Proper nuclear positioning is important to cell function in many biological processes during animal development. In certain cells, the KASH-domain-containing proteins have been shown to be associated with the nuclear envelope, and to be involved in both nuclear anchorage and migration. We investigated the mechanism and function of nuclear anchorage in skeletal muscle cells by generating mice with single and double-disruption of the KASH-domain-containing genes Syne1 (also known as Syne-1)and Syne2 (also known as Syne-2). We showed that the deletion of the KASH domain of Syne-1 abolished the formation of clusters of synaptic nuclei and disrupted the organization of non-synaptic nuclei in skeletal muscle. Further analysis indicated that the loss of synaptic nuclei in Syne-1 KASH-knockout mice significantly affected the innervation sites and caused longer motor nerve branches. Although disruption of neither Syne-1 nor Syne-2 affected viability or fertility, Syne-1; Syne-2 double-knockout mice died of respiratory failure within 20 minutes of birth. These results suggest that the KASH-domain-containing proteins Syne-1 and Syne-2 play crucial roles in anchoring both synaptic and non-synaptic myonuclei that are important for proper motor neuron innervation and respiration.

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 are evenly distributed at the periphery of each cell, with 3-8 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) have been shown to interact with KASH domain-containing proteins (KASH proteins) at the nuclear envelope (NE), but their roles in nuclear positioning in mice are unknown. Here we show that the synaptic nuclear anchorage is partially perturbed in Sun1, but not in Sun2, knockout mice. Disruption of 3 or all 4 Sun1/2 wild-type alleles revealed a gene dosage effect on synaptic nuclear anchorage. The organization of nonsynaptic nuclei is disrupted in Sun1/2 doubleknockout (DKO) mice as well. We further show 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.KASH domain ͉ Nesprin ͉ neuromuscular junction ͉ nuclear envelope protein ͉ Syne-1 P roper nuclear positioning relative to the cell body is important for many cellular processes during animal development. Mammalian skeletal muscle fibers are giant syncytial cells, each containing more than 100 nuclei. Each muscle fiber contains a neuromuscular junction (NMJ) that starts to form at approximately embryonic day 14 (E14) in mice (1). Following a multistep process involving the function of the agrin-MuSK-rapsynAChR pathway, the motor nerve terminal and the postsynaptic membrane become highly differentiated, ensuring reliable terminal transmission. The nuclei in the muscle fibers are positioned in a nonrandom manner, with 3-8 nuclei (synaptic nuclei) clustered beneath each NMJ and other nuclei (nonsynaptic nuclei) distributed evenly along the cell membrane (1, 2). How synaptic and nonsynaptic nuclei properly position themselves within each muscle fiber and the consequences of disrupting the nuclear positioning are attractive problems to investigate. Recent studies have indicated that KASH (Klarsicht/ANC-1/Syne homologue) domain-containing and SUN domain-containing proteins (KASH/SUN proteins) form a complex at the nuclear envelope (NE) for various cellular functions (3-6). Whereas a mammalian KASH protein has been found to have a critical function in myonuclear anchorage (7-9), the roles of SUN proteins in this process remain unclear.The SUN domain was first defined as a domain of shared homology between Sad1 in Schizosaccharomyces pombe and UNC-84 in Caenorhabditis elegans (10). SUN proteins are conserved inner nuclear membrane proteins containing at least 1 transmembrane domain and a C-terminal SUN...