Illuminating SUN2/KASH Hetero-Complex Formation Within the Nuclear Envelope of Living Cells with Dual-Color Time-Shifted MSQ

Abstract

The nuclear envelope (NE) is a cellular compartment consisting of an inner and outer nuclear membrane separated by the perinuclear space. Research on cellular mechanotransduction identified the nucleus as a mechanosensor, which responds to the mechanical properties of the extracellular environment. Force transmission across the NE into the nucleoplasm is facilitated by linker of nucleoskeleton-and-cytoskeleton (LINC) complexes, which are conserved molecular bridges formed by the transluminal physical interaction of the outer and inner nuclear membrane KASH and the SUN proteins, respectively. Mammals encode six KASH proteins and five SUN proteins. In vitro, KASH proteins bind to the domain interfaces of trimeric SUN2 proteins resulting in the formation of a SUN2/KASH hetero-hexamer. Recently, we extended the application of single-color fluorescence fluctuation spectroscopy (FFS) to quantify protein oligomerization within the NE of living cells by developing mean segmented Q-factor (MSQ) and time-shifted MSQ (tsMSQ) to address experimental challenges unique to the environment of the NE. Using FFS, we demonstrated that SUN2 trimerizes in vivo, whereas the luminal SUN-binding KASH peptide of the KASH protein nesprin-2 (KASH2) remains monomeric. To be able to identify the oligomerization of SUN2/KASH within the NE of living cells, we describe here the development of dual-color (DC) tsMSQ. We first validated DC tsMSQ using nuclear envelope proteins tagged with EGFP and mCherry as model systems. Finally, we applied DC tsMSQ to detect the formation of hetero-complexes of EGFP-tagged SUN2 and mCherry-tagged KASH2. The development of DC tsMSQ will enable future efforts aimed at the mechanistic dissection of LINC complex assembly and its regulation within the NE as well as investigations of binary protein systems within this important sub-cellular compartment. This work has been supported by a grant from the NIH (R01 GM64589).