Mouse Sun Protein (sun1, Sun2) And Kash (syne-1, Syne-2) Protein In Muscle Nuclei Anchor, Brain Cells Migration, Retinal Development And Functional Studies As Well As In Response To Dna Damage

Nuclear migration and positioning are essential for many biological processes. Prior to our studies, several studies have shown the functions of microtubule and actin cytoskeletal system in nuclear migration and positioning. However, the roles of nuclear envelope proteins in these processes were largely unknown. SUN proteins and KASH proteins are two families of nuclear envelope proteins, which locate through the inner nuclear membrane and outer nuclear membrane, respectively. Although studies in yeast, worms, flies, zebrafish and cultured mammalian cells have revealed their roles in nuclear migration and positioning, their roles in mammalian development remained to be further studied. During the research for my Ph.D. thesis, I carried out genetic analysis in mouse and uncovered that SUN1, SUN2, Syne-1/Nesprin-1 and Syne-2/Nesprin-2 play critical roles in myonuclear anchorage, neurogenesis, neuronal migration, and DNA damage response.How the nuclei in mammalian skeletal muscle fibers properly position themselves relative to the cell body is an interesting and important cell biology question. In the syncytial skeletal muscle cells, more than 100 nuclei, named non-synaptic nuclei, are evenly distributed at the periphery of each cell, with 3-8 nuclei, named synaptic nuclei, anchored beneath the neuromuscular junction (NMJ). Our previous studies revealed that the KASH domain-containing Syne-1/Nesprin-1 protein plays an essential role in anchoring both synaptic and nonsynaptic myonuclei in mice. SUN domain-containing proteins (SUN proteins) had been shown to interact with KASH domain-containing proteins (KASH proteins) at the nuclear envelope (NE), but their roles in nuclear positioning in mice were unknown. As described in Chapter 2, I collaborated with Xiaochang and found that the synaptic nuclear anchorage is partially perturbed in Sun1, but not in Sun2, knockout mice. Disruption of 3 or ail 4 Sun 1/2 wild-type alleles revealed a gene dosage effect on synapiic nuciear anchorage. The organization of nonsynaptic nuclei was also shown to be disrupted in Sun 1/2 doubleknockout (DKO) mice. We further showed that the localization of Syne-1 to the NE of muscle cells is disrupted in Sun1/2 DKO mice. These results clearly indicate that SUN1 and SUN2 function critically in skeletal muscle cells for Syne-1 localization at the NE, which is essential for proper myonuclear positioning.Nuclear movement is critical during neurogenesis and neuronal migration, which are fundamental for mammalian brain development. Although dynein, Lisl, and other cytoplasmic proteins are known for their roles in connecting microtubules to the nucleus during interkinetic nuclear migration (INM) and nucleokinesis, the factors connecting dynein/Lis1 to the nuclear envelope (NE) remain to be determined. As described in Chapter 3, I collaborated with Xiaochang Zhang to find that the SUN-domain proteins SUN1 and SUN2 and the KASH-domain proteins Syne-1/Nesprin-1 and Syne-2/Nesprin-2 play critical roles in neurogenesis and neuronal migration in mice. We also showed that SUN1 and SUN2 redundantly form complexes with Syne-2 to mediate the centrosome-nucleus coupling during both INM and radial neuronal migration in the cerebral cortex. Syne-2 is connected to the centrosome through interactions with both dynein/dynactin and kinesin complexes.Nuclear movement relative to cell bodies is a fundamental process during certain aspects of mammalian retinal development. During the generation of photoreceptor cells in the cell division cycle, the nuclei of progenitors oscillate between the apical and basal surface of the neuroblastic layer (NBL). This process is termed as interkinetic nuclear migration (INM). Furthermore, newly formed photoreceptor cells migrate and form the outer nuclear layer (ONL). As described in Chapter 4, I collaborated with Juehua Yu to demonstrate that a KASH domain-containing protein, Syne-2/Nesprin-2, as well as SUN domain-containing proteins, SUN1 and SUN2, play critical roles during INM and photoreceptor cell migration in the mouse retina. A deletion mutation of Syne-2/Nesprin-2 or double mutations of Sun1 and Sun2 caused severe reduction of the thickness of ONL, mislocalization of photoreceptor nuclei, and profound electrophysiological dysfunction of the retina characterized with the reduction of a-and b-wave amplitudes. We also provided evidence that Syne-2/Nesprin-2 forms complexes with either SUN1 or SUN2 at the nuclear envelope to connect the nucleus with dynein/dynactin and kinesin molecular motors during the nuclear migrations in the retina. These key retinal developmental signaling results have advanced our understanding of the mechanism of nuclear migration in mammalian retina.DNA damage response and repair is critical for normal development and disease pathyology. Defects in DNA damage response and repair processes may cause gene mutation and genomic instability which could lead to immunodeficiency, cancer and premature aging. Chapter 5 describes my analysis of the functions of SUN1 and SUN2 in DNA damage response (DDR). I found that the Sun1 Sun2 double knockout MEFs grows slowly, and that the DDR signals were decreased in Sun1-/-Sun2-/- MEFs. In addition, I found that SUN1 and SUN2 interact with a Calcium-binding protein, Reticulocalbin-2 (Rcn2) for this function. I propose that through binding with Rcn2, SUN1 and SUN2 activate the ERK/MAPK pathway, which then phosphorylates the DDR proteins such as ATM andγ-H2AX. I am carrying out experiments to test this hypothesis. This study will help us understand the molecular mechanism involved in a group of human diseases called Laminopathy, which are caused by the mutations in nuclear envelope proteins.