Nuclear migration and anchorage are central to many cellular events. We uncovered a conserved network of nuclear envelope proteins and force generators that mediate nuclear positioning. LINC (linker of nucleoskele- ton and cytoskeleton) complexes, which we discovered, maintain nuclear envelope architecture, mark the surface of nuclei distinctly from the contiguous ER, and were instrumental in the early evolution of eukaryotes. We address four gaps in our knowledge of the mechanisms regulating nuclear positioning. (1) How is the developmental switch between nuclear migration and anchorage mediated? We hypothesize that different LINC complexes are required for a nucleus to switch from migrating to being anchored. We propose that an intermolecular disulfide bond, which could be regulated by protein disulfide isomerases and/or the AAA+ ATPase torsin, is central to the switch. We further hypothesize that LINC directly interacts with the outer nuclear membrane to optimize the transfer of forces across the nuclear envelope. (2) How are nuclei anchored in large syncytial cells? It is important for nuclei to be evenly spaced so that multi-nucleated syncytia are able to act as a single unit. We recently found that ANC-1 anchors syncytial nuclei and mitochondria through unknown, LINC-independent mechanisms, and hypothesize that ANC-1 organizes the cytoplasm through microtubules. (3) How do nuclei favor one microtubule motor over another at different stages of development? The KASH protein UNC-83 mediates nuclear movements toward plus or minus ends of microtubules at differ- ent stages of development. We hypothesize that the choice is regulated by alternative isoforms of UNC-83 that differentially activate kinesin-1 motor activity. (4) How do nuclei deform to migrate through narrow spaces? Our data support a model where LINC complexes function parallel to branched actin networks to deform nuclei as they squeeze through narrow constrictions. Our experimental system is innovative because we can view live nuclei throughout development, including a tissue where 139 nuclei are in a single hypodermal syncytium and a second tissue where nuclei migrate through narrow constrictions as a normal part of development. Further- more, we have developed reagents essential to our future plans, including an array of point mutants in LINC complexes that separate function, cell-specific markers, a tissue-specific auxin-induced degron system, and over ten mutant lines from a forward genetic screen for defects in nuclear migration through constrictions. To complement our C. elegans genetic approaches, we also collaborate to confirm our findings in mammalian tissue culture cells and an in vitro microtubule motor assay with TIRF microscopy. Our studies are expected to determine how LINC complexes are regulated at molecular and biophysical levels, how the outer nuclear membrane is involved in force transmission, how giant KASH proteins organize the global cytoskeleton and position organelles, how UNC-83 mediates the choice between dynein and kinesin-directed nuclear move- ments throughout development, and how actin helps nuclei squeeze through constricted spaces.
Defects in nuclear positioning disrupt development in many mammalian tissues and nuclear migration through constricted spaces is important for metastasizing cancer cells and during inflammation. LINC complexes play many important cellular functions including nuclear positioning, homolog pairing in meiosis, DNA damage repair, wound healing, spermatogenesis, and the formation of nuclear pore complexes. The proposed research is relevant to public health because nuclear positioning and mutations in LINC components are associated with a wide variety of human diseases including muscular dystrophies, neurological disorders, Progeria, aneurysms, hearing loss, blindness, sterility, and multiple cancers. Thus, our studies are significant for understanding the molecular underpinnings of a wide variety of cell and developmental processes, and for elucidating how LINC complexes contribute to disease.
