Monday, December 30, 2024
12/30/2024
PROJECT ABSTRACT The nuclear envelope is a distinct domain of the endoplasmic reticulum (ER) that serves to surround and protect the genome. The unique identity of the nuclear envelope is determined by a subset of integral membrane proteins that execute its distinct functions. After each open mitosis, the nuclear envelope must regain its identity as it forms from ER-derived membranes. In the current funding period, we showed that regulation of the phosphatidic acid phosphatase lipin by the nuclear envelope-enriched protein phosphatase CTDNEP1 restricts ER membrane biogenesis, maintains the lipid composition of the nuclear envelope and contributes to the sealing, size and protein identity of nuclear envelope. In this project, we want to understand how the lipid composition of the nuclear envelope is established to control its formation and functions. Our proposed work uses vertebrate tissue culture cells to define (1) how CTDNEP1 itself is targeted to the nuclear envelope to establish the lipid content and promote the formation of the nuclear envelope, (2) how local lipid metabolism orchestrates membrane remodeling during nuclear formation and (3) how membrane sensing by nuclear envelope-associated proteins contributes to the formation and function of the nuclear envelope. Aim 1 will characterize the nanoscale organization of CTDNEP1 at the nuclear rim and determine the mechanism that promotes its selective targeting, retention and stabilization at nuclear envelope subdomains. This will determine how CTDNEP1 recognizes the nuclear envelope to drive lipid identity. To understand how lipid metabolism in turn controls nuclear envelope formation, we will determine the lipid species (eg head group and acyl chain composition) necessary for nuclear membrane remodeling using lipid biosensors, advanced imaging, lipidomics, and candidate screens of lipid modifying enzymes. Aim 2 builds on our preliminary data in which we uncovered putative membrane-targeting amphipathic helices (AHs) in known and previously uncharacterized nuclear envelope-associated proteins. We will directly follow up on two proteins with membrane binding amphipathic helices to determine the significance of membrane binding to their functions at the nuclear envelope. We will also complete the screen to determine the putative amphipathic helices for each protein that bind membranes in vitro and the inner nuclear membrane in vivo. This will allow us to determine if there is a unique, shared code for inner nuclear membrane binding via amphipathic helices. The completion of this work will elucidate how a unique lipid composition in established at the nuclear envelope and how the distinct functions of the nuclear envelope are modulated by lipid metabolism. Defects in the formation of the nuclear envelope from ER-derived membranes leads to genome damage common to cancer. Mutations in nuclear envelope proteins cause human dystrophies including muscular dystrophy and progeria. Thus, an understanding of how the nuclear envelope establishes and maintains its distinct structure and identity will inform on mechanisms of diseases that range from cancer to accelerated aging.
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