comet tails in infected PtK cells (A and B), brain cytosol that produces a comet tail consisting of branched filaments and parallel bundles (C), and brain cytosol that produces only branched filaments (D). (E) Comet tails formed initially in brain cytosol that forms branched and bundled filaments, then switched to the same cytosol containing CA. (F) Comet tails formed initially in brain cytosol that forms only branched filaments, then switched to the same cytosol containing CA. Bars: (A, B, E, and F) 1 μm; (C and D) 0.75 μm.
Figure 3.
Isolation of fascin as the only motility factor required in addition to actin for movement in the absence of Arp2/3. (A) Separation of Listeria propulsion into three steps: (step 1) preincubation step in cytosol; (step 2) nucleation phase with Arp2/3, capping protein, and Alexa® 488–labeled actin; and (step 3) elongation phase in CA, TMR-labeled actin, and fascin purified from bovine brain. (B) SDS-PAGE summarizing the purification of fascin using the three-step assay. (C) Result of a three-step assay using bacterially expressed, recombinant fascin in step 3. The comet tail assembled in step 2 (nucleation in Arp2/3) is labeled green, and the portion assembled in step 3 (elongation in fascin) is labeled red. Bars: (A) 5 μm; (C) 1 μm.
Figure 4.
Fascin localizes to comet tails in _L. monocytogenes_–infected cells. Infected BSC-1 cells (A and B) or XTC cells (C and D) were costained for actin (A and C) and fascin (B and D). Bar, 10 μm.
Figure 5.
Different bundling proteins can mediate Arp2/3- independent motility. (A) Coomassie-stained gel of the bundling proteins used for the elongation reactions. Fascin is a fascin–thioredoxin fusion protein causing it to run at 68 kD. (B–E) Arp2/3-independent motility using either fascin (B), fimbrin (C), α-actinin (D), or filamin (E) in the elongation step. All reactions were performed using the three-step protocol described in Fig. 3. Green actin marks the portion of the comet tail assembled in the presence of Arp2/3. Red actin marks the portion of the comet tail assembled during the elongation phase in the presence of the indicated bundling protein and CA. Bar, 10 μm.
Figure 6.
Thin-section electron micrographs showing segregation of actin filament organization by experimentally separating nucleation-driven propulsion from elongation-driven propulsion. (A) Branched actin cloud generated by Arp2/3 and actin alone. (B) Branched actin comet tail generated by motility during the Arp2/3-mediated nucleation phase (corresponding to step 2 of Fig. 3). (C) Comet tails after fascin-dependent elongation-driven motility (corresponding to step 3 of Fig. 3). (D) Comet tail organization generated by the nucleation reaction. (E) Comet tail organization generated by the elongation reaction. Bars, 1 μm. Bar in B applies to A and B. Bar in E applies to D and E.
Figure 7.
The fascin-mediated elongation phase is independent of Arp2/3-nucleating activity. (A) CA inhibits Arp2/3-dependent nucleation, but does not affect fascin-mediated elongation. (B) AMPPNP inhibits Arp2/3-dependent nucleation, but does not affect fascin-mediated elongation. Each point in A and B represents the average of at least 66 bacteria from two experiments ± SD. (C) Arp2/3 localizes to the portion of the comet tail assembled during the nucleation phase, but is not found on the tail assembled during elongation or at the bacterial surface. Bar, 10 μm.
Figure 8.
L. monocytogenes motility in the fascin-dependent elongation phase. (A) Distance traveled over time in the presence or absence of fascin. Each point is the average of at least 57 bacteria ± SD. Every bacterium is included in each time interval, even if it stopped moving at an earlier time. (B) The fraction of the total population moving over time in the presence or absence of fascin.
Figure 9.
Rates of L. monocytogenes motility as a function of G-actin concentration under nucleation and elongation conditions. Bacteria were preincubated in either VASP or brain cytosol (step 1). To initiate motility, the preincubation solution was replaced with Arp2/3, actin, and capping protein (step 2). For motility in the absence of Arp2/3, the nucleating solution in step 2 was replaced with a solution containing actin, fascin, and CA (step 3). Motility rates in the Arp2/3-dependent reaction were analyzed in step 2. Rates in the fascin-dependent elongation reaction were analyzed in step 3. Each point is the average of at least 30 bacteria from three separate experiments ± SD.
Figure 10.
A Brownian ratchet model for bundling-mediated motility of L. monocytogenes. Individual filaments form weak attachments with the bacterial surface. However, incorporation of filaments into bundles is incompatible with their binding to the bacterial surface. Moving the bacteria to the right maximizes the number of favorable bonds because it can attach to growing individual filaments while leaving bundled filaments behind it. The bacteria is blue. Green lines represent actin and red lines newly polymerized actin. Bundling proteins are represented as black ovals.
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