Containing The Kash And Sun Function Of Structure And Function Domain Protein (drosophila And Mice),

KASH and SUN proteins are two kinds of nuclear envelope(NE) proteins bridging the cytoskeletons and nuclear envelope(NE),which play critical roles in nuclear migration and anchorage during multiple biological processes.In the past decade,the extensive investigation on KASH and SUN proteins has been carried out from lower to higher organisms using various systems from in vitro tissue culture cell systems to pathological analysis with mouse models.The knowledge of the biological functions and the molecular mechanisms of these two protein families are increasing very fast.Using msp-300^sz75 mutant fly and gene knockout mice for KASH proteins Syne-1 and Syne-2,SUN proteins SUN1 and SUN2,we have studied their essential functions in nuclear anchorage and migration during multiple biological processes from Drosophila to mouse.The investigation of Drosophila KASH proteins focused on the functional analysis of MSP-300 protein in oogenesis.During late stages of Drosophila oogenesis,the cytoplasm of nurse cells in the egg chamber is rapidly transferred("dumped") to oocytes,while the nurse cell nuclei are anchored by a mechanism that involves the actin cytoskeleton.This thesis reports that maternal MSP-300 plays an important role in the actin-dependent nuclear anchorage during the cytoplasmic transport.MSP-300 is distributed throughout the cytoplasm and accumulates at the nuclear envelope of nurse cells and the oocyte with an antibody against the C-terminus of MSP-300.A GFP fusion protein containing the C-terminal region of MSP-300 is sufficient to localize the protein on the nuclear envelope in oocytes.To eliminate the maternal effect during oogenesis,we generated homozygous germ-line clones of a msp-300 loss-of-function mutation in otherwise heterozygous mothers.In the mutant egg chambers that develop from such clones,cytoplasmic dumping of nurse cells is severely disturbed.The nuclei of nurse cells and the oocyte are mislocalized and the normally well-organized actin structures are severely disrupted.The investigation of mammalian KASH and SUN proteins is mainly focused on the functional analysis of Syne-2,Sun1 and Sun2 knockout mice in retinal development.Retinal progenitor cells can form all kinds of retinal neurons,and all of the proliferation and differentiation take place in a well-organized temporal sequence.Studies on Drosophila KASH/SUN proteins Klarsicht/Klaroid and zebrafish KASH protein syne2a have shown that these proteins play important roles in nuclear positioning and migration during retinal development.Studies performed in this thesis indicate that Syne-2,SUN1 and SUN2 proteins mediate the rod cell nuclear positioning and cone cell nuclear migration.In addition,SUN1 and SUN2 proteins might play a redundant function in cone cell nuclear migration during retinal development.Staining of the retinal sections with Syne-2,SUN1 and SUN2 antibodies,we found that Syne-2,SUN1 and SUN2 proteins are localized to the NE of most cell types in the mouse retina.Both HE and immunofluorescent staining revealed a severe loss of photoreceptors in the outer nuclear layer(ONL) and a group of mislocalized nuclei lying between the inner plexiform layer(IPL) and inner nuclear layer(INL).The mislocalized nuclei were identified to belong to rods both in Syne-2^-/- and Sun1^-/- mice with immunofluorescent staining. Physiological ERG test also implied potential problems in photoreceptors and some other cell types in adult Syne-2^-/- and Sun1^-/- mice.Immunofluorescent staining of specific cell markers in the retina displayed a severe loss of outer segment(OS) in both rods and cones in the retina of Syne-2^-/- mice while only a loss of rod OS only in the retina of Sun1^-/- mice.Furthermore,the normal cone cell nuclear migration is found to be blocked in the retinas from Syne-2^-/- and Sun1^-/-;Sun2^-/-;NSE-SUN1 mice.This thesis also describes the success generation of both Syne-1 and Sun1 conditional knockout mice.Both Syne-1/2 and Sun1/2 double knockout mice died soon after birth.These conditional knockout mice would be helpful for the further investigation of the KASH and SUN protein functions in various tissues and different organs.The Syne-1^Del/Del mice from Syne-1 targeted alleles exhibited myonuclear mislocalization defects in skeletal muscle.The Syne-1^Del/Del;Syne-2^-/- mice died immediately after birth as expected,but no obvious defects were observed on the Syne-1^Flox/Flox;Syne-2^-/- mice.Meanwhile, Sun1^Del/Delmice from Sun1 targeted alleles exhibited sterility phenotype similar to that of Sun1^-/- mice.Sun1^Del/Del;Sun2^-/- mice died shortly after birth,while no gross abnormalities were observed on Sun1^Flox/Flox;Sun2^-/- mice.Sun1 and Sun2 conditional double knockout mice were generated for study of the interatction between the two genes.Strikingly,the Sun1^Flox/-;Sun2^-/-;Nestin-Cre mice died in one or two days after birth due to a lack of the instinct of milk sucking.Histological analysis revealed that the cerebral cortex of Sun1^Flox/-;Sun2^-/-;Nestin-Cre mice displayed severe laminary defects,which was similar with those of Sun1^-/-;Sun2^-/- mice.Our studies on Drosophila MSP-300 and mouse Syne-1/2 and SUN1/2 proteins discovered their crucial functions in nurse cell and oocyte nuclei anchorage during cytoplasmic transport process in fly oogenesis and in photoreceptor cell nuclear migration during retinal development and neuronal migration of brain cortex in mice.These results would advance our knowledge on the molecular mechanism of nuclear positioning and migration during multiple processes,and provide new insights into the development of retina, brain and related diseases. Dentatorubral-pallidoluysian atrophy(DRPLA)is a dominant hereditary neurodegenerative disorder caused by the expansion of a poly-glutamine (poly-Q) repeat in Atrophin-1 protein.Ectopic expression of a poly-Q expanded human Atrophin-1 protein is sufficient to induce DRPLA phenotypes in transgenic mice.However,it is still unclear whether the dominant effect of poly-Q expansion is due to its functional interference with wild-type Atrophin-1 proteins,which are present in both human patients and transgenic mouse models.Here,we report the generation and analysis of an Atrophin-1 gene targeting allele.Homozygous mutant mice exhibit growth retardation and progressive male infertility,but no obvious signs of neurodegeneration. Disruption of wild-type Atrophin-1 gene neither blocked nor enhanced the neurodegenerative phenotypes caused by a poly-Q expanded transgene.Our result supports the model that the DRPLA disease is induced by poly-Q expanded proteins in a manner independent of any functional interaction with the non-poly-Q expanded proteins present in patients.