Figure 3.
Rap2 is dynamically trafficked through the endocytic pathway. (A) Colocalization of mCherry-Rap2a and YFP-Rab4 WT. MDA-MB-231 cells were transiently cotransfected with mCherry-Rap2a and YFP-Rab4 WT, followed by fixation and staining for DAPI (blue). Arrows indicate examples of mCherry-Rap2a and YFP-Rab4 WT overlap. Arrowheads point to mCherry-Rap2a organelles that are not Rab4 positive. Scale bars, 10 and, 2 µm. (B) Colocalization of mCherry-Rap2a and YFP-Rab11a WT. MDA-MB-231 cells were transiently cotransfected with mCherry-Rap2a and YFP-Rab11a WT, followed by fixation and staining for DAPI (blue). Arrows indicate examples of mCherry-Rap2a and YFP-Rab11a WT overlap. Arrowheads point to mCherry-Rap2a organelles that are not Rab11a positive. Scale bars, 10 and, 2 µm. (C) Rap2a colocalization analysis with Rab4 WT and Rab11a WT. 3i SlideBook6 software was used to calculate the percent of total mCherry-Rap2a that colocalizes with Rab4 or Rab11a (see Materials and methods). One biological replicate was performed. Mean ± SD. n = 10 for mCherry-Rap2a/YFP-Rab4 WT, n = 9 for mCherry-Rap2a/YFP-Rab11a WT. (D) Model for Rap2 endocytic trafficking. (1) Rap2 is internalized at the lamellipodia plasma membrane via a pinocytosis-like mechanism. (2) Internalization mediates the delivery of Rap2 to EEA1/Rab5-positive early endosomes, where it is sorted into different fates, either recycling to the plasma membrane via the Rab4/Rab11 pathways or targeting to lysosome for degradation. We hypothesize that Rap2 recycling back to the leading edge is needed to sustain cell migration.

Rap2 is dynamically trafficked through the endocytic pathway. (A) Colocalization of mCherry-Rap2a and YFP-Rab4 WT. MDA-MB-231 cells were transiently cotransfected with mCherry-Rap2a and YFP-Rab4 WT, followed by fixation and staining for DAPI (blue). Arrows indicate examples of mCherry-Rap2a and YFP-Rab4 WT overlap. Arrowheads point to mCherry-Rap2a organelles that are not Rab4 positive. Scale bars, 10 and, 2 µm. (B) Colocalization of mCherry-Rap2a and YFP-Rab11a WT. MDA-MB-231 cells were transiently cotransfected with mCherry-Rap2a and YFP-Rab11a WT, followed by fixation and staining for DAPI (blue). Arrows indicate examples of mCherry-Rap2a and YFP-Rab11a WT overlap. Arrowheads point to mCherry-Rap2a organelles that are not Rab11a positive. Scale bars, 10 and, 2 µm. (C) Rap2a colocalization analysis with Rab4 WT and Rab11a WT. 3i SlideBook6 software was used to calculate the percent of total mCherry-Rap2a that colocalizes with Rab4 or Rab11a (see Materials and methods). One biological replicate was performed. Mean ± SD. n = 10 for mCherry-Rap2a/YFP-Rab4 WT, n = 9 for mCherry-Rap2a/YFP-Rab11a WT. (D) Model for Rap2 endocytic trafficking. (1) Rap2 is internalized at the lamellipodia plasma membrane via a pinocytosis-like mechanism. (2) Internalization mediates the delivery of Rap2 to EEA1/Rab5-positive early endosomes, where it is sorted into different fates, either recycling to the plasma membrane via the Rab4/Rab11 pathways or targeting to lysosome for degradation. We hypothesize that Rap2 recycling back to the leading edge is needed to sustain cell migration.

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