Single-Molecule Displacement Mapping Unveils Sign-Asymmetric Protein Charge Effects on Intraorganellar Diffusion

Using single-molecule displacement/diffusivity mapping (SMdM), an emerging super-resolution microscopy method, here we quantify, at nanoscale resolution, the diffusion of a typical fluorescent protein (FP) in the endoplasmic reticulum (ER) and mitochondrion of living mammalian cells. We thus show that the diffusion coefficients D in both organelles are & SIM;40% of that in the cytoplasm, with the latter exhibiting higher spatial inhomogeneities. Moreover, we unveil that diffusions in the ER lumen and the mitochondrial matrix are markedly impeded when the FP is given positive, but not negative, net charges. Calculation shows most intraorganellar proteins as negatively charged, hence a mechanism to impede the diffusion of positively charged proteins. However, we further identify the ER protein PPIB as an exception with a positive net charge and experimentally show that the removal of this positive charge elevates its intra-ER diffusivity. We thus unveil a sign asymmetric protein charge effect on the nanoscale intraorganellar diffusion.

Automated summary

Using single-molecule displacement/diffusivity mapping (SMdM), we analyzed the diffusion of a typical fluorescent protein (FP) in living mammalian cells at nanoscale resolution. We found that the diffusion coefficients in the endoplasmic reticulum (ER) and mitochondrion are about 40% of that in the cytoplasm, with higher spatial inhomogeneities in the cytoplasm. Furthermore, we discovered that positive net charges impede diffusions in the ER lumen and mitochondrial matrix, while negative net charges do not.