Biophysical Society Thematic Meeting | Singapore

Mechanobiology of Disease

Poster Abstracts

47-POS Board 47 LINC Complex Assembly Within The Nuclear Envelope Of Living Cells Jared Hennen 2 , Cosmo A. Saunders 1 , Jochen D. Mueller 1 , GW Gant Luxton 2 . 1 University of Minnesota, Minneapolis, MN, USA, 2 University of Minnesota, Minneapolis, MN, USA. LINC (linker of nucleoskeleton and cytoskeleton) complexes enable the direct transmission of mechanical forces across the nuclear envelope and into the nucleoplasm, which is required for wholesale positioning of meiotic chromosomes and nuclei. LINC complexes conserved molecular bridges that span the nuclear envelope and consist of the inner and outer nuclear membrane KASH and SUN proteins, respectively. KASH proteins interact with the cytoskeleton within the cytoplasm, while in the nucleoplasm SUN proteins interact with nuclear lamins and chromatin. Within the PNS (perinuclear space), KASH and SUN proteins directly interact to form the core of the LINC complex. Recent structural studies revealed that SUN2 forms a mushroom-like homo-trimeric structure with a stalk of three coiled-coils and a globular head that recruits three KASH proteins that bind in three deep hydrophobic grooves formed between adjacent SUN2 protomers. Despite this significant structural insight into LINC complex assembly, the in vivo relevance and conservation of SUN homo-trimerization remain unclear. Here, we apply z-scan FFS (fluorescence fluctuation spectroscopy), a biophysical imaging-based approach with single-molecule sensitivity, to quantify interactions within the PNS of living cells by brightness analysis. Consistent with in vitro studies, we demonstrate the existence of SUN2 homo-trimers within the PNS and identify structural requirements for SUN2 homo-trimerization. We then show that within the PNS, SUN1 can form higher-order homo-oligomers than SUN2. Finally, we uncover environment-dependent differences in SUN protein oligomerization suggestive of previously unidentified mechanisms for the regulation of LINC complex assembly and function. Taken together, our results establish z-scan FFS as a powerful tool for studying LINC complex assembly within the nuclear envelope of living cells. This information will be critical for understanding the functional consequences of mutations in KASH and SUN proteins associated with human diseases including cancer and muscular dystrophies.

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