Anisotropic Diffusion of Proteins in the Sarcoplasmic Reticulum of Skeletal Muscle

Abstract

The way proteins move inside crowded organelles is determined by both the connectivity of the organellar lumen and the mutual interactions between molecules. To probe diffusional movement inside the SR we used FRAP of three proteins expressed in mouse paw muscles: the biosensor D1ER and two fluorescently tagged forms of calsequestrin (Casq1). Longitudinal diffusion (parallel to the fiber axis) was evaluated by the rate of fluorescence recovery in a rectangular bleached region, 10 μm wide and long enough to span the fiber width (∼40 μm). Diffusion in the transversal direction was evaluated with a bleached region of the same dimensions, placed longitudinally, right at the myofiber axis. The FRAP rates measured thus for D4cpV-Casq1 in resting myofibers corresponded (by theory and simulations) to an effective longitudinal diffusion coefficient DL of 0,012 μm2/s, while the transversal coefficient DT was 0,026 μm2/s. The difference was statistically significant. Faster transversal diffusion was also found for EYFP-Casq1. Additionally, the measurements evinced the presence of a large fraction of immobile Casq1, presumably polymerized within terminal cisternae. As reported elsewhere, the immobile fraction of Casq1 decreased, while the diffusional mobility of Casq1 and D1ER increased upon Ca2+ depletion in the SR. Electron microscopic images of resting myofibers demonstrate a greater connectivity of SR cavities in the transversal direction (i.e. along terminal cisternae) than longitudinally, to an extent consistent with and probably explaining in full the observed diffusional anisotropy. Regardless of the SR Ca2+ content, the diffusion coefficient values found for these proteins are much lower than the lowest values reported for proteins inside the ER, which probably reflects the intricacy of the SR structure and the polymeric state of calsequestrin in resting skeletal muscle. Supported by NIH grants AR049184 and GM111254.