Nesprin-1/-2 Ortholog ANC-1 Regulates Organelle Positioning and Subcellular Crowding in C. elegans

Organelle positioning is important in many cellular and developmental processes, especially for the function of polarized and spatially extended cells. For example, in mammalian myofibers, most nuclei are evenly spaced out at the periphery of the fiber, except for a few that group into small clusters anchored under the neuromuscular junction. Disruption of nuclear positioning is found in many muscular diseases. Nuclear positioning is mediated by the conserved bridge termed the LINC (Linker of nucleoskeleton and cytoskeleton) complex, which is formed by the interaction between inner nuclear membrane SUN domain proteins and the outer nuclear membrane KASH domain proteins. The SUN proteins pair with different KASH proteins which contains various cytoskeleton interacting domains, to perform distinct functions. For example, in Caenorhabditis elegans, SUN protein UNC-84 either interacts with the KASH protein UNC-83 to move nuclei during development, or the KASH protein ANC-1 to anchor nuclei in the hyp7 syncytium. Interestingly, UNC-83 KASH is shorter and lacks the conserved cysteine predicted to be important to stabilize SUN/KASH interaction by structural and molecular dynamic simulations. In collaboration with other lab mates, we showed that the conserved intermolecular disulfide bond plays important roles for UNC-84/ANC-1 mediated nuclear anchorage but is dispensable for UNC-84/UNC-83 mediated nuclear migration. In addition, the longer KASH containing the DDEY residues predicted to be associated with the membrane also is necessary for ANC-1 KASH to function. ANC-1 has been proposed to physically tether nuclei to the actin cytoskeleton through its C-terminal KASH domain and the N-terminal calponin homology (CH) actin-binding domains. However, I found that deletion of the KASH domain causes less severe nuclear anchorage defects than null mutations. Moreover, the short isoform anc-1b lacking CH domains is sufficient for hyp7 nuclear anchorage. It was completely unknown how ANC-1 functions without CH and KASH domains. Interestingly, other than nuclear anchorage defects, the ER, mitochondria, and lipid-droplets were also severely unanchored and sometimes moved throughout the cytoplasm in anc-1 but not unc-84 null mutants. Unlike most KASH proteins which are enriched on the nuclear envelope, I found that GFP-tagged ANC-1 was mostly colocalized with the ER. To understand the mechanism by which ANC-1 regulates organelle positioning, I used CRISPR/Cas9 gene editing to delete different domains of ANC-1 and found that the spectrin-like repeat structures and the transmembrane domain play important roles in organelle anchorage. Deletion of the spectrin-like repeat region disrupted the protein’s ER-localization. These results led to a cytoplasmic integrity model where ANC-1 localizes to the ER and functions in positioning nuclei, the ER, mitochondria, and likely other organelles in place. The cytoplasm is a crowded environment filled with macromolecules. To test if ANC-1 anchors organelles through regulating the intracellular crowding, I expressed and analyzed the diffusion of 40nm GEMs (genetically encoded multimeric nanoparticles) in C. elegans hypodermal and intestine tissues. The diffusion of the 40nm GEMs is significantly increased in the cytoplasm of anc-1 null mutants in both tissues, indicating that ANC-1 may regulate the crowding of the cytoplasm. Ribosome concentration has been reported to play an important role in cytoplasmic crowding. RNAi of small ribosome protein rps-15 or rps-18 caused nuclear anchorage defects. In addition, in anc-1 mutants, the ribosome distribution is altered. These results indicate that ANC-1 may regulate the biophysical properties of the cytoplasm through the ER and ribosomes