The centrosome/nuclear membrane coalition and its impact on bouquet formation in premeitotic germ cells

The centrosome/nuclear membrane coalition

In the early stages of meiosis, all the telomeres in the cell attach to the nuclear envelope and gather near the centrosome. This polarized chromosomal arrangement is known as the „chromosomal bouquet“ and is highly conserved among eukaryotes. Bouquet formation is a two step process involving the dispersed attachment of telomeres to the nuclear envelope and their subsequent movements towards the cluster site, which, in animals, is defined by the centrosome. During zygotene/early pachytene the bouquet dissolves in most organisms. The bouquet has been suggested to facilitate pairing of homologous chromosome ends within a limited volume in the nucleus. The mechanisms by which cells collect telomeres from disparate regions of the nucleus and pull each chromosome into a bouquet have remained elusive as well as the molecules that link the telomere chromatin complex to the centrosome. However, investigations in divers model organisms have identified proteins which may be involved in bouquet formation and the attachment of telomeric chromatin to the centrosome. For example in fission yeast bouquet formation depends on the telomere binding proteins Taz1 and Rap1. The telomere chromatin complex then is linked to the spindle pole body (SBP; the centrosome equivalent in yeast) via protein-protein interactions including Rap1 (at the telomere) and Sad1 (in the SBP). Sad1 shares a SUN domain with Unc-84, a protein required for the positioning of the nucleus in C. elegans. SUN domain containing proteins are also found in mammals. Two of them, SUN1 and SUN2 are inner nuclear membrane proteins. In C. elegans a germ-line specific SUN-domain nuclear membrane protein has been described which is essential for germ cell development. In mammals a closely related protein seems to exist but has not been investigated in detail. We have isolated the cDNA encoding this SUN-domain protein from mice and could show that the protein is found in the nuclear membrane. Its molecular analysis is currently under investigation.
The goal of the project is to identify proteins which are involved in anchoring of telomeric chromatin complex to the centrosome in mammalian premeiotic germ cells. Methods which will be applied are expression analyses by RT-PCR and Northern blots, generation of antibodies and immunocytological investigations, DNA transfection in cultured cells, identification of interacting proteins by YTH screen, pull down experiments and immunoprecipitation, functional analyses by generation of knock out mice as well as rescue experiments.