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

  • ️Sun Jan 01 2023

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

Limin Xiang et al. Nano Lett. 2023.

Abstract

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 ∼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.

Keywords: diffusion coefficient; intraorganellar diffusion; nanoscale diffusivity mapping; protein net charge; single-molecule imaging; super-resolution microscopy.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.

High-content single-molecule statistics and super-resolution mapping of intraorganellar diffusivity. (a) Schematics: Tandem excitation pulses of τ = 500 μs duration are applied across paired camera frames at a Δt = 1 ms center-to-center separation, and this scheme is repeated ~104 times to enable local statistics. (b) Example single-molecule images captured in a tandem frame pair, for ER-Dendra2 diffusing in the ER lumen of a live COS-7 cell. (c) Two-dimensional plot of the vectorial 1-ms single-molecule displacements accumulated for an ER-tubule segment [white arrows in (e,f)], from which a principal direction θ of 141° is assessed. (d) Distribution of the displacements in (c) projected along θ. Blue curve: maximum likelihood estimation (MLE) fit to a one-dimensional diffusion model, yielding D = 8.9 μm2/s. (e) Color map presenting the calculated local θ across the imaged COS-7 cell. (f) pSMdM super-resolution D map via local fitting along the local principal directions. Inset: Pooled distributions of the 3.1×105 single-molecule displacements in the sample, each projected along its local θ. Blue curve: MLE fit yielding D = 8.5 μm2/s.

Figure 2.
Figure 2.

pSMdM quantification of intra-ER diffusion and charge effects. (a,b) Representative pSMdM super-resolution D maps for ER-Dendra2 in the ER lumen of live COS-7 cells, after Brefeldin A treatment (a) or ATP depletion (b), respectively. (c) pSMdM-determined D values of ER-Dendra2 in untreated (Org), Brefeldin A-treated (BA), and ATP-depleted (-ATP) COS-7 cells. (d) pSMdM-determined D values for the different charge-varied versions of ER-Dendra2 diffusing in the ER lumen of live COS-7 cells. In (c,d), each circle presents the holistic D value extracted from an individual cell. Squares + bars: averages and standard deviations. (e) Representative pSMdM super-resolution D maps of the differently charged ER-Dendra2 versions. Note that different color scales are used in (a,b,e) to present the different D ranges.

Figure 3.
Figure 3.

SMdM quantification of diffusion in the mitochondrial matrix and effects of protein net charge. (a) Representative SMdM super-resolution D map of mito-Dendra2 in the mitochondrial matrix of a live COS-7 cell, alongside a zoom-in of the boxed region. (b) Two-dimensional plots of the vectorial 1-ms single-molecule displacements accumulated for the high-D (top) and low-D (bottom) regions indicated by the arrow and arrowhead in (a), respectively. The distributions show no strong directional preferences, with principal directions θ of 61° and 91° evaluated. (c) Distributions of the displacements in (b) after projecting along their respective θ directions. Blue curves: MLE fits to a one-dimensional diffusion model, yielding D = 15.6 and 7.1 μm2/s, respectively. (d) Distributions of the scalar magnitude of single-molecule displacements in (b). Blue curves: MLE fits to a two-dimensional isotropic diffusion model, yielding D = 15.0 and 7.4 μm2/s, respectively, comparable to that found in (c) with the one-dimensional diffusion model. (e) SMdM-determined D values for the different charge-varied versions of mito-Dendra2 diffusing in the mitochondrial matrix of live COS-7 cells. Circles: holistic D values of individual cells. Squares + bars: averages and standard deviations. (f) Representative SMdM super-resolution D maps of the differently charged mito-Dendra2 versions, on the same color scale as (a).

Figure 4.
Figure 4.

Implications for a positively charged endogenous ER protein. (a) Surface charge models for the +6-charged wild-type (top) and 0-charged mutant (bottom) PPIB, generated using PyMOL (Version 2.0; Schrödinger, Inc.) based on the PPIB structure (PDB ID: 3ICH). Red and blue indicate negative and positive charges, respectively. (b,c) Representative pSMdM super-resolution D maps for the two proteins diffusing in the ER lumen of live COS-7 cells. Both proteins are tagged with the 0-charged version of Dendra2 FP. (d) Statistics of pSMdM-determined D of the two proteins in COS-7 cells. Circles: holistic D values of individual cells. Squares + bars: averages and standard deviations.

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