UC Davis

Confined cell migration is a critical part of many biological processes. It is essential in developmental events such as neurogenesis, it’s important during the immune response as immune cells must migrate through endothelial tissue, and it is also a hallmark of cancer metastasis. The rate of confined cell migration is usually limited by the rate of nuclear migration as the nucleus is usually the largest and most rigid organelle of the cell. There are several factors that contribute to nuclear deformability such as lamin composition, heterochromatin levels, and the cytoskeletal network. Much of what we know about confined nuclear migration and the factors that regulate this process comes from in vitro studies. Most of these in vitro experiments utilize fabricated 3D matrices with constrictions that cultured cells can migrate through. While these experiments have been vital in our understanding of how this process is regulated, it is still unclear how this process is modulated in vivo.Our lab has developed an in vivo model to study nuclear migration through constricted spaces by observing hypodermal precursor cells called P cells in Caenorhabditis elegans. During the early L1 larval stage, P-cell nuclei have to move from a lateral position to a ventral position by migrating through a narrow constriction that is about 5% the diameter of the nucleus. If this process is successful, the P cells are able to divide and develop into vulval cells and GABA neurons. The canonical pathway for P-cell nuclear migration is through the Linker of the Nucleoskeleton and Cytoskeleton (LINC), which is made up of the SUN protein (UNC-84) found at the inner nuclear membrane and the KASH protein (UNC-83) found at the outer nuclear membrane. UNC-84 binds to UNC-83 which is able to interact with microtubule motor proteins. I show that conditional knockdown of DHC-1 leads to a significant nuclear migration defect and therefore dynein is the main microtubule motor protein involved. Our lab has shown that disruption of the LINC complex leads to a temperature sensitive phenotype, suggesting there is an alternative pathway that functions parallel to the LINC complex pathway. From a forward-genetics screen, we were able to identify several putative actin regulators that are involved in this alternative pathway, highlighting the major role that actin plays in P-cell nuclear migration. One of the genes identified is cgef-1, which codes for a GEF that is able to activate CDC-42. I hypothesized that CGEF-1 activates CDC-42, which indirectly activates the Arp2/3 complex to generate branched actin. I show that CDC-42, the Arp2/3 complex, and non-muscle myosin II (NMY-2) are required for P-cell nuclear migration in the absence of the LINC complex at the restrictive temperature. I also show constitutively active CDC-42 is able to partially rescue the nuclear migration defect of the cgef-1(-); unc-84(-) double mutant. I propose future work to determine if other GEFs are involved in regulating CDC-42 and if CDC-42 regulates both the Arp2/3 complex and NMY-2. It would also be informative to observe localization of CGEF-1, CDC-42, the Arp2/3 complex, and NMY-2 to further understand the mechanism by which these proteins function. An area of interest for further studies would be to determine how much nuclear actin plays a role in confined nuclear migration.