Megraw Lab Research

Productive cell division depends upon the accurate segregation of chromosomes at mitosis. The regulation and orchestration of this process is critical during growth and development, and its failure can lead to aneuploidy, resulting in cell death or oncogenesis. Key to this process, the accurate assembly of the bipolar spindle apparatus ensures that the cytoskeletal machinery necessary to accomplish this task is in place. In most animal cells, the centrosome is the primary microtubule-organizing center (MTOC). Because they act as dominant organizers of microtubules, centrosome duplication must be tightly regulated during the cell cycle or else chromosomal instability, al leading cause of tumor progression, will result. Centrosomes are also very important regulators of cell polarity for example in stem cell division and during cell migration. The structure of the centrosome and the proteins that comprise it are only recently becoming elucidated. MTOCs playother roles in the cell in addition to spindle assembly. Notably, in the organization of the oocyte during its development and during male meiosis and spermiogenesis.

Our group is focused on the application of genetic, genomic, molecular and cell biological approaches to address the important questions in this field. Drosophila melanogaster provides an excellent model organism for molecular genetic analysis of important problems in cell biology. With the completed sequencing of the Drosophila genome and the introduction of new techniques to study gene function such as RNAi and targetted gene knockouts, the fly is indeed a powerful model organism.

The centrosome is the most characterized MTOC in animal cells. Proteins that are known structural components of the centrosome are few. Even fewer have been analyzed genetically and their in vivo functions characterized. The centrosomin (cnn) gene encodes a centrosomal protein in Drosophila. Cnn is found at the mitotic centrosomes of all cell types examined (see Fig 1 for example). Our current research is focused on the functions of Cnn, the role of the centrosome as an organizer of both microtubules and actin, and the roles of MTOCs during oogenesis and spermatogenesis.

Figure 1. cnn mutants lack mitotic centrosomes and spindles appear meiotic in character. Wild type (a) and cnn mutant (b) neuroblasts were stained for microtubules (green), Cnn (red) and DNA (blue).

Cnn is a 1148 amino acid core centrosomal protein. Upon disruption of the microtubule array with drugs such as colchicine, Cnn is still localized to the mitotic centrosome. This feature distinguishes Cnn from MAP proteins or motors, which bind to microtubules and are sometimes also localized to centrosomes, but in a microtubule-dependent manner. Immuno-electron microscopy revealed that Cnn is a component of the electron-dense pericentriolar material that surrounds the centrioles.

Mutations in cnn prevent the assembly of mitotic centrosomes, and centrosomal proteins are not found at mitotic spindle poles in the mutants (1). Gamma tubulin, normally localized to both interphase and mitotic centrosomes, is not localized to cnn mutant spindle poles at mitosis, but is still localized to interphase centrosomes. Surprisingly, cnn mutant animals can develop into adults. Thus, zygotic development can be accomplished in the absence of functional mitotic centrosomes (2,3). This result was unexpected considering that centrosomes are found in all animals. Without centrosomes, mitotic spindles are organized and assembled by an alternative 'anastral' pathway. During anastral mitosis, microtubules grow initially on the chromosomes shortly following nuclear envelope breakdown, and then become organised into a bipolar spindle apparatus with focused spindle poles. This anastral pathway for mitosis was observed in cnn mutant animals and were efficiently phenocopied in cell culture using RNAi (2,4).

We had initially observed that cnn mutant cells appear meiotic in character with respect to their spindle assembly and cell divisions (Fig 1). In normal female meiosis, spindles assemble in the absence of centrosomes and, at anaphase, the smaller achiasmate fourth chromosomes segregate precociously. In cnn mutant cells the spindles assemble without centrosomes and the smaller fourth chromosomes are readily observed to move toward the spindle poles ahead of the other chromosomes at anaphase (Fig 1). In more recent experiments using mutations in proteins involved normally in meiotic spindle assembly, our data show that a meiotic division pathway may indeed be co-opted in the absence of centrosomes. These findings show that, although there is a limited requirement for mitotic centrosomes and meiotic motors in cell division, affecting both mechanisms for spindle assembly is deleterious. Thus, there is an alternative 'anastral' pathway for mitosis that is invoked upon disruption of mitotic centrosome function. We are currently dissecting the components of both pathways.

In oogenesis, Cnn is localized to the MTOC during oocyte development (5). The MTOC is poorly understood at the molecular level, and mutations that affect the MTOC specifically have not been described. Overexpression of Cnn disrupts the oocyte MTOC function. This disruption is likely a direct affect on the oocyte MTOC and can serve as a tool to further characterize this structure.