The nuclear envelope is a dynamic boundary that separates the nuclear and cytoplasmic compartments of the cell, as well as the host of an integrated protein network responsible for regulating nuclear morphology, stability, positioning, migration, and genome structure. Failure of the nuclear envelope or its associated proteins, the nuclear lamina, to coordinate nuclear movement or respond to force leads to developmental defects and disease through mechanisms that remain unclear. Here, I investigate intranuclear dynamics during nuclear migration through confined spaces using Caenorhabditis elegans and characterize the roles of nuclear envelope-associated proteins in cell migration and dystrophic disease.

Cells migrating through confined spaces are limited by the movement of their nuclei, which undergo massive deformations to squeeze through constrictions. The force used to move nuclei is transmitted from molecular motors to the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, formed when cargo-adapter KASH-domain proteins at the outer nuclear membrane and lamina-bound SUN-domain proteins at the inner nuclear membrane bind within the perinuclear space. LINC complexes transduce this force to nuclear lamins, the meshwork of intermediate filaments that interact with inner nuclear membrane-associated proteins and chromatin to regulate nuclear stiffness, genome organization, and gene expression. Mutations in either LINC or lamin causes a series of heterogeneous tissue-specific disorders known as laminopathies. Laminopathies have a wide range of phenotypes, and many of their causative mutations and mechanisms remain to be characterized.

I developed diagnostic models for identifying and characterizing disease variants of striated muscle laminopathies by editing pathogenic missense variants or variants of uncertain clinical significance found in human lamin into homologous sites within the single C. elegans lamin gene. Pathogenic variants and one variant of uncertain clinical significance caused various defects in viability, motility, nuclear migration, and nuclear envelope integrity that resemble human dystrophic disease phenotypes. I also identified a separation-of-function allele with a severe nuclear migration defect that fails to recruit LINC complex components to the nuclear envelope. I developed a scoring system for classifying variants based on the number and severity of phenotypic defects in order to predict variant pathogenicity. Overall, these studies provide C. elegans models in which to interrogate nuclear dynamics in human laminopathic disease and propose potential mechanisms involved in lamin-associated striated muscle disorders.

In vitro models for cell migration and laminopathic disorders show dynamic changes in both lamin and chromatin, and have revealed roles for each in attuning nuclear deformability, migration rate, and nuclear envelope integrity. However, there is no evidence that chromatin regulates nuclear mechanics independently of lamin during cell migration in vivo. I used C. elegans hypodermal precursor cells, or P cells, which migrate into a constriction between the muscle and cuticle that comprises a fraction of the nuclear diameter, as an in vivo model for confined nuclear migration. To test whether chromatin was important for P-cell nuclear migration, I targeted CEC-4, an inner nuclear membrane protein responsible for anchoring condensed heterochromatin to the nuclear periphery. Knocking out CEC-4 in the absence of other nuclear migration pathways resulted in P-cell nuclear migration failure in all conditions assayed. Because knockout of CEC-4 does not affect gene expression, this data indicates a mechanical role for peripheral heterochromatin in constrained nuclear migration in vivo. Removing either the MET-2 or SET-25 methyltransferase, which each deposit repressive markers necessary for chromatin to interact with CEC-4, produced a similar failure in nuclear migration in the absence of other P-cell nuclear migration pathways, as did ablating one copy of the JMJD-1.2 demethylase. I therefore identified a role for epigenetic machinery in regulating nuclear dynamics during confined nuclear migration and established the importance of heterochromatin localization at the nuclear envelope in nuclei squeezing through constrictions. All together, this data demonstrates multiple roles for inner nuclear envelope components, including LINC, lamin, and heterochromatin, in regulating nuclear mechanics during normal developmental events such as cell migration and in disease.