Presentation description

While abnormal nuclear morphology is a hallmark of cancer and other diseases, nuclear membranes are also dynamically remodeled to perform vital functions under physiological conditions. Inner and outer nuclear membranes form the nucleus and comprise the nuclear envelope (NE). The inner nuclear membrane (INM) protein LEM2 is active in constraining deformations at the NE, and de novo point mutations in this protein result in human diseases, including heart disease and Progeria, a rapid aging disease. However, the mechanism by which LEM2 constrains NE deformations remains unknown. To elucidate mechanisms through which LEM2 regulates NE morphology, I built and tested a biotin-based proximity labeling system to identify interactors of LEM2 to gain insight into its function. I created a LEM2 construct fused to TurboID, an engineered biotin ligase that produces a highly reactive biomolecule, resulting in neighboring proteins becoming biotinylated. I confirmed that my fusion construct localized with endogenous LEM2 and other INM proteins, resulting in a biotinylation pattern that overlaps with LEM2 by immunofluorescence microscopy. Specific proteins were biotinylated by my LEM2-TurboID fusion protein compared to the non-targeting control. To identify the specific protein interactors of the LEM2-TurboID fusion protein, I performed a pull-down to isolate and enrich biotinylated proteins and tested for known and putative interactors of LEM2 by western blot analysis. To gain a comprehensive list of LEM2 interacting partners, my samples will be sent for mass spectrometry, a technique that allows the identification of all enriched proteins present. Ultimately, my assay will yield unique biological clues and identify interactors of LEM2 that may function in regulating NE morphology, which could represent potential therapeutic targets. For instance, if specific proteins interacting with LEM2 play roles in pathogenesis, they could be targeted for drug development, leading to treatments for conditions associated with NE dysfunction.