Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy.

Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with the cytoskeleton and mediate force transmission into the nucleoplasm. Despite their critical roles in fundamental cellular processes such as meiotic chromosome and nuclear positioning, the mechanism of LINC complex assembly in cells remains unclear. To begin to address this deficit, we recently developed z-scan fluorescence fluctuation spectroscopy (FFS) and brightness analysis as a method for quantifying the oligomeric states of fluorescent protein-tagged NE proteins including nesprins and SUN proteins. Since the homo-oligomerization of SUN2 is critical for its ability to interact with nesprins within the perinuclear space, the knowledge obtained through quantitative brightness experiments reveals important insights into the in vivo mechanisms of LINC complex assembly. Here we describe the procedure we use to determine the brightness of proteins in the NE of living cells. In addition to the measurement procedure, we discuss the instrumentation requirements and present the results of applying this procedure to measure the brightness of nesprin-2 and SUN2.