Brightness Characterization of SUN1 and SUN2 by Z-Scan Fluorescence Fluctuation Spectroscopy

Abstract

Fluorescence fluctuation spectroscopy (FFS) has been used to quantify the stoichiometry of soluble proteins in the cytoplasm of cells by brightness analysis. The development of z-scan FFS broadened the capability of brightness analysis to include proteins distributed across stratified layers, such as the cytoplasm and the plasma membrane. Here, we use z-scan FFS to study proteins that reside in the nuclear envelope/perinuclear space (PNS). Experimentally, we place a 20 amino acid signal-sequence in front of EGFP to drive translation into the lumen of the NE. The brightness of EGFP is determined by performing z-scan FFS measurements where corrections are applied for both the thin-layer geometry and coexcitation of adjacent layers. We then generated a tandem dimeric EGFP protein to establish a model for calibrating brightness and stoichiometry. Finally, we utilize our technique to study the assembly of the inner-nuclear membrane proteins SUN1 and SUN2 within the PNS. SUN proteins are transmembrane proteins that interact with nuclear lamins in the nucleoplasm and with cytoskeletal-interacting, outer nuclear-membrane nesprin proteins within the PNS. Together, SUN and nesprin proteins form the linker of nucleoskeleton and cytoskeleton complex, which is critical for nuclear positioning and movement. The crystal structure of SUN2 reveals a trimer composed of SUN domains and a stalk of coiled-coils. Each SUN trimer binds three nesprin proteins. Currently, evidence for this structure in living cells does not exist nor does stoichiometric information for SUN1. Here, we show that the lumenal regions of both SUN1 and SUN2 oligomerize in a regulated manner within the PNS. SUN2 oligomerizes into trimers while SUN1 forms even higher order oligomers. Taken together, our results establish z-scan FFS as a powerful method for the biochemical and biophysical analysis of nuclear membrane proteins within living cells.