UCSF

The nuclear lamina is an essential cellular structure that protects and organizes the genome. The lamina is a cage-like meshwork of intermediate filament lamin proteins and associated nuclear transmembrane proteins. Together, these proteins maintain the mechanical integrity of the nucleus and shape gene expression through development. While all human cells possess a nuclear lamina, mutations to its component proteins only affect some cell types. The molecular mechanisms that underlie these tissue-specific phenotypes are not understood, largely because we do not understand the function or regulation of nuclear lamina proteins across cell types. This dissertation investigates how the primary sequence of these proteins determines their stability and turnover, and how these properties are shaped by cellular context.We first examine emerin, a tail-anchored inner nuclear membrane protein, and show that its unique transmembrane domain determines its localization and expression. Through targeted mutagenesis, we discover sequences in the emerin protein that direct or oppose displacement from the ER into the secretory system, and find that C-terminal tagging artificially accelerates secretory trafficking—a cautionary lesson for interpreting the behavior of tagged proteins. To facilitate the stable expression of protein variants across cell types, we develop cassette exchange-based landing pads in human stem cells. This technology enables rapid and efficient single-copy integration of transgenes into the genome, which we leverage to perform the first saturation-scale mutagenesis screen in differentiated cardiac cells. We evaluate the effects of ~15,000 mutations on Lamin A protein abundance, revealing that destabilization is a predominant consequence of pathogenic mutations and that lamin protein stability relates to its polymerization capacity. We uncover cell type-specific consequences of destabilizing mutations to lamin polymerization interfaces, finding that cardiomyocyte nuclei are specifically deformed by the disruption of head-to-tail lamin assembly. Together, our results demonstrate that variant effects depend on both the structural impact on protein function and cellular context.