A combined in vitro and in vivo study of the assembly mechanism of actin bundles in Drosophila bristles.

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Parallel actin bundles are composed of tightly packed filaments that all have the same polarity and are crosslinked by an actin-bundling protein and constitute a key component of eukaryotic cytoskeleton structures. Their function is to reinforce the cell against mechanical deformation and to allow localized force generation. One prominent actin bundle organization is found in Drosophila bristle cells. The bristle actin bundles are membrane-bound, triangular-shaped, and are organized into modules along their length. They are evenly spaced around the outer perimeter of the bristle, extending from the base of the bristle to its tip with well-defined spaces between modules. The formation of bristle actin bundles has already revealed a fascinating machinery of the growth process. However, the molecular mechanism determining and controlling the length and the shape of bristle actin bundles is still unknown. In this proposal we plan an inter-disciplinary study to identify the mechanism of Drosophila bristle formation by combining in vivo and in vitro experiments. We will identify the essential genes that are part of the bristle actin bundle machinery and use high-resolution light microscopy using transgenic flies, to reveal the growth rate limiting steps. Our hypothesis is that an intricate interplay between actin nucleation, crosslinking and depolymerization proteins, set the observed actin bundles length. To test this mechanism, an in vitro system of actin bundle formation will be developed. We will test how the triangular shape of the bundles depends on the longitudinal association of spontaneously nucleated filaments in the cytosol. Both in vivo as in in vitro experiments will be used to pin down the mechanism of the lateral growth and geometry of the bristles. As it is believed that common mechanisms are responsible for control of bundle shapes in a variety of systems, such as microvilli or stereocilia, the results of this study will affect our understanding well beyond the formation of bristles bundles.