Choi AA et al. (2024), Single-molecule diffusivity quantification in X...

XB-ART-61110

Proc Natl Acad Sci U S A 2024 Dec 10;12150:e2411402121. doi: 10.1073/pnas.2411402121.

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Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm.

Choi AA , Zhou CY , Tabo A , Heald R , Xu K .

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The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SMdM) with physiologically active extracts prepared from Xenopus laevis eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused ~50% slower, while diffusion of positively charged proteins was reduced by ~80 to 90%. Adding increasing concentrations of salt progressively alleviated the suppressed diffusion observed for positively charged proteins, signifying electrostatic interactions within a predominately negatively charged macromolecular environment. To investigate the contribution of RNA, an abundant, negatively charged component of cytoplasm, extracts were treated with ribonuclease, which resulted in low diffusivity domains indicative of aggregation, likely due to the liberation of positively charged RNA-binding proteins such as ribosomal proteins, since this effect could be mimicked by adding positively charged polypeptides. Interestingly, in extracts prepared under typical conditions that inhibit actin polymerization, negatively charged proteins of different sizes showed similar diffusivity suppression consistent with our separately measured 2.22-fold higher viscosity of extract over water. Restoring or enhancing actin polymerization progressively suppressed the diffusion of larger proteins, recapitulating behaviors observed in cells. Together, these results indicate that molecular interactions in the crowded cell are defined by an overwhelmingly negatively charged macromolecular environment containing cytoskeletal networks.

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Species referenced: Xenopus laevis
Genes referenced: actc1 actg1 ahcy aldoa cgl.2 cirbp ckb eno1 fabp4 ftmt gapdh gpi grhpr.2 gstm1 hba-l5 hbz hpgds ldhb mdh1 nme2 pgk1 pkm plin2 rpl17 rpl18 rpl19 rpl22 rpl23a rpl32 rpl4 rplp0 rplp2l rps11 rps12 rps16 rps17 rps18 rps19 rps27a rps3 rps4x rps6 serpina6 tpi1

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	Fig. 1. SMdM-based protein diffusivity quantification in Xenopus egg extract versus in PBS. (A) Schematic: Cytoplasmic extracts from Xenopus eggs. (B) Schematic: In SMdM, paired excitation pulses are repeatedly applied across tandem camera frames, so that transient single-molecule displacements are captured in the wide field for the time window defined by the separation between the paired pulses, Δt. (C) Example single-molecule images of Cy3B-labeled bovine carbonic anhydrase diffusing in the extract, shown as a magenta-green overlaid image for a tandem pair of frames at Δt = 1 ms. (D) Example distributions of SMdM-recorded 1-ms single-molecule displacements for ~200 pM Cy3B-labeled HEWL diffusing in PBS (Top) versus in extract (Bottom). Blue curves: fits to our single-mode diffusion model, with resultant apparent diffusion coefficients D and 95% CI marked in each plot. (E) Similar to (D), but for succinylated HEWL (sHEWL). (F) SMdM-determined D values for 15 proteins of varied sizes and charges (SI Appendix, Table S1), in PBS (hollow symbols) and extract (filled symbols). Red squares: Negatively charged proteins. Blue circles: Positively charged proteins with >+5 net charges. Light-blue circles: Weakly positively charged proteins with ~+2 net charges. Each data point is an average of at least three SMdM measurements from two or more extract samples. Solid curve: Expected D in PBS at room temperature according to the Young−Carroad−Bell (YCB) model. Dashed curve: The PBS YCB values divided by 2
	Fig. 2. Net-charge effects on protein diffusion in Xenopus egg extract. (A) SMdM-determined diffusion coefficients D in the extract relative to those in PBS for different proteins, as a function of their net charges in the range of −25 to +25. (B) Blue and red symbols: Relative in-extract D values normalized to in-PBS values, plotted as a function of added NaCl, for the positively charged HEWL-Cy3B (blue) and the negatively charged BCA-CF647 (red). Values were obtained through sequential SMdM in two color channels. Error bars: Sample SD between results from two or three SMdM measurements at each data point. Black diamonds (y-axis on the Right): Ratio between the PBS-normalized D values of the two proteins.
	Fig. 3. SMdM diffusivity mapping of HEWL in RNase-treated egg extracts further underscores the charge-sign asymmetry of the cytoplasmic environment. (A) Color-coded SMdM diffusivity map of Cy3B-labeled HEWL in an extract sample 20 min after RNase A treatment at room temperature. (B) Distributions of single-molecule displacements for the data in (A). Blue line: fit to the SMdM diffusion model, with resultant apparent diffusion coefficient D and 95% CI noted. (C) SMdM diffusivity map of the same sample after 3 h. (D) Distributions of single-molecule displacements for regions outside the low-diffusivity domains in (C). (E) Photos of extract samples taken immediately (Left) and after 3 h (Right), after RNase A treatment or the addition of 1 mg/mL polylysine, HEWL, or polyglutamic acid. (F) Color-coded SMdM D map of Cy3B-labeled HEWL in an extract sample supplemented with 1 mg/mL polylysine. (G) Distribution of single-molecule displacements for regions outside the low-diffusivity domains in (F).
	Fig. 4. Importance of the actin cytoskeletal network in molecular size-dependent diffusion suppression. (A) 3D-STORM superresolution images of phalloidin-labeled actin filaments in actin-preserved (Top) and actin-supplemented (Bottom) Xenopus egg extracts. Color presents axial (depth) information. (B) SMdM-determined D values in the extract relative to in PBS, for the ~30 kDa BCA (black) and the ~660 kDa thyroglobulin (red), in actin-inhibited, actin-intact, and actin-supplemented samples. Each data point corresponds to one independent SMdM measurement for a different sample region, from ~3 samples under each condition.
	Figure S1. SMdM diffusivity mapping of Cy3B-labeled HEWL in untreated and BSA-supplemented Xenopus egg extracts. (a) Color-coded SMdM diffusivity map for an untreated sample, presented on the same color scale as Fig. 3. (b) Distribution of the 1-ms single-molecule displacements from (a). Blue curve: fit to our single-mode SMdM diffusion model, with resultant apparent diffusion coefficients D and 95% confidence intervals marked. Note that in this sample, a higher concentration of Cy3B-labeled HEWL (~1 nM) was used to increase the detected single- molecule density to facilitate spatial mapping. While this led to a higher background in the displacement distribution (when compared to Fig. 1d), the D value obtained with our fitting model (Eqn. 1) stays consistent, in agreement with what we have analyzed and demonstrated previously (6). (c) Color-coded SMdM diffusivity map for another sample to which BSA was added at 1 mg/mL. (d) Distribution of the 1-ms single-molecule displacements from (c) and fit to our model.
	Figure S2. Two-component D fit to the distribution of SMdM-recorded 1-ms single-molecule displacements of Cy3B-labeled HEWL diffusing in extract as shown in Fig. 1d. Histogram: displacements; Red curve: fast component of the fit (D1 = 21.3 μm2/s, fraction f1 = 0.65); Blue curve: slow component of the fit (D2 = 3.4 μm2/s, fraction f2 = 0.35); Black curve: sum of the red and blue curves. While the two-component fit improves over the single-component model (Fig. 1d), the system may be better assumed as a continuous distribution of different transient states.
	Figure S3. Diffusion of Cy3B-labeled HEWL in extracts supplemented with NaCl, KCl, and 1,6-hexanediol. (a) SMdM-determined relative in-extract D values of Cy3B-labeled HEWL normalized to in-PBS values, for two samples separately added with NaCl (red) and KCl (black) to different concentrations. Error bars: Sample standard deviations between results from three SMdM measurements at each data point. (b) SMdM-determined D values of Cy3B-labeled HEWL in two extract samples, before and after separately adding 500 mM KCl or 3% 1,6-hexanediol. Error bars: Sample standard deviations between results from three SMdM measurements.
	Figure S4. SMLM super-resolution images of Cy3B-labeled HEWL in RNase-treated extract, as generated from the single-molecule localizations of the SMdM data. (a) Same field of view as the SMdM data of Fig. 3c. (b) Zoom- ins of two regions. (c) Distribution of single-molecule positions for overlaid 4 small nanoclusters like those indicated by the red arrows in (b), in the X (top) and Y (bottom) directions, respectively. Gaussian fits (red curves) give standard deviations of 27 and 32 nm.
	Figure S5. Diffusion of the negatively charged BCA in RNase-treated extract. (a,b) SMLM images of Cy3B-labeled HEWL (a) and CF647-labeled BCA (b) in an RNase-treated extract sample. Vertical stripe patterns are attributed to local lensing effects from the high refractive indices of the aggregates under our inclined illumination scheme (7). (c) SMdM diffusivity map of the CF647-labeled BCA. Arrows point to aggregates, where diffusivity reduction was accompanied by local increases and decreases in the abundances of HEWL and BCA, respectively. These results may be interpreted as that as the RNase-released positively charged proteins interact with the negatively charged cytoplasm environment, the resultant aggregates are overwhelmed by the latter on the surface. Positively and negatively charged tracer proteins are thus respectively attracted to and repelled by the aggregates, with the former further likely participating in the aggregate cores.
	Figure S6. Aggregation assays for the ribosome-depleted high-speed extracts (HSEs). Photos are shown for HSEs at 0 h (left) and 3 h (right), without or with the addition of RNase or 1 mg/mL polylysine. Whereas polylysine addition generated immediate clouding, RNase treatment did not induce clouding over 3 h, consistent with our model that in the crude, ribosome-containing cytoplasm extract, RNase degradation of rRNA releases positively charged ribosomal proteins to cause aggregation.
	Figure S7. Additional data related to actin filaments. (a,b) Typical epifluorescence (a) and STORM (b) images of phalloidin-AF647 in actin-inhibited Xenopus egg extracts, showing no resolvable structures. (c) SMdM-determined D values in the extract relative to in PBS, for the positively charged HEWL in actin-inhibited and actin-intact extracts. Each data point corresponds to one independent SMdM measurement for a different sample region, from 5 actin-inhibited and 3 actin-intact samples, respectively.
	Figure S8. SMdM-determined D values of CF647-labeled BCA and Cy3B-labeled HEWL in extracts with different ATP levels. “Extract”: Typical samples added with 1x energy mix (4 mM creatine phosphate, 0.5 mM ATP, 0.05 mM EGTA, and 0.5 mM MgCl2). “-ATP”: Samples obtained in the same preparation but without addition of the energy mix. “- -ATP”: “-ATP” samples further subjected to ATP depletion by apyrase [New England Biolabs M0398S; incubated at 1:40 (12.5 units/mL) for 20 min], a condition known to abolish energy-dependent processes in extract, e.g., cell-like compartmentalization (8) and DNA compaction (9). Error bars: Sample standard deviations between results from three SMdM measurements.
	Figure S9. Size-exclusion chromatography elution volumes of four of the protein samples used in this study (red circles) compared to calibration standards (Bio-Rad 1511901; black squares), plotted versus the expected molecular weight. Separation was performed at 4 °C in PBS. 50 μL of protein solution (1 mg/mL) was injected into an ӒKTA pure micro chromatography system (Cytiva 29302479) equipped with a Superdex 200 Increase 3.2/300 column (Cytiva 28990946) at a flow rate of 0.025 mL/min. Sample elution was monitored by absorbance at 280 nm.