Gamma-Tubulin Is Required for Bipolar Spindle Assembly and for Proper Kinetochore Microtubule Attachments during Prometaphase I in Drosophila Oocytes

Abstract

In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles.

Normal Segregation of a Foreign-Species Chromosome During Drosophila Female Meiosis Despite Extensive Heterochromatin Divergence

“…These tethers appear to be built from pericentromeric heterochromatin and are proposed to establish tension between chromosomes not held together by chiasmata, thus allowing homologous coorientation to be established (Hughes et al 2009(Hughes et al , 2011. Similar tethers have been inferred by micromanipulation experiments in grasshopper spermatocytes (LaFountain et al 2002) and by PICH localization to DNA threads connecting mitotic sister kinetochores in mammalian cultured cells (Baumann et al 2007).…”

“…This was determined by whether there was at least a 50% dip in background-subtracted fluorescent intensity, measured on the 4 and the space between the 4 and the adjacent chromosome using the line ROI tool. Oocytes with both 4s on the same side of the spindle, with additional nonexchange chromosomes, or with chromosomes in the "slippage" configuration (Hughes et al 2011) were counted as having chromosomes out on the spindle, but their 4-4 distances were not included in the analysis. Plots and t-tests were then done in R.…”

“…We were able to detect them by both a phospho-specific histone antibody that can highlight threads (Hughes et al 2011) and by FISH of an AATAT probe (Figure 2). However, during this experiment, we noticed that it was much more difficult to find oocytes that had their chromosome 4s positioned far enough out on the spindle to have detectable threads, in both D. simulans and introgressed sim-IV females.…”

“…To quantify this, we did preps under tightly controlled aging and dissection conditions and measured the 4-4 distances for oocytes from pure-strain D. melanogaster, introgressed sim-IV heteroand homozygotes, and pure-strain D. simulans (Figure 3). To limit consideration to 4-4 distances under comparable conditions of prometaphase and congression, we excluded those oocytes where other chromosomes besides the 4 were spontaneously nonexchange, as well as oocytes that were fixed while chromosomes were in transient configurations such as having both homologs on the same side of the spindle (Hughes et al 2009) or in the slippage configuration where the chiasmate autosomes are positioned end to end (Hughes et al 2011).…”

The Chromosomal Passenger Complex Is Required for Meiotic Acentrosomal Spindle Assembly and Chromosome Biorientation

“…However, an alternative model incorporates an important role for kinetochore microtubules (Dumont et al 2010). Kinetochore microtubules in oocytes have been inferred by Hughes et al (2011) and could be the cold-resistant karyosome-associated microtubules that we have observed (Salmon and Begg 1980;Rieder 1981). Whether the microtubules connect to the chromosomes though traditional endon kinetochore attachments or lateral attachments, we propose that these microtubules are bundled with central spindle microtubules to achieve biorientation.…”