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Steps and fluctuations of Listeria monocytogenes during actin-based motility

Nature volume 407, pages 1026–1029 (2000)Cite this article

Abstract

The actin-based motility of the bacterium, Listeria monocytogenes, is a model system for understanding motile cell functions involving actin polymerization1. Although the biochemical and genetic aspects of Listeria motility have been intensely studied2,3,4,5, biophysical data are sparse6. Here we have used high-resolution laser tracking to follow the trailing ends of Listeria moving in the lamellae of COS7 cells. We found that pauses during motility occur frequently and that episodes of step-like motion often show pauses spaced at about 5.4 nm, which corresponds to the spatial periodicity of F-actin7. We occasionally observed smaller steps (<3 nm), as well as periods of motion with no obvious pauses. Clearly, bacteria do not sense cytoplasmic viscoelasticity because they fluctuate 20 times less than adjacent lipid droplets. Instead, bacteria bind their own actin ‘tails’, and the anchoring proteins can ‘step’ along growing filaments within the actin tail. Because positional fluctuations are unusually small, the forces of association and propulsion must be very strong. Our data disprove the brownian ratchet model8 and limit alternative models, such as the ‘elastic’ brownian ratchet9 or the ‘molecular’ ratchet4,10.

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Figure 1: Response of laser-tracking signals with bacterial position.
Figure 2: Actin-based motility in COS7 lamellae.
Figure 3: Power spectra of laser-tracking signals.
Figure 4: Comparisons of fluctuations.

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Acknowledgements

We thank L. Machesky for J774 cells, and D. Portnoy and J. Skoble for Listeria strains and general advice. We also thank the National Science Foundation, the National Institutes of Health and the Whitaker Foundation for their support. J.L.M. was supported as a BME Distinguished Postdoctoral Fellow, partially funded by the Whitaker Foundation.

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  1. Department of Biomedical Engineering, The Johns Hopkins University, 720 Rutland Avenue, Baltimore, 21205, Maryland , USA

    Scot C. Kuo & James L. McGrath

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Correspondence to Scot C. Kuo.

Supplementary information

41586_2000_BF35039544_MOESM1_ESM.jpg

Figure 1. Instance of faster motility. a, High-resolution trajectory (x: RMS 7.1 nm; linear-fit slope 7 mm/min). With transposed axes, the left panel shows the bacterium’s trajectory and a contour plot of its positional frequencies (bins 1´1 nm; local maxima >6 ms/nm2 filled red). The right panel shows bacterial position vs. time. All grid lines are spaced 5.4 nm and green dashed lines denote the intercalation size for G-actin (linear fit ±2.7 nm). Spatial frequency analysis of step-size: b, Normalized histogram (bins 0.5 nm) of pairwise displacements shows a strong ~6 nm periodicity (grid spacing 5.4 nm). c, Fourier analysis (power spectrum) of pair-wise displacement histogram in c show a major component at 0.17 nm-1 (6 nm) and minor components at 0.24 nm-1 (4.2 nm), 0.27 nm-1 (3.7 nm), and 0.35 nm-1 (2.9 nm). (JPG 73 kb)

41586_2000_BF35039544_MOESM2_ESM.jpg

Figure 2. Model summarizing observations. There are three relevant classes of actin filaments: flexed/free, flexed/tethered, and stretched taut/tethered. Pressure at the bacterial surface originates from the restoring forces of flexed filaments (their bending is highly exaggerated in this cartoon) and pressure is maintained by polymerization at their uncapped barbed ends. Free, flexed filaments could occur by either the elastic Brownian ratchet [Mogilner, 1996] or by release from the molecular ratchet. Tethered, flexed filaments are consistent with the molecular ratchet [Laurent, 1999; Egile, 1999] and may grow if tethering proteins were sufficiently flexible and large. Because filament bending would shorten apparent steps, taut filaments are responsible for steps spaced 5.4 nm. Tethering proteins bound to the sides of filaments can slide to reveal this periodicity. Because actin monomers are staggered in filaments, growth of tethered filaments (probably in steps of 2.7 nm) are not necessarily coordinated with sliding. The anchoring proteins stretching these taut filaments are probably the same proteins involved in the molecular ratchet. Details about capped filaments, angled filaments, dendritic crosslinking and actin nucleation caused by ARP2/3 are omitted for clarity. (JPG 65 kb)

41586_2000_BF35039544_MOESM3_ESM.mov

Movie. Spontaneous detachment during actin-based motility. A rod-shaped Listeria bacterium in COS7 lamellae is undergoing actin-based motility, when its motility stops. Note the large increase in the magnitude Brownian fluctuations, including fish-tailing, of the bacterium. Adjacent spherical lipid droplets show comparably large Brownian fluctuations. Because the local viscoelasticity of lamellae allow much larger fluctuations, bacteria must bind their tails during motility. Movie is best viewed with QuickTime 4. (MOV 682 kb)

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Kuo, S., McGrath, J. Steps and fluctuations of Listeria monocytogenes during actin-based motility. Nature 407, 1026–1029 (2000). https://doi.org/10.1038/35039544

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