Orbit/CLASP Is Required for Myosin Accumulation at the Cleavage Furrow in Drosophila Male Meiosis

Abstract

Peripheral microtubules (MTs) near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR) in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II was first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs made contact with the equatorial cortex, myosin II was concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appeared on the equatorial cortex, simultaneously with myosin accumulation. We found that the accumulation of myosin did not require centralspindlin, but was instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit was also localized in a mid-zone of peripheral MTs, and was concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. We also showed that the myosin heavy chain was in close proximity with Orbit in the cleavage furrow region. Centralspindlin was dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex was required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit was also necessary for the maintenance of the CR. Our data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.

C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion

“…spindle microtubules that normally promote contractile ring assembly [51,52]. Additionally, human CLASP proteins have been shown to associate with actin filaments and may cross-link microtubules and filamentous actin [25], and mammalian CLASPs have been proposed to link microtubule plus ends with the cell cortex [53,54].…”

Plk4 triggers autonomous de novo centriole biogenesis and maturation

“…Table 3). Homozygous mNeonGreen-Plk4 and pUb–RFP– β 2-Tubulin flies (gift from Yoshihiro Inoue, (Kitazawa et al, 2014)) were crossed, establishing a stable stock.…”

Mutations in <i>mxc</i> Tumor-Suppressor Gene Induce Chromosome Instability in <i>Drosophila</i> Male Meiosis

“…To investigate genetic interactions with mxc, we used following mutants; Lsm10 f06616 (Godfrey et al, 2009), snRNA:U7 EY11305 (Godfrey et al, 2006), coil B220 (Liu et al, 2009), mute EY22147 and FLASH LL01602 (Rajendra et al, 2011). GFP-βTubulin, RFP-βTubulin, and mRFP-Histone H2Av were used as a normal control for cytological imaging and time-lapse observations (Inoue et al, 2004;Kitazawa et al, 2014;Tanabe et al, 2017). Ub-GFP-cid (Talbert et al, 2018) was used to visualize centromeres.…”

“…The samples were mounted with VEC-TASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Microtubules were visualized by expression of GFP-βTubulin (Inoue et al, 2004) or RFP-βTubulin (Kitazawa et al, 2014) and image acquisition was performed as described above.…”