The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo - PubMed (original) (raw)
The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo
Sophie Quintin et al. EMBO Rep. 2003 Dec.
Abstract
The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule-related processes. In a screen for mutants affecting the first cell division, we isolated a temperature-sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk-2, a conserved serine/threonine kinase. The microtubule-related defects are due to the postmeiotic persistence of MEI-1, a homologue of the microtubule-severing protein katanin. In addition, P-granule distribution is abnormal in mbk-2 mutants, consistent with genetic evidence that mbk-2 has other functions and with the requirement of mbk-2 activity at the one-cell stage. We propose that mbk-2 potentiates the degradation of MEI-1 and other proteins, possibly via direct phosphorylation.
Figures
Figure 1
mbk-2 encodes a serine/threonine kinase required for microtubule-based processes in the one-cell Caenorhabditis elegans embryo. (A) Time-lapse differential interference contrast series from recordings of wild-type (WT), mbk-2(dd5ts) and mbk-2(RNAi) embryos. In this and subsequent figures, anterior is to the left and the bar represents 10 μm. Time (s) is relative to nuclear envelope breakdown (NEB). (a) Maternal (m) and paternal (p) pronuclei at opposite sides of the cell. (b,c) In WT, pronuclei meet before NEB (t_=0) whereas the male pronucleus undergoes NEB and sets up the spindle before pronuclear meeting in mbk-2(−). In most cases, the maternal pronucleus is eventually captured by the spindle (32/37 in dd5ts; 14/15 in RNAi embryos). (d) Unlike WT, the spindle forms transversely in the posterior in mbk-2(−). Arrowheads indicate spindle poles. (e) At the two-cell stage, multiple nuclei and ectopic furrows are visible in mbk-2(−). (B) Fixed embryos stained for α-tubulin (red) and DNA (blue) at prometaphase in WT and_mbk-2(dd5ts). The spindle axis is transverse and MTs less frequently reach the cellular cortex in mbk-2(dd5ts). (C) Average length of astral MTs in WT and mbk-2(dd5ts). MTs were visualized as in (B). The ten longest MTs from each centrosome were measured in five embryos of each genotype. (D) Schematic structure of the MBK-2.A protein. The grey box indicates the serine/threonine kinase domain, where the dd5ts mutation is located (asterisk).
Figure 2
Centrosome organization is normal in mbk-2 mutants but chromosome segregation is impaired. (A) Time-lapse GFP series from four-dimensional analyses of wild-type (WT) and mbk-2(dd5ts) embryos expressing histone and γ-tubulin::GFP. Time (s) is relative to nuclear envelope breakdown (NEB). Embryos are shown at corresponding stages to those in Fig. 1A. In the mutant, centrosome labelling (arrows) appears unchanged, but lagging chromosomes are visible from early anaphase to the two-cell stage (arrowheads). (B) Snapshots taken shortly after NEB from time-lapse recordings of WT and mbk-2(dd5ts) embryos expressing TAC-1::GFP. The mutant shows normal GFP expression. (C) Late-anaphase embryos stained for ZYG-9 (green) and DNA (blue). In the mutant, ZYG-9 expression is unaffected, but a failure in chromosome segregation is visible. (D) Distance between centrosomes (μm) in one-cell embryos versus time, observed in embryos expressing γ-tubulin::GFP as shown in (A). Four embryos per genotype were tracked. DIC, differential interference contrast.
Figure 3
mbk-2 microtubule defects result from persistence of MEI-1/katanin during mitosis. (A) Images from time-lapse movies of wild-type (WT), mel-26(RNAi), mbk-2(dd5ts) and mbk-2(dd5ts); mei-1(RNAi) anaphase embryos expressing histone::GFP and β-tubulin::GFP. Note the similarity between mel-26(RNAi) and_mbk-2(dd5ts)_ embryos, which both exhibit abnormal spindle positioning and lagging chromosomes. These defects are entirely suppressed in_mei-1(RNAi); mbk-2(dd5ts)_ embryos (bottom, right panel in A). Enlargement of the polar body indicates a meiosis failure in these embryos (left arrow), characteristic of mei-1(RNAi). The arrow at the right points to meiotic chromosomes that have been abnormally captured by the microtubules. (B) WT and mbk-2(dd5ts) early-anaphase embryos expressing MEI-1::GFP. The corresponding differential interference contrast (DIC) images are shown to the left. Note the presence of ectopic MEI-1::GFP on the spindle in the mutant.
Figure 4
mbk-2 activity is required for the proper localization of P granules. (A) Fixed embryos stained for microtubules (red), DNA (blue) and P granules (anti-PGL-1, green), in wild-type (WT), mbk-2(dd5ts),zyg-9(b244), mel-26(RNAi) and mbk-2(dd5ts); mei-1(RNAi) embryos. The arrowhead points to the enlarged polar body due to meiotic failure in the latter. The table shows the number of embryos of each genotype with a given type of P-granule distribution. (B) WT and mbk-2(dd5ts) embryos expressing PAR-2::GFP and PAR-6::GFP. The distribution of the cortical markers is unaffected in the mutant. DIC, differential interference contrast.
References
- Bellanger J.M. & Gonczy P. ( 2003) TAC-1 and ZYG-9 form a complex that promotes microtubule assembly in C. elegans embryos. Curr. Biol., 13, 1488–1498. - PubMed
- Bowerman B. ( 1998) Maternal control of pattern formation in early Caenorhabditis elegans embryos. Curr. Top. Dev. Biol., 39, 73–117. - PubMed
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