Jaguar physically intereacts with Miranda. Anti-Miranda antibodies were used to extract protein complexes from Drosophila embryos and more than 90% of Miranda was immunoprecipitated. In addition to a quadruplet of Miranda isoforms identified by mass spectrometry, two proteins of approximately 250 and 140 kDa were reproducibly immunoprecipitated with anti-Miranda antiserum. Mass spectrometry determination of tryptic peptide sequences and a database search identified these proteins as the nonmuscle myosin II Zip and the unconventional myosin VI Jar. Immunoprecipitation with anti-Jar antibodies further verified that Jar is associated with Miranda. In addition to Jar, Staufen, Prospero, and prospero mRNA were also coimmunoprecipitated with Miranda using anti-Miranda antibodies, demonstrating in vivo association of Miranda with myosin VI Jar as well as several components that require Miranda for basal localization (Petritsch, 2002).
To further examine the role of Jar in asymmetric division, its distribution was compared with the dynamic Miranda localization in neuroblasts. Miranda appears around the apical cortex and in the cytoplasm in early prophase, in the cytoplasm and on the cortex in late prophase, and is translocated at metaphase to a tight basal crescent, as well as to puncta around the aster microtubules and a faint lining of the microtubules of the mitotic spindle. In telophase, Miranda is mainly inherited by the ganglion mother cell (Petritsch, 2002).
Jar is localized in small particles mainly in the cytoplasm and less frequently at the cortex in a dynamic pattern. The density of Jar particles in neuroblasts is highest in prophase and metaphase, coinciding with Miranda translocalization and spindle rotation. Jar particles accumulate preferentially to the basal half in 45% of metaphase neuroblasts, whereas they are more homogeneously distributed in the rest. In telophase, Jar particles are inherited by the ganglion mother cell preferentially but are also seen in the neuroblast. This dynamic pattern is reminiscent of the linear movement of Jar-containing particles in syncytial blastoderm embryos (Mermall, 1994). The partial overlap between Jar and Miranda puncta in the cytoplasm in prophase and in the cytoplasm and at the cortex in metaphase and telophase is consistent with a role for Jar in the basal translocation of Miranda. Given that Jar interacts with a number of proteins besides Miranda (Buss, 2001; Geisbrecht, 2002), it is perhaps to be expected that Jar does not colocalize with Miranda to a greater extent. Jar has never been seen concentrated to a tight basal crescent, suggesting that Jar itself is most likely not an anchor for the cell fate determinants at the basal cortex. The transient accumulation of Jar particles on the basal side in metaphase neuroblasts is consistent with its involvement in the transport of Miranda to the basal pole (Petritsch, 2002).
A 195-kD protein coimmunoprecipitates with Jaguar. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homolog identified of cytoplasmic linker protein (CLIP)-170, a protein that links endocytic vesicles to microtubules. This protein has been named CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between Drosophila CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. Jaguar and CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They also colocalize at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homolog of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, suggests that these two proteins may functionally link the actin and microtubule cytoskeletons (Lantz, 1998).
Because 95F myosin has been implicated in transport and the vertebrate homolog of Drosophila CLIP-190, CLIP-170, is suspected of being involved in transport, the colocalization in axons, where vesicle/organelle transport along both actin and microtubules has been observed, is particularly intriguing. The particulate distribution of both Jaguar and CLIP-190 in nerve processes in the embryo and also in cultured cells from Drosophila embryos is consistent with these structures being vesicles. CLIP-170 has been suggested to participate in loading endocytic vesicles on to the plus ends of microtubules. No previous data exist that suggest an interaction of CLIP-170 with the actin cytoskeleton. However, the plus ends of peripheral microtubules to which the CLIP-170-associated vesicles appear to bind are adjacent to the actin filament-rich cortex. One function for CLIP-190 and Jaguar in the same structures may be to coordinate the transport of specific cargoes along both microtubules and actin filaments to facilitate their proper localization in the cell (Lantz, 1998).
Myosin VI localizes to the leading edges of growth-factor-stimulated fibroblast cells and has been suggested to be involved in cell motility. There has been no direct test of this hypothesis, however. Drosophila melanogaster MyoVI is expressed in a small group of migratory follicle cells, known as border cells. Depletion of MyoVI specifically from border cells severely inhibits border cell migration. Similar to MyoVI, E-cadherin is required for border cell migration. E-cadherin and Armadillo (Arm, Drosophila beta-catenin) protein levels are specifically reduced in cells lacking MyoVI. In addition, MyoVI protein levels are reduced in cells lacking DE-cadherin or Arm. MyoVI and Arm co-immunoprecipitated from ovarian protein extracts. These data suggest that MyoVI is required for border cell migration where it stabilizes E-cadherin and Arm. Mutations in MyoVIIA, another unconventional myosin protein, also lead to deafness, and MyoVIIA interacts with E-cadherin through a membrane protein called vezatin. Multiple biochemical mechanisms may exist, therefore, for cadherins to associate with diverse unconventional myosins that are required for normal stereocilium formation or maintenance (Geisbrecht, 2002).
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