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.

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The actin cytoskeleton plays a crucial role in pollen tube growth. In elongating pollen tubes the organization and arrangement of actin filaments (AFs) differs between the shank and apical region. However, the orientation of AFs in pollen tubes has not yet been successfully demonstrated. In the present work we have used myosin II subfragment 1 (S1) decoration to determine the polarity of AFs in pollen tubes. Electron microscopy studies revealed that in the shank of the tube bundles of AFs exhibit uniform polarity with those close to the cell cortex having their barbed ends oriented towards the tip of the pollen tube while those in the cell center have their barbed ends oriented toward the base of the tube. At the subapex, some AFs are organized in closely packed and longitudinally oriented bundles and some form curved bundles adjacent to the cell membrane. In contrast, few AFs are dispersed with random orientation in the extreme apex of the pollen tube. Our results confirm that the direction of cytoplasmic streaming within pollen tubes is determined by the polarity of AFs in the bundles.

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca(2+)-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen-pistil interactions, apart from its postulated function in cellular Ca(2+) homeostasis, is discussed.

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca2+-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca2+ storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma–style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis.