Photodamage was achieved by local illumination of a sample containing 5% Alexa Fluor 594-labeled actin and 5% Alexa Fluor 488-labeled actin with a helium–neon 1-mW laser (543 nm) iterated 200–400 times (corresponding to a few seconds).
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Surface-rendered 3D reconstructions of confocal slices were performed by using volumej software ( 21) with filtered images ( metamorph, Median Filter) and enhanced contrast. To limit convective flux, 0.5–1 μM α-actinin was added.
Actin–Alexa Fluor 488 was observed with an ion–argon 25-mW laser (488 nm). Confocal microscopy experiments were carried out with a Zeiss confocal microscope with a ×63 (1.40 numerical aperture) oil-immersion objective and controlled by lsm 510 meta software. 5, which is published as supporting information on the PNAS web site). Both methods gave the same results for the thickness as a function of time (see Fig. We either followed one single bead for a certain time or took pictures of many different, randomly chosen beads. Measurement of the thickness of the actin gel (the edge of the gel was taken as the point at which the fluorescence intensity decayed to half the maximum intensity) and the bead velocity were performed by using meta-morph software (Universal Imaging). Fluorescence microscopy was performed by using an Olympus inverted microscope with a ×100 oil-immersion objective. The total volume of sample was such that the spacing between slide and coverslip was at least three times the bead diameter (5 μl for beads with radii of R = 1, 1.4, and 2.3 μm 6 μl for beads with a radius of R = 3 μm 10 μl for beads with a radius of R = 5 μm and 16 μl for beads with a radius of R = 8 μm).īead Observation and Data Processing. (Note that the same results were obtained for twice the amount of beads.) The sample was placed between a glass slide and coverslip (18 × 18 mm) sealed with Vaseline/lanolin/paraffin (1:1:1). At time 0, a small volume of bead suspension was diluted 30 times in motility medium and mixed gently with a pipette. Unless otherwise indicated, the motility medium contained 10 mM Hepes (pH 7.5), 0.1 M KCl, 1 mM MgCl 2, 0.15 mM CaCl 2, 1.8 mM Mg♺TP, 6 mM DTT, 0.13 mM diazabicyclooctane (Dabco an antiphotobleaching agent), 7.44 μM F-actin (10% labeled with Alexa Fluor 594 or Alexa Fluor 488 for confocal microscopy), 0.05 μM Arp2/3, 0.2 μM gelsolin, 3 μM ADF/cofilin, 0.3 μM profilin, and 10 mg/ml BSA. We propose a theoretical model based on elasticity and fracture mechanics to explain the experimental results. This process is reminiscent of the fracture observed in brittle solids or gels. We show experimental evidence that symmetry breaking occurs through a release of elastic energy in the actin gel, which can be triggered locally by disrupting the region of accumulated stress. We varied the composition of the motility medium and added actin filament crosslinking agents to obtain a precise description of the physical and biochemical parameters that govern the growth of actin gels and the characteristics of symmetry breaking.
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In this article, we examine how symmetry breaking arises on beads coated with verprolin/cofilin/acidic domain (VCA) (an Arp2/3 activator derived from WASP) in a mix of commercially available proteins. Except in cases of preexisting asymmetry, the initial actin gel that grows around the beads is homogeneous and must undergo symmetry breaking to generate a comet that can push forward ( 3, 4, 17– 19). The motility of Listeria can be reproduced in a medium containing a minimum set of purified proteins that can also support the movement of solid beads coated with activating factors of actin polymerization ( 4, 15, 16).