Research in the laboratory is directed at understanding meiosis in Drosophila melanogaster females. Our studies include several important aspects of meiotic pathway including: i) the pairing of homologous chromosomes, ii) the initiation of meiotic recombination (DSBs), iii) the repair of DSBs, and iv) the mechanism of homologous chromosomes segregation. Many of the important genes in this process are also involved in DNA repair or the fidelity of chromosome division in other cell types. Therefore, these studies will likely provide insights into the factors affecting genome stability in mitotic cells.
The SC is intimately involved in the process of meiotic recombination in most organisms, but its exact role remains enigmatic. Two putative SC proteins have been identified in Drosophila, C(3)G and C(2)M, that colocalize to meiotic chromosomes by immunofluorescence. Mutations in either gene cause defects in SC structure, DSB formation and meiotic crossing over. While neither gene is well-conserved at the amino acid level, the predicted secondary structure of C(3)G is similar to that of transverse filament proteins, and C(2)M is a distantly related member of the – kleisin family that includes Rec8, a meiosis-specific cohesin protein. We used immunogold labeling of SCs in Drosophila ovaries to localize C(3)G and C(2)M at the electron microscopic level (Figure 1). Using antibodies specific to the ends of each protein, we confirmed that C(3)G and C(2)M are components of the SC and that the orientation of C(3)G within the SC is similar to other transverse filament proteins. The N-termini of the C(3)G dimers are located in the middle of the SC and are connected by interacting coiled-coil segments to the C-termini that are located near or in the lateral elements. The C(2)M protein may be located intermittently along the SC, with its N-terminus located near the inner side of the lateral element close to the C-termini of C(3)G proteins. This distribution of C(2)M is consistent with the observation that in the absence of C(2)M, C(3)G localizes is discontinuous patches.
To detect meiotic DSBs, we have used antibodies that detect the phosphorylation of a Drosophila histone H2 variant, HIS2AV. HIS2AV is phosphorylated to maximum levels within minutes in Drosophila mitotic cells in response to DSB formation. The rapid onset of the phosphorylation makes g-HIS2AV a useful cytological marker for DSB formation. The foci can be counted to determine how many breaks are made in a wild-type meiotic cell (~20 per cell or 5 per chromosome arm). We have found that the g-HIS2AV foci are only present for a limited time during early prophase, indicating there is tight regulation of DSB formation. Formation of the majority of DSBs requires the SC proteins C(2)M and C(3)G.
Genetic and cytological data have shown that 60-80% of DSBs do not become crossovers, suggesting that the outcome of DSB repair is not random. To study the relationship between the number of DSBs and crossing over, we counted DSBs and scored crossing over in different mei-P22 mutants which express altered levels of MEI-P22 protein and exhibit different levels of DSBs formation. We found a linear relationship between DSB formation and crossing over, suggesting that a decrease in DSB formation is not compensated by the increasing the probability of a single break becoming a crossover. Hence, to ensure cross over formation sufficient numbers of DSBs have to be formed.
In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. We have found that a region similar to the anaphase central spindle functions beginning at prometaphase to organize acentrosomal spindles. This region is populated by interpolar microtubules, those which originate at the spindle-poles and engage in antiparallel interactions between the two half -spindles. These conclusions are based on the analysis of subito, which encodes a kinesin-like protein and ortholog of mammalian MKLP2. subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito localizes to the meiotic central spindle where antiparallel microtubules overlap. The meiotic central spindle appears during prometaphase and includes the precocious localization of passenger proteins such as AurB and Incenp. In the absence of Subito, central spindle formation is defective, AurB and Incenp fail to properly localize and the spindles are disorganized. These results indicate that Subito is required for establishing and/or maintaining the central spindle. We propose that the central spindle in Drosophila oocytes substitutes for the role of centrosomes in directing formation of the bipolar spindle.
Figure 1. EM micrographs of frontal sections of Drosophila SC labeled with antibodies to the (A) N-terminal domain of C(3)G, (B) C-terminal end of the coiled-coil of C(3)G (C) C-terminal globular domain of C(3)G, or (D) HA tag on the N-terminus of C(2)M. Some of the dark gold/silver particles are indicated by arrowheads. The light formaldehyde fixation necessary for immunolabeling is not as good for cellular preservation as traditional EM fixation techniques. Bar is 100 nm. E) Model of SC structure in Drosophila females.
Figure 2. Model for acentrosomal spindle formation in Drosophila oocytes. A) The chromosomes enter prometaphase clustered together in a ball and capture microtubules. At this time, SUB protein accumulates on antiparallel microtubules that are adjacent to the chromosomes. B-C) Elongation of the spindle could occur via microtubule capture and bundling, microtubule sliding or microtubule polymerization at the plus ends. The direction of microtubule elongation/bundling/sliding is dictated by the metaphase central spindle, which provides a “backbone” structure to the spindle. Organization of the kinetochore microtubules by the central spindle could occur through cross-linking interactions, examples of which are shown by arrows. Additional proteins bundle parallel microtubules and stabilize the poles. C)
Lab Members:
Dr. Kim S. McKim
Associate Professor
Janet Jang
Laboratory Researcher
Eric Joyce
Laboratory Technician
Taslima Rahman
Laboratory Technician
Sonam Mehrotra
Graduate Fellow
Jeffry Cesario
Graduate Student
Bethany Redding
Undergraduate Student
Nishit Shah
Undergraduate Student
Vinod Singaram
Undergraduate Student