Figure 5.
Rap2 activation is closely linked with its subcellular localization. (A) GFP-Rap2a-G12V localization. MDA-MB-231 cells stably expressing GFP-Rap2a-G12V were fixed and stained with phalloidin (magenta) and DAPI (blue). Scale bars, 50 and 5 µm. (B) GFP-Rap2a-S17N localization. MDA-MB-231 cells stably expressing GFP-Rap2a-S17N were fixed and stained with phalloidin (magenta) and DAPI (blue). Scale bars, 50 and 5 µm. (C) Intracellular to whole-cell fraction quantification, Rap2a-G12V versus Rap2a-S17N. To quantify GFP-Rap2a localization changes (i.e., decreased Eos-Rap2a at the plasma membrane in S17N background), intracellular/whole-cell fractions were defined and calculated as the total fluorescence intensity of the intracellular GFP-Rap2a pool divided by the total fluorescence intensity of the whole-cell GFP-Rap2a pool (see Materials and methods). Two biological replicates were performed for each cell line. In each biological replicate, five fields of view were imaged, with 3 cells analyzed from each field (total of 15 cells per n). The two shades of blue represent each set of 15 cells from n1 and n2. Each cell was treated as its own data point, and statistical analysis (unpaired t test) was performed with n = 30 total for each condition. Mean ± SD. G12V versus S17N, P < 0.0001. (D) Colocalization of mCherry-Rap2a and GFP-RBDRalGDS. MDA-MB-231 cells were transiently transfected with mCherry-Rap2a and GFP-RBDRalGDS, then fixed. Arrow points to example of mCherry-Rap2a and GFP-RBDRalGDS overlap at the plasma membrane. Arrowheads point to mCherry-Rap2a intracellular organelles, where GFP-RBDRalGDS is not present. Scale bars, 10 and 2 µm. (E) Quantification of GFP-RBDRalGDS enrichment at the leading edge. In addition to the experiment in 3D, where cells were transfected with mCherry-Rap2a and GFP-RBDRalGDS, another experiment was performed side by side. MDA-MB-231 cells were transfected with mCherry-Rap2a and free GFP (not depicted) to control for free FP localization at the leading edge. Fluorescence intensity ratios (leading edge/cell body, i.e., enrichment at ruffles) were calculated for individual cells (see Materials and methods) in both conditions. One biological replicate was performed, n = 7 cells for GFP-RBDRalGDS condition and n = 8 cells for free GFP condition. Mean ± SD. The two conditions are separated by the dotted line in the center of the graph. Green dots represent GFP-RBDRalGDS or free GFP calculations; magenta represents mCherry-Rap2a. One-way ANOVA with Tukey’s multiple comparisons test. GFP-RBDRalGDS versus GFP alone, P = 0.0020. (F) Live imaging of mCherry-Rap2a and GFP-RBDRalGDS in MDA-MB-231 cells. Cells were transiently transfected with both mCherry-Rap2a and GFP-RBDRalGDS, plated on a collagen-coated glass dish, and imaged every 3 s using a 63× objective. Widefield microscope. Arrowheads point to an example of an mCherry-Rap2a–positive organelle being internalized from the lamellipodia. GFP-RBDRalGDS overlaps with mCherry-Rap2a primarily at the plasma membrane and is not found on the internalized organelle. See Video 7. Scale bars, 10 µm.

Rap2 activation is closely linked with its subcellular localization. (A) GFP-Rap2a-G12V localization. MDA-MB-231 cells stably expressing GFP-Rap2a-G12V were fixed and stained with phalloidin (magenta) and DAPI (blue). Scale bars, 50 and 5 µm. (B) GFP-Rap2a-S17N localization. MDA-MB-231 cells stably expressing GFP-Rap2a-S17N were fixed and stained with phalloidin (magenta) and DAPI (blue). Scale bars, 50 and 5 µm. (C) Intracellular to whole-cell fraction quantification, Rap2a-G12V versus Rap2a-S17N. To quantify GFP-Rap2a localization changes (i.e., decreased Eos-Rap2a at the plasma membrane in S17N background), intracellular/whole-cell fractions were defined and calculated as the total fluorescence intensity of the intracellular GFP-Rap2a pool divided by the total fluorescence intensity of the whole-cell GFP-Rap2a pool (see Materials and methods). Two biological replicates were performed for each cell line. In each biological replicate, five fields of view were imaged, with 3 cells analyzed from each field (total of 15 cells per n). The two shades of blue represent each set of 15 cells from n1 and n2. Each cell was treated as its own data point, and statistical analysis (unpaired t test) was performed with n = 30 total for each condition. Mean ± SD. G12V versus S17N, P < 0.0001. (D) Colocalization of mCherry-Rap2a and GFP-RBDRalGDS. MDA-MB-231 cells were transiently transfected with mCherry-Rap2a and GFP-RBDRalGDS, then fixed. Arrow points to example of mCherry-Rap2a and GFP-RBDRalGDS overlap at the plasma membrane. Arrowheads point to mCherry-Rap2a intracellular organelles, where GFP-RBDRalGDS is not present. Scale bars, 10 and 2 µm. (E) Quantification of GFP-RBDRalGDS enrichment at the leading edge. In addition to the experiment in 3D, where cells were transfected with mCherry-Rap2a and GFP-RBDRalGDS, another experiment was performed side by side. MDA-MB-231 cells were transfected with mCherry-Rap2a and free GFP (not depicted) to control for free FP localization at the leading edge. Fluorescence intensity ratios (leading edge/cell body, i.e., enrichment at ruffles) were calculated for individual cells (see Materials and methods) in both conditions. One biological replicate was performed, n = 7 cells for GFP-RBDRalGDS condition and n = 8 cells for free GFP condition. Mean ± SD. The two conditions are separated by the dotted line in the center of the graph. Green dots represent GFP-RBDRalGDS or free GFP calculations; magenta represents mCherry-Rap2a. One-way ANOVA with Tukey’s multiple comparisons test. GFP-RBDRalGDS versus GFP alone, P = 0.0020. (F) Live imaging of mCherry-Rap2a and GFP-RBDRalGDS in MDA-MB-231 cells. Cells were transiently transfected with both mCherry-Rap2a and GFP-RBDRalGDS, plated on a collagen-coated glass dish, and imaged every 3 s using a 63× objective. Widefield microscope. Arrowheads point to an example of an mCherry-Rap2a–positive organelle being internalized from the lamellipodia. GFP-RBDRalGDS overlaps with mCherry-Rap2a primarily at the plasma membrane and is not found on the internalized organelle. See Video 7. Scale bars, 10 µm.

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