Nanoscopic Spatial Association between Ras and Phosphatidylserine on the Cell Membrane Studied with Multicolor Super Resolution Microscopy

Abstract

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane.

Keywords: DNA-PAINT; Ras GTPases; Ras dimers; actin cytoskeleton; colocalization analysis; membrane nanodomains; nanoclusters; phosphatidylserine; super resolution microscopy.

Figure A1

Anti-GFP-nanobody purification and conjugation with DNA oligos. (A) Schematic of GFP-Nb purification workflow. (B) SDS-PAGE (NuPAGE Bis-Tris 4–12%) gel of GFP-Nb purified by Ni-chelating chromatography after Coomassie staining. The GFP-Nb was detected at an expected band size of 15 kDa. E1 = first elution and E2 = second elution. (C) SDS-PAGE (NuPAGE Bis-Tris 4–12%) gel of purified GFP-Nb after conjugation to DS labeled with Cy3 via clicking of AzPhe after Coomassie staining (top) and Cy3 in-gel fluorescence (bottom) detected by excitation with green light and filter F595 (for 595 nm). (D) Fluorescence images of U2OS cells transiently transfected with GFP-tubulin (green) labeled with GFP-Nb conjugated to DS1-Cy3 (yellow). (E) DNA-PAINT image of U2OS cells expressing GFP-tubulin stained with GFP-Nb.

Figure A2

Independence of DNA-PAINT image signal intensity measured using Image J and colocalization coefficients obtained from Coloc-Tesseler. (A) The scatterplot shows the whole cell intensity of the PS channel normalized to area plotted against both the Manders A (circles) and B (squares) colocalization coefficients of eight KRas-G12D U2OS cells with PS (each symbol represents one cell in the plot). (B) The scatterplot shows the whole cell intensity of the KRas channel normalized and plotted in the same way. Both plots show no relationship between intensity levels of either PS or KRas, and colocalization coefficients describing their co-organization, which rules out that increased co-organization is simply due to higher levels of probes.

Figure A3

Multiplexed imaging of PS and caveolae using exchange-PAINT and qualitative assessment of co-organization with Coloc Tesseler. (A) Single DNA-PAINT image of PS and caveolae in U2OS cells and magnified regions from (A) namely (a) and (b) showing the distinct molecular distribution of PS (green) and caveolae (cyan). (B) Colocalization analysis with Coloc-Tesseler of region (A) with the same magnified areas namely (a’) and (b’) showing the Voronoi diagram for PS (blue) and caveolae (yellow). Magnifications show classification of molecule coordinates into five orthogonal classes dependent on their local pair-normalized localization densities (cyan = dense PS; yellow = dense caveolae; red = caveolae overlap with PS; blue = PS overlap with caveolae). It can be seen that caveolae directly co-organize with patches of PS (indicated by the red color).

Figure A4

STORM imaging of U2OS cells, showing the effect of actin depolymerization. (A) U2OS untreated control cells were fixed with 3.7% PFA, stained with Alexa Fluor™ 647 Phalloidin (#A22287), and imaged in a standard STORM imaging buffer with 100mM MEA, as previously described [50,51]. Magnifications (a) and (b) show intact F-actin fibers. (B) U2OS cells treated with 2 μM LA for 10 min stained with Alexa Fluor™ 647 Phalloidin and imaged with STORM. Magnifications (c) and (d) clearly show a significant reduction in large fibers that are characteristic of depolymerization of the actin cytoskeleton. The experiment was performed twice with similar results (N = 2), and included three cells for each condition.

Figure 1

Super resolution imaging of plasma membrane PS in U2OS cells using a combination of the Lact-C2-GFP sensor with DNA-PAINT. (A) Structure of the GFP-enhancer nanobody (GFP-Nb) complex (based on PDB ID 3K1K). The location of the artificial amino acid (AzPhe) is indicated by a red asterisk. (B) Schematic for the conjugation of the GFP-Nb with a single-stranded DNA oligonucleotide via site-specific click chemistry between AzPhe and dibenzocyclooctyne (DBCO); shown here is the reaction with DBCO-modified docking strand 1 (DS1). (C) Schematic of DNA-PAINT imaging showing complementary imager strands (IS1) diffusing above the sample. Binding of IS1 (originally in solution) to DS1 (immobilized on target) and subsequent unbinding generate transient (~0.1 s) single-molecule signals, which accumulate over time to probe all DS1 on the sample. (D) Example super resolution image of PS in U2OS cells from the DNA-PAINT process depicted in (C). Lact-C2-GFP was transiently expressed in U2OS cells and labeled with the Nb-DS1 prior to imaging. The imaging was performed under total internal reflection to limit the detection volume to the ventral membrane. The image on the left is an overview, with serial magnifications shown in (a–c).

Figure 2

Multiplexed imaging of PS and KRas-G12D using exchange-PAINT and assessment of colocalization with Coloc-Tesseler. (A) Schematic of dual-color imaging with fluorescently conjugated imaging strands, each targeting the corresponding docking strand. DS1 conjugated to the GFP-nanobody (Nb-DS1, grey) binds to the transiently transfected PS sensor Lact-C2-GFP (green) and is recognized by IS1-Atto643. DS2 conjugated to the SNAP-substrate reacts with SNAP tag to form a covalent linkage and is recognized by IS2. The two targets are probed sequentially in two imaging cycles with a washing step in between to remove the imager strand used in the previous cycle, allowing the same (bright) fluorophore to be used for imaging both targets. (B) Example dual-color DNA-PAINT image of PS (green) and KRas-G12D (red) in U2OS cells. All cells in the field of view expressed SNAP-KRas-G12D, and only the cell in the middle was transiently transfected with Lact-C2-GFP. (C) Flow chart for the image analysis. (D) Magnified region from (B) with further magnified areas (a) and (b) showing distinct molecular distribution of PS (green) and KRas-G12D (red). (E) Colocalization analysis of magnified areas namely (a’) and (b’) that correspond to the same regions shown in (a) and (b) of region (D) using Coloc-Tesseler, showing the Voronoi diagrams for PS (green mesh) and KRas-G12D (purple mesh). Regions of molecular enrichments are shown as dots in distinct colors. (F) Magnification of the Voronoi diagram of PS (green) and KRas-G12D (purple), including classification of molecule coordinates into four orthogonal classes dependent on their local pair-normalized localization densities (cyan = PS enriched alone; yellow = KRas-G12D enriched alone; red = KRas-G12D co-enriched with PS; and blue = PS co-enriched with KRas-G12D). Non-enriched localizations in the PS or KRas channels are not shown. (G) Molecule coordinates divided into the four orthogonal classes without Voronoi diagrams. (H) Plot of Manders A (fraction of KRas-G12D co-enriched with PS of all enriched KRas-G12D, or # of red dots/# of red plus yellow dots) versus Manders B (fraction of PS co-enriched with KRas-G12D of all enriched PS, or # of blue dots/# of blue plus cyan dots) coefficients based on the classifications shown in (E–G). The experiment was performed in triplicate (N = 3) and included eight cells overall, with 55 analyzed regions of interest (ROIs).

Figure 3

Dual color super resolution imaging of PS and HRas-G12V using exchange-PAINT and comparison of the spatial associations between PS and HRas-G12V or KRas-G12D. (A) Example dual-color DNA-PAINT image of transiently transfected Lact-C2-GFP (bound to PS) and stably expressed Halo-HRas-G12V in U2OS cells. Halo-HRas-G12V was imaged using a similar strategy to that for SNAP-KRas-G12D shown in Figure 2. Magnified views (a–c) show the molecular distributions of both targets. (B) Quantitation of Manders coefficients A vs. B based on the dual-color DNA-PAINT images. The experiment was performed in triplicate (N = 3) and included seven cells overall with 29 analyzed ROIs. (C) Violin plots of Manders coefficients for the associations of KRas-G12D or HRas-G12V with PS (A) and PS with either KRas-G12D or HRas-G12V (B). Experiments performed in triplicate (N = 3) including eight and seven cells for each KRas-G12D and HRas-G12V, with 55 and 29 analyzed ROI, respectively, were used for this graph.

Figure 4

Assessment of the role of the actin cytoskeleton in Ras-PS interactions through DNA-PAINT imaging. (A) Impact of actin perturbations on the spatial distribution of PS on the plasma membrane of U2OS cells. Example 4 µm × 4 µm areas of DNA-PAINT images taken on U2OS cells transiently transfected with Lact-C2-GFP and treated as indicated. (B) U2OS cells stably expressing SNAP-KRas-G12D were transfected with Lact-C2-GFP and treated with a vehicle (15 μM Jasplakinolide (Jas) for 30 min or 2 μM Latrunculin A (LA) for 10 min) before imaging. Molecule localizations were analyzed with Coloc Tesseler and violin plots were prepared showing Manders A (left) and Manders B (right) coefficients for the treated or untreated cells. (C) U2OS cells stably expressing Halo-HRas-G12V were treated similarly; the violin plots show Manders A (left) and Manders B (right) coefficients. Control conditions included eight and seven cells for both KRas-G12D and HRas-G12V expressing cells, and experiments were performed in triplicate (N = 3) with 55 and 29 ROIs analyzed, respectively. The Jas treatment group included seven KRas and six HRas cells, and was performed in duplicate (N = 2) with 52 and 44 ROIs analyzed, respectively. The LA group included eight KRas and six HRas cells, and was performed in two independent experiments with 54 and 34 ROIs analyzed, respectively.