Date of Award
5-2-2025
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Nucleocytoplasmic transport fundamentally regulates cellular phenotype and function across tissues in the body. In epithelial tissues, various processes—including embryogenesis, tissue repair, and cancer invasion—require cells to go from a homeostatic, mostly stationary and solid group to a fluidlike migratory collective. Underlying this tissue destabilization are two physical transitions: the epithelial-mesenchymal transition (EMT) and the unjamming transition (UJT). EMT comprises the loss of cell-cell junctions, front-back cell repolarization, and elevated migration. In contrast, UJT preserves cell-cell junctions, but cell elongation and migration fluidity more significantly increase. While nucleocytoplasmic transport forms the basis of EMT, UJT has only recently gained recognition, and its underlying nucleocytoplasmic transport mechanisms are still being revealed. Nuclear export inhibition (NEI) offers a conducive method for studying these transitions by directly interfering with nucleocytoplasmic transport. NEI works by binding to a nuclear export receptor, thereby inhibiting the transport of its cargos out of the nucleus. In this way, NEI facilitates study of the retained cargos and their impact on cellular outcomes. NEI commonly targets the exportin CRM1, which has over 370 known cargo proteins, many of which regulate epithelial cell phenotypes and cancer progression. While CRM1-based NEI has been studied as a cancer therapeutic for its effects on growth and proliferation, reports covering effects in healthy cells are limited. Preliminary experiments for this body of work indicated that CRM1-based NEI induces EMT and apparent UJT in healthy cells. Therefore, subsequent studies leveraged NEI as a tool to explore fundamental EMT and UJT questions in different contexts. EMT is regulated by the nucleocytoplasmic transport of core (e.g., SNAIL, TWIST, ZEB) and affiliated EMT factors (e.g., YAP, IκBα). Multiple nucleocytoplasmic proteins have been individually characterized as switches, either promoting or inhibiting EMT. Yet, the combined interaction of opposing switch-like proteins has not been investigated. Chapter 2 explores how these opposing E-M switch-like proteins interrelate, using NEI to understand their combined influence on cell phenotype. UJT arises from increased cellular propulsion during conditions of maintained cell-cell adhesion. However, the intracellular processes that enable increased migration fluidity during UJT are not fully understood. Chapter 3 applies NEI to study the physical and biological mechanisms underlying fluidlike migration. Experiments investigate whether decreasing cytoplasmic viscosity facilitates migration fluidity, with particular consideration for potential connections to glycolytic metabolism. Measurements further examine how glycolytic metabolism might be linked to actin fiber formation to drive fluidlike migration, surveying involvement of the FMRP/CYFIP1/eIF4E/cofilin signaling axis. Finally, the analyses explore whether Fragile X Messenger Ribonucleoprotein (FMRP) could exhibit mechanosensitive nuclear localization, potentially triggering the fluidlike adaptation of cells to their physical environment. Together, these experiments reveal new biophysical mechanisms regulating epithelial migration. Chapter 2 demonstrates that NEI induces concurrent epithelial-mesenchymal states, leading to an expanded model of EMT. Chapter 3 classifies highly fluid migration that does not align with the standard protein and morphology markers for EMT or UJT. Results indicate that this fluidity might stem from increasing cellular deformability, potentially regulated by mechanosensitive FMRP signaling. Clinically, both studies highlight significant off-target effects of NEI, raising additional considerations for therapeutic use. Overall, the findings enhance our understanding of epithelial transitions by expanding traditional migration models, and the analyses introduce new questions concerning epithelial function in health and disease.
Language
English (en)