The Cohesion Protein MEI-S332 Localizes the Condensed Meiotic and Mitotic Centromeres until Sister Chromatids Separate
Daniel P. Moore, Andrea W. Page, Tracy Tzu-Ling Tang, Anne W. Kerrebrock, and Terry L. Orr-Weaver. 1998 J. Cell Biol. 140: 1003-1012.
Reviewed by Anne Ford
Previous studies demonstrated that MEI-S332 is a protein essential to the cohesion of sister chromatids during Drosophila melanogaster spermatogenesis. Sister chromatid cohesion is extremely important for the proper division of genetic material between daughter cells. Other research located MEI-S332 on the chromosomes of meiotic dividing spermatocytes during the first meiotic division until anaphase I and during metaphase II. The protein is no longer found after metaphase II. The authors of this paper deemed several aspects in male Drosophila meiosis atypical. Therefore, predicting that female meiosis will be more pertinent to the study of other eukaryote's meiotic processes, they decided to investigate the presence and location of MEI-S332 during Drosophila female meiosis.
In order to determine when and where MEI-S332 isfound in female Drosophila meiosis, the experimenter's created a chimeric mei-S332-GFP gene which expresses a fusion protein with Green Fluorescence Protein at the amino terminus of the MEI-S332 protein. This allowed the authors to determine the location of MEI-S332 protein throughout meiosis in Drosophila oocytes. The DNA in the oocytes was also stained and appeared red when it fluoresced. Figure 1 shows that MEI-S332 is located on the chromosomes of the oocyte from metaphase I until the metaphase II - anaphase II transition. The position of the fluorescence pattern is also indicative of the MEI-S332 being in the centromeric position.
The first panel (A) clearly shows two green dots on the opposite sides of the chromatin during metaphase I. During early anaphase, MEI-S332 was found as eight green dots on the leading edge of the separating chromatin (Fig. 1B). This strengthen the argument that MEI-S332 is in the centromeric region because 1) there are eight pairs of sister chromatids at this stage (Drosophila 2n = 8) and 2) the leading edge is where the centromeres should be as the pairs of sister chromatids are pulled apart. MEI-S332 was present in the oocytes until the end on metaphase II and was not found on the chromosomes in anaphase II or post meiotic interphase (Fig. 1C-G). The results from this first figure are fairly strong. However, they could be outstanding if a positive control was used to demonstrate normal centromeric patterns.
The authors then dissected ovaries from transgenic flies to determine the location of MEI-S332 before metaphase I. The results in Figure 2 indicate that MEI-S332 first appears in several spots during prometaphase I and more specifically developmental stage 13, which coincides with spindle formation (Fig. 2A). The MEI-S332 then appears to congregate at the pole along the spindle in late stage 13 and in stage 14 (Fig. 2B-C). This figure was very well presented. The leftmost column for each stage shows the fluorescing patterns on the DNA, the MEI-S332 and the tubulin. The second, third and forth columns present the separate staining patterns of the DNA, MEI-S332 and tubulin respectively. It seems clear from the data presented that as the spindle elongates MEI-S332 becomes more concentrated as caps aligned with the poles.
After it was determined when, where and how MEI-S332 was located during the early stages of female Drosophila meiosis, the next question ask was what happened to MEI-S332 after the sister chromosomes separated in anaphase II. Specifically, the authors ask if MEI-S332 is degraded or just disassociated at the metaphase II - anaphase II transition. To determine the amount of MEI-S332 present in ovaries and embryos of Drosophila, western blot was performed using an anti MEI-S332 polyclonal antibody as the probe. Figure 3B presents the results. The authors claim that this banding pattern indicates that the amount of MEI-S332 does not decrease as ovaries mature and the amount of MEI-S332 even increase in 2-4 hour old embryos. This figure had a very well designed negative control. A nonsense mutation of the mei--S332 gene called mei-S3327 should lack the epitope for the polyclonal antibodies. The authors claim that for both the mei-S3327 ovaries and oocytes there was no banding. It appears to me that there might be a faint band in the mei-S3327 ovaries lane. Another weakness in this figure is the lack of a positive control band in each lane. Without this it is difficult to conclude that the negative control is working or that there is any relative difference in the amount of MEI-S332 in each lane. Without such a positive control the authors conclude that MEI-S332 is not completely degraded after metaphase II. I agree with the authors' interpretations, but I would like better controls.
The next step was to determine if the degradation of some MEI-S332 was been mask by continued translation of the protein. The authors, therefore, incubated oocytes in cyclohexamide, a translation blocker, and compared these to oocytes that were not exposed to the drug. These results, found in Fig. 3C, suggest that MEI-S332 is not been degraded because the level of MEI-S332 did not drop, even when translation was blocked. It appears that in this figure the authors decided to use a positive control in each lane which strengthens their claims that MEI-S332 is delocalized instead of degraded at the metaphase II- anaphase II transition.
The authors then continued to follow MEI-S332 through embryo development. Figures 4 and 5 do an adequate job of using fluoresence to demonstrate that the MEI-S332 protein is found on polar body rosettes, localized during mitotic metaphase, and no longer detectable at the metaphase - anaphase transition. This leads to the question, what is MEI-S332 doing during mitosis?
The authors address this question in the discussion section. They conclude that a mitotic phenotype would be the best tool to investigate the role of MEI-S332 in mitosis. Until this phenotype is found, I would suggest investigating the protein-protein interacts of MEI-S332 during mitosis. The best way to accomplish this would be to use a two hybrid system. With this system, the any protein that interacts with MEI-S332 would be purified. This could give researchers a better understanding of the mechanism by which MEI-S332 is localized to the centromeric region, delocalized at anaphase and how it functions to in sister chromatid cohesion in mitosis. This system might also give clues to other previously unknown functions of MEI-S332.
The two hybrid system should also be performed using cDNA libraries from every stage of oocyte meiosis. Again, this system would isolate the proteins that interact with MEI-S332. I would suspect that MEI-S332 interacts with a protein to move it to and from the centromeric region and several other proteins once it is there. Therefore, it would be very important to study the protein interactions at each stage of development. This system might also indicate what causes the MEI-S332 to dissociate from the centromeric region. The authors suggest that phosphorlization or an inhibitor might cause the cohesion release. The results of this test could give indication of the mechanism.
Finally, I would suggest that RT-PCR be used to study the expression rates of MEI-S332 throughout meiosis and mitosis. It would be interesting to see if mei-S332 was transcribed throughout development or only at the beginning of each division.
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