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This dissertation, Characterization of Sad1/UNC-84 domain protein 2 (SUN2) by Ying, Liang, 梁瑛, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of Thesis Entitled Characterization of Sad1/UNC-84 Domain Protein 2 (SUN2) Submitted by Liang Ying for the Degree of Doctor of Philosophy at The University of Hong Kong in June 2006 The human SUN2 protein is a nuclear envelope protein with a conserved Sad1/UNC-84 domain at the C-terminus. SUN2 has also been reported to localize in endosomes stained with rab5, a small GTPase regulating the early endocytic pathway. To gain insights into the functions of SUN2, the subcellular localization and interacting partners of SUN2 were investigated in my study. By using an affinity purified antibody, SUN2 was detected at molecular weights of around 80 and 85 kDa and mainly localized on the nuclear envelope. An aberrant distribution of SUN2 in the cytoplasm was observed in the lamin A/C-deficient cells. The proper localization of SUN2 on the nuclear envelope was restored in lamin A/C-deficient cells transfected with lamin A. Therefore, SUN2 required lamin A for its nuclear envelope localization. By cotransfection and immunoprecipitation, SUN2 was shown to bind rab5. Overexpression of rab5 or its dominant-positive mutant, but not dominant-negative mutant, led to redistribution of SUN2 to early endosomes. Thus, recruitment of SUN2 to endosomal compartments was affected by rab5-GTP. The translocation of SUN2 to endosomal/lysosomal compartments was also induced by nutrient starvation. Furthermore, overexpression of SUN2 stimulated the transferrin uptake while suppression of SUN2 expression attenuated the process. In summary, our data suggested that SUN2 may participate in the endosomal/lysosomal pathway in addition to the early endosome stage and enhance the transferrin-receptor mediated endocytosis. Yeast two-hybrid screening was used to identify a novel interacting partner of SUN2, the major histocompatibility complex class II-associated invariant chain which contains two short endosomal-targeting motifs. The interaction between SUN2 and invariant chain was further confirmed by immunoprecipitation. Overexpression of the major isoform of invariant chain Iip33 led to SUN2 redistribution to endosomal compartments. Our data suggested that SUN2 specifically interacted with invariant chain not only in yeast but also in mammalian cells. By immunoblotting, SUN2 was detected in the mouse brain, heart and lung. Immunohistochemistry staining showed that SUN2 was specifically distributed in the Purkinje cells, hippocampal pyramidal neurons, and smooth muscle cells in the tunica media layer of vessels and internal organs. Using C2C12 myoblast cells, the expression of SUN2 was found to decrease upon skeletal muscle cell differentiation. Immunohistochemistry and immunoblotting analyses showed that SUN2 was expressed in cardiac, skeletal and smooth muscles during embryonic day 8.5 to day 15.5. After birth, the expression of SUN2 diminished in the heart and became restricted to the smooth muscles of great cardiac vessels during postnatal day 0 to day 42. The specific temporal-spatial distribution pattern of SUN2 suggested that SUN2 may play a role during muscle differentiation. In summary, SUN2 might serve as a structural and functional link between the nuclear architecture and the endosomal/lysosomal machinery. The effects of SUN2 on endocytosis and its temporal-spatial specific expression suggest the multiple roles of SUN2 in neurons and muscles. (461 words) DOI: 10.5353/th_b3661651 Subjects:

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