Determining roles of the SUN domain proteins klaroid and Dspag4 in Drosophila development
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In eukaryotes, the process of nuclear migration is critical in fusion of haploid pronuclei after fertilization, in separation of daughter nuclei during mitosis, and in nuclear positioning in interphase cells. Experiments in several organisms have identified the basic protein requirements for nuclear migration and positioning: molecular motors that provide motive force; the cytoskeleton along which motors move nuclei, or to which the nuclei are anchored; and proteins of the outer and inner nuclear envelopes. These nuclear membrane proteins interact with the motors, the nuclear lamina and each other to effect nuclear migration and positioning. Proteins containing a SUN domain, which were first characterized in S. pombe Sad1 and C. elegans UNC-84, are inner nuclear envelope linkers of the nucleus to the cytoskeleton. In fungi, C. elegans, D. discoideum and vertebrates, these proteins are required not only for nuclear positioning, but also for maintaining the connection of the nucleus to the MTOC, for centrosomal duplication, for homologous pairing of chromosomes in meiosis, for distribution of nuclear pore complexes and for connecting the centrosome to chromatin to ensure genomic stability. The D. melanogaster genome has two genes, CG18584 and CG6589, which encode SUN domain proteins. The specific aims of my dissertation research were to generate null mutants in these genes, to characterize their null phenotypes, and to analyze where the genes are expressed. CG18584 = klaroid mutants are grossly normal, but adult eyes are mildly rough due to a defect in nuclear positioning that occurs during larval eye development. Klaroid protein is perinuclear in every cell of the eye, and functions by localizing the MTOC connector Klarsicht to the outer nuclear envelope. CG6589 = dspag4 null mutants are male sterile. In mature sperm, Dspag4 protein localizes rostrally to the sperm centriole. In the absence of Dspag4, most steps of gametogenesis occur normally, however, prior to the final steps of sperm maturation, the sperm nucleus dissociates from its centriole. Klaroid and Dspag4 thus have cellular roles typical for SUN domain proteins, and Dspag4 is unique in that its function is to attach nuclei to centrioles exclusively in maturing spermatids in the male germline.