RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells

Abstract

It has become increasingly clear that protein-protein interactions (PPIs) are compartmentalized in nanoscale domains that define the biochemical architecture of the cell. Despite tremendous advances in super-resolution imaging, strategies to observe PPIs at sufficient resolution to discern their organization are just emerging. Here we describe a strategy in which PPIs induce reconstitution of fluorescent proteins (FPs) that are capable of exhibiting single-molecule fluctuations suitable for stochastic optical fluctuation imaging (SOFI). Subsequently, spatial maps of these interactions can be resolved in super-resolution in living cells. Using this strategy, termed reconstituted fluorescence-based SOFI (refSOFI), we investigated the interaction between the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the pore-forming channel subunit ORAI1, a crucial process in store-operated Ca(2+) entry (SOCE). Stimulating SOCE does not appear to change the size of existing STIM1/ORAI1 interaction puncta at the ER-plasma membrane junctions, but results in an apparent increase in the number of interaction puncta.

Figure 1. Characterization of an enhanced cpDronpa variant cpDMVF-181 and development of its BiFC

(A) Reversible photoswitching of Dronpa and cpDMVF upon illumination with 488 and 405 nm laser light. (B) Fluorescence time-course of the renaturation of denatured Dronpa and cpDMVF. The decrease of measured fluorescence from cpDMVF, observable after approximately 200 seconds, is caused by fluorescence off-switching. (C) Fluorescence time-course of the renaturation of denatured and reduced Dronpa and cpDMVF. (D) Schematic representation of the DMVF-based constructs used in this study. (E) Time-course of DMVF reconstitution in HeLa cells upon rapamycin (Rap, 100 nM) induced heterodimerization of Lyn-FRB-DMVF-181-N and FKBP-DMVF-181-C. The maturation process is reflected by the normalized fluorescence intensity of reconstituted DMVF (rcDMVF) in responding cells (n≥13; data shown as mean ± s.e.m.). (F) Comparison of the fluctuation behavior of Lyn-targeted Dronpa (Lyn-FRB*FKBP-Dronpa) and reconstituted DMVF (Lyn-FRB*FKBP-rcDMVF) 4h after induction with rapamycin by means of the normalized 2^nd order SOFI value (n=15 for Dronpa and n=12 for rcDMVF; data shown as mean ± s.e.m.). (G) Scatter plot showing the comparison of the basal signal and the signal after rapamycin-induced reconstitution (4 h) of DMVF in individual HeLa Cells expressing FKBP-DMVF-181-C and Lyn-FRB-DMVF-181-N or LifeAct-FRB-DMVF-181-N, respectively, normalized to the fluorescence of TagRFP-T-CAAX serving as an expression marker (Lyn: n=133 for negative and n=130 for positive control; LifeAct: n=123 for negative and n=151 for positive control; black horizontal line indicates mean ± s.e.m.).

Figure 2. Reconstituted DMVF for refSOFI

(A) Schematic representation of the underlying principle of refSOFI. Two proteins of interest (POIs) are fused to non-fluorescent fragments of a FP suitable for refSOFI. Upon interaction, the FP is reconstituted, exhibits intensity fluctuations and can be imaged in super-resolution by acquiring an image sequence and applying pcSOFI analysis. (B) Representative pcSOFI image (3^rd order analysis, triple pixel density) of Lyn-FRB/FKBP/reconstituted DMVF complexes in HeLa cells. (C) to (F) are expansions of (B), displaying a side by side comparison of a conventional TIRF image (C), a 2^nd order pcSOFI image (double pixel density, D), a 3^rd order pcSOFI image (E) and the final deconvolved image (F), illustrating the increase in spatial resolution, enhanced contrast and a reduction of background signal. (G) Representative pcSOFI image (3^rd order analysis, triple pixel density) of LifeAct-FRB/FKBP/reconstituted DMVF complexes in HeLa cells. (H) to (K) are expansions of (G) analogous to (C) to (F). (Scale bar: 2 μm in all expansions) See also Figure S1, S2 and Movie S1.

Figure 3. Super-resolution imaging using Venus

(A) Schematic representation of the Venus-constructs used for super-resolution imaging. (B) Representative pcSOFI TIRF image (3^rd order) of a HeLa cell expressing Venus-CAAX. (C) and (D) show expansions of an area of (B) allowing a side-by-side comparison of the average fluorescence image and the corresponding pcSOFI image, which features triple pixel density. (E) Representative pcSOFI TIRF image (3^rd order) of a HeLa cell expressing LifeAct-Venus. (F) and (G) display expansions of (E) analogous to (C) and (D). (H) Representative pcSOFI image (near-TIRF illumination, 3^rd order) of a nucleus of a NIH3T3 cell expressing histone H2B-Venus. (I) and (J) display expansions of (H) analogous to (C) and (D). (Scale bar: 1 μm in all expansions) See also Figure S3.

Figure 4. Reconstituted Venus for refSOFI

(A) Schematic representation of the Venus-based constructs used in this study. (B) Representative pcSOFI TIRF image (3^rd order) of Lyn-FRB*FKBP/reconstituted Venus in HeLa cells 2 h after rapamycin treatment. (C) and (D) show expansions of (B) allowing a side-by-side comparison of the average fluorescence image and the corresponding pcSOFI image. (E) Representative pcSOFI TIRF image (3^rd order) of LifeAct-FRB*FKBP/reconstituted Venus in Hela cells 2 h after rapamycin treatment. (F) and (G) display expansions of (E) analogous to (C) and (D). (Scale bar: 1 μm in all expansions) (H) Time-course of average intensity and normalized pcSOFI value of cells expressing FKBP-VC and Lyn-FRB-VN after addition of rapamycin (100 nM at × = 0). The data were normalized to the respective average values at the time × = 0 (n≥9, data shown as mean ± s.e.m.). It has to be noted that the observed broad increase of the apparent normalized value mainly results from the initial occurrence of fluctuations and the high impact of noise due to the low population of intact Venus molecules at the start. See also Figure S4 and Movie S2.

Figure 5. Applying refSOFI to investigate the interactions between STIM1 and ORAI1

(A) Representative pcSOFI image (2^nd order) of STIM1/ORAI1/reconstituted Venus complexes in an unstimulated HeLa cell expressing STIM1-VC, VN-ORAI1 and ER-rsTagRFP (not shown). (B) Image shown in (A) scaled to the color scale of (C) for direct comparison. (C) Image of the cell shown in (B) 30 min after treatment with 1 μM Thapsigargin (TG). (D) Schematic representation of the constructs used in these experiments. (E) Multicolor pcSOFI image displaying the localization of STIM1/ORAI1 complexes (green) and the ER (red) in this cell 30 min after TG addition. (F) Expansion of an area of the average florescence image for comparison with the corresponding pcSOFI image in (G) (Scale bar: 1 μm). All images were acquired under TIRF conditions. (H) Bar graph displaying the interaction puncta density of individual cells before and 30 min after treatment with 1 μM TG (n=26), illustrating the difference between the analysis of standard TIRF images and the corresponding refSOFI images. (I) Analogous bar graph showing the average interaction puncta size of individual cells under the indicated conditions. Data shown as mean + s.e.m.. (J) Normalized frequency of the sizes of the individual puncta detected in HeLa cells using refSOFI presented in (I), before (n=3301) and 30 minutes after stimulation with TG (n=4173). A bin size of 0.025 μm² is used and 90% of the dataset is displayed for clarity. See also Figure S5.