Mapping the Proteome of the Outer Nuclear Envelope — 13p — Margaret Ludwig, Danielle May

The nuclear envelopathies are a group of genetic disorders caused by mutations in the genes  encoding lamins and nuclear envelope-associated proteins that lead to a variety of rare  diseases including muscular dystrophies, lipodystrophies, neuropathies and progeria. An  understanding of the proteome of the nuclear envelope is essential to uncover the cellular  mechanisms of these diverse diseases, information that is critical to develop targeted  therapeutics. While we have well-developed approaches to map the proteome of the inner  nuclear membrane (INM), there have been limited options to map the proteome of the outer  nuclear membrane (ONM). The majority of proteins known to enrich on the OMN are the KASH  domain constitutions of the LINC complex that are selectively retained on the ONM by  association with the INM SUN domain proteins and function to mechanically link the  nucleoskeleton to the cytoskeleton. We have developed an approach to identify ONM proteins  using protein proximity labeling. Fusion of an ONM-targeted TurboID biotin ligase to the KASH  domain protein Nesprin-3 that lacks its cytoplasmic binding domain can biotinylate proteins on  the ONM for subsequent isolation and identification. A control TurboID-Nesprin 3 that lacks the  KASH domain and cannot enrich on the ONM allows us to identify proteins enriched on the  ONM. Using this tool in mouse myoblast cells we have identified all five of the known ONM  enriched proteins. We have also identified centrosomal proteins that likely mediate the  association of this structure with the NE. Finally, we have identified a few proteins not known to  accumulate at the ONM. We have also developed tools to probe the proteome of the ONM in  diverse cell types, including human skin fibroblasts, lung cancer derived cells and  neuroblastoma cells. Using these new tools, we will be able to assess the protein constituency  of a broader range of cell types with the goal to identify novel mechanisms by which the ONM  contributes to normal cellular functions and nuclear envelope-associated diseases.

Sanford Research
Dr. Kyle Roux