XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes

Abstract

During the maturation of Xenopus oocytes, a transient microtubule array (TMA) is nucleated from a novel MTOC near the base of the germinal vesicle. The MTOC-TMA transports the meiotic chromosomes to the animal cortex, where it serves as the precursor to the first meiotic spindle. To understand more fully the assembly of the MTOC-TMA, we used confocal immunofluorescence microscopy to examine the localization and function of XMAP215, XKCM1, NuMA, and cytoplasmic dynein during oocyte maturation. XMAP215, XKCM1, and NuMA were all localized to the base of the MTOC-TMA and the meiotic spindle. Microinjection of anti-XMAP215 inhibited microtubule (MT) assembly during oocyte maturation, disrupting assembly of the MTOC-TMA and subsequent assembly of the first meiotic spindle. In contrast, microinjection of anti-XKCM1 promoted MT assembly throughout the cytoplasm, disrupting organization of the MTOC-TMA and meiotic spindle. Finally, microinjection of anti-dynein or anti-NuMA disrupted the organization of the MTOC-TMA and subsequent assembly of the meiotic spindles. These results suggest that XMAP215 and XKCM1 act antagonistically to regulate MT assembly and organization during maturation of Xenopus oocytes, and that dynein and NuMA are required for organization of the MTOC-TMA.

Cdc6 is required for meiotic spindle assembly in Xenopus oocytes

“…Similar phenotypes were observed when antibodies against NuMA or cytoplasmic dynein were injected into oocytes during maturation. 27 The presence of more than one bipolar spindle in Cdc6-depleted samples in MI may have resulted from the splitting of the MTOC-TMA. 27 Further, monasters associated with chromosomes perhaps represent a very early or deformed MTOC-TMA, which is incapable of spindle assembly.…”

Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

“…Beads were washed, eluted with sample buffer, and immunoblotted for pADPr, NuMA, and CH-TOG ( Figure 5). CH-TOG, also known as XMAP215 and CKAP5, is enriched at spindle poles and has an important role in microtubule nucleation and elongation (Becker et al, 2003). It was indentified by LC/MS as a potential pADPr-binding protein in early experiments, but after refining our protocol it was less evident.…”

Visualization of the Cytoskeleton in Xenopus Oocytes and Eggs by Confocal Immunofluorescence Microscopy

“…For the procedure, 1. Rinse samples three times with TBS to remove bleach or detergent from previous steps.…”