CV Science

thomas rubin PhD Researcher

Course and current status

Scientific summary

We are studying the potential role of the cytoplasmic cytoskeleton in regulating chromosome organization during the early steps of meiosis in the germarium.

Our lab has a longstanding interest in investigating the relationships between microtubules and the early stages of meiosis 18. How can this cytoplasmic cytoskeleton influence nuclear organization? A direct link between the cytoplasmic and nuclear sides remains mysterious. A first cue came very recently when we found that homologous chromosomes start to pair when reaching the nuclear envelope in the mitotic zone.  Once at the membrane, centromere movements are highly dynamic and we recently demonstrated that these movements are caused by rotations of the entire nucleus driven by microtubules and the SUN/KASH complex in females (Christophorou et al, Nature Cell Bio, 2015).

Meiosis is a two-step cell division process (meiosis I and meiosis II) generating gametes. During meiosis I, each chromosome has to find its homologue in order to pair, synapse and initiate recombination and chromatin exchange between homologues. These events lead to the correct segregation of each homologue into separate daughter cells. Errors in any of these processes can result in aneuploidy, which leads to severe birth defects and miscarriages. In C. elegans, chromosome pairing depends on movements driven by cytoplasmic forces that act via members of the SUN and KASH domain proteins at the nuclear membrane (Penkner et al., 2009; Sato et al., 2009). Following homologue chromosomes pairing, synapsis takes place, a process that involves the formation of the synaptonemal complex (SC), a proteinaceous structure that forms between chromosomes and reinforces pairing. Drosophila is a powerful genetic system and the initiation of meiosis in germinal cells oogenesis is a useful model to study the mechanisms regulating homologue chromosome pairing and synapsis. In the germarium a series of mitotic divisions occur that give rise to a cyst of 16 cells interconnected by a cytoplasmic structure called the fusome (de Cuevas and Spradling, 1998). Among the cyst cells, one of them will differentiate as the oocyte while the rest of cells will adopt a nurse cell fate (Huynh and St Johnston, 2004). When the cyst has formed, meiosis, as seen by the formation of the SC is initiated in all 16 cells of the cyst (Carpenter, 1975; Huynh and St Johnston, 2000; King, 1970). Gradually meiosis becomes restricted to the future oocyte, while the 15 sister cells become nurse cells and lose the SC.

We have recently shown that meiosis starts during the preceding mitoses in the fly germarium (Christophorou et al. PLoS Genetics, 2013)! We showed that in contrast to every cell types described so far in Drosophila, homologous chromosomes are not paired in germline stem cells. We further showed that during the differentiation of the daughter cell of the stem cell, homologous chromosomes become progressively paired. One surprising result is that this pairing occurs during the four mitosis preceding the entry into meiotic prophase. So, these mitoses “pre-pair” chromosomes for meiosis.

In addition, we uncovered parts of the molecular mechanisms underlying this novel process by identifying two components of the synaptonemal complex (i.e. considered specific to meiosis) expressed during the four mitoses (Christophorou et al. PLoS Genetics, 2013). These two components (C(3)G/Zip1 and Corona) localize at the chromosome centromeres in the mitotic region, and homologues pairing is greatly affected in their absence. Our results thus demonstrate that there is an active and de novo pairing of homologous chromosomes before entry in meiosis. In germ cells, meiosis is thus intimately linked with mitosis. Our results challenge current models and should change people’s thinking of chromosome organization during entry in meiosis.

Cell biologists tend to study cytoplasmic and nuclear organizations separately. However, increasing evidence suggests that cytoskeletal forces can directly influence chromosome organization within the nucleus. The pairing of meiotic chromosomes is a perfect case study to address this biological question. Indeed, at this stage, each chromosome needs to move within the nucleus to pair with their unique homologue, while the nuclear envelope is still intact. This question has been investigated in a few model organisms and has revealed a bewildering diversity of mechanisms such as actin-driven telomere movements in budding yeast, microtubule-driven horsetail motion in fission yeast, and very recently telomere rotations in mouse spermatocytes (Shibuya et al., 2014, Nat. Cell Bio). Surprisingly, this question remains completely unexplored in Drosophila, despite being a leading model system to study meiosis. One reason is that meiotic pairing was not known to even exist in Drosophila, as it was believed that homologous chromosomes were always paired in somatic and germline cells.

We and others have shown very recently that instead chromosomes were not paired in germline stem cells, and required pairing before entering meiosis (Christophorou et al. PLoS Genetics, 2013 and see also Cahoon and Scott Hawley, PLoS Genetics, 2013). It opened a complete new field to understand the underlying cellular mechanisms. We have developed fast and ultra-fast live-imaging of these stages, based on techniques we had pioneered (Fichelson et al., 2009, Nat. Cell Bio), to investigate pairing in Drosophila. We found that meiotic nuclei performed cycles of complete rotations required for chromosome pairing. We were able to photo-tag with a laser any subpart of the nucleus and characterize quantitatively that nuclei were rotating as units, which is a novel phenomenon in meiosis to our knowledge. In addition, we have uncovered the molecular mechanisms required for nuclear looping and chromosome pairing.

We demonstrated that:

1) the force-generators are the microtubules and the motor Dynein;

2) the force-transmitters at the nuclear envelope are the Drosophila SUN/KASH homologues, Klaroid and Klarsicht respectively. We showed that they link chromosome centromeres to the microtubule cytoskeleton;

3) we showed that the dynein- interacting protein Mud (NuMA in vertebrates) colocalizes with KASH and Dynein at the nuclear envelope and that Mud is required to maintain the integrity of the nuclear envelope and to assemble the synaptonemal complex. We thus identified a novel function for Mud/NuMA, which is to resist mechanical forces exerted on the nuclear envelope during nuclear rolling.

Overall, our study reveals a novel cellular phenomenon, which is rolling nuclei in Drosophila, and uncovers a novel pathway (centromere/SUN/KASH/Mud/Dynein), which transmits and resists cytoplasmic forces during meiosis. (Christophorou et al. Nature Cell Biology, 2015)