Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.

During spermatid individualization in Drosophila, actin structures (cones) mediate cellular remodeling that separates the syncytial spermatids into individual cells. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. We show here that the two domains form separately in time, are regulated by different sets of actin-associated proteins, can be formed independently, and have different roles. Newly forming cones were composed only of bundles, whereas the meshwork formed later, coincident with the onset of cone movement. Polarized distributions of myosin VI, Arp2/3 complex, and the actin-bundling proteins, singed (fascin) and quail (villin), occurred when movement initiated. When the Arp2/3 complex was absent, meshwork formation was compromised, but surprisingly, the cones still moved. Despite the fact that the cones moved, membrane reorganization and cytoplasmic exclusion were abnormal and individualization failed. In contrast, when profilin, a regulator of actin assembly, was absent, bundle formation was greatly reduced. The meshwork still formed, but no movement occurred. Analysis of this actin structure's formation and participation in cellular reorganization provides insight into how the mechanisms used in cell motility are modified to mediate motile processes within specialized cells. INTRODUCTIONOne of the best-studied roles of the actin cytoskeleton is mediating cell and intracellular motility (Carlier et al., 2003;Ridley et al., 2003;Carlier and Pantaloni 2007). Several different types of actin structures are known to contribute to motility. Filopodia, which contain parallel bundles of actin filaments, are thought to be important for exploring the cells' environment and making initial contacts with substrate for movement in a particular direction. Lamellipodial meshwork is proposed to push the leading edge of the cell forward using the force generated by addition of actin monomers at the barbed ends of the filaments, which face the membrane (Pollard and Borisy 2003). Behind the leading edge, a lamellar region consisting primarily of bundles that contain tropomyosin and myosin II plays an important role in cell movement (Gupton et al., 2005). In addition, stress fibers, composed of antiparallel actin filament bundles connected to adhesion complexes, are important for traction and contractile forces that release the cell-substrate attachments so that forward movement can occur.The different actin structures and domains are regulated by different sets of actin-associated proteins. Much is known about control of assembly of the two main types of actin organizations, meshwork and bundles, from biochemical analysis in vitro and studies of motile cells in vivo. Actin meshwork is nucleated by the Arp2/3 complex, and the branched organization relies on the ability of the complex to bind to the side of an actin filament and nucleate a new filament (Goley and Welch, 2006). Parallel bundles are nucleated by formins, which associate with the barbed ends, but...

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Localization of pectins in the style of Petunia hybrida before and after pollination was investigated by immunocytochemistry using two primary monoclonal antibodies specific to highly (JIM7) and weakly (JIM5) methylesterified pectins. In the unpollinated style, esterified pectins occurred mainly in the cell walls of cortex tissue, while unesterified pectins were present mainly in the extracellular matrix (ECM) of the transmitting tract. After pollination no remarkable differences were found in pectin distribution in the ground tissue of the style. On the other hand, in the transmitting tract a reduction in the quantity of unesterified pectins was observed. Unesterified pectins in the extracellular regions of the transmitting tissue decreased before the penetration of the pollen tubes, indicating that pollination induces a reduction in the amount of unesterified pectins in the transmitting-tract ECM. The correlation between the degradation of strongly Ca2+-binding pectins and the growing level of those ions in the extracellular regions of the transmitting tract in the pollinated pistil of P. hybrida (M. Lenartowska et al. 1997) suggests that this process may constitute a mechanism for creating an optimum calcium medium for in vivo-growing pollen tubes. Both pectin categories were localized in pollen tubes. Esterified pectin epitopes were localized mainly in the vesicles of the tip cytoplasm. Unesterified pectin epitopes were found in the external fibrillar wall of pollen tubes.