Supplemental Materials

This article contains the following supporting material:

• Supplemental Video 1 - Rab11a vesicles are observed traveling along Rab8a tubules in vivo. HeLa cells expressing mCerulean-tagged Rab8a (green) and mVenus-tagged Rab11a (red) were analyzed live using a Zeiss LSM510 confocal microscope. Sixty images were captured over a period of 419 seconds. In the video, small mVenus-Rab11a-positive vesicles are observed traveling along mCerulean-Rab8a-labeled tubules.
• Supplemental Table S1 - Interactions between Rab8a or Rab11a and Myosin Vb tail truncations were analyzed by a yeast two-hybrid assay. The interaction between Myosin Vb tail and Rab8a is lost if Myosin Vb tail is truncated, either from the N- or C-terminus. In contrast, the interaction between Myosin Vb tail and Rab11a is unaffected until greater than the first 139 amino acids are removed.
• Supplemental Figure S2 - Rab4a and Rab5a do not co-localize with Rab8a tubules. (A) HeLa cells were transiently transfected with mRFP-tagged human Rab5a and labeled with anti-Rab8a polyclonal antibody and an Alexa-488-labeled secondary antibody. Confocal images reveal that mRFP-Rab5a is localized to punctate spots around the cell, while Rab8a is localized to long tubules. (B) Similarly, cells were transiently transfected with mRFP-tagged human Rab4a were labeled with anti-Rab8a polyclonal antibody and an Alexa-488-labeled secondary antibody. As seen with mRFP-Rab5a, mRFP-Rab4a did not localize with Rab8a, but instead was present in puntate spots. Scale bars represent 10 µm.
• Supplemental Figure S3 - Myosin Vb tail, but not Myosin Va tail, is able to FRET with Rab8a: an example of negative FRET data. Either mVenus-tagged Myosin Va tail or Myosin Vb tail were co-transfected with mCerulean-tagged Rab8a-Q67L. (A) The FRET efficiency was calculated as in Figure 4. Error bars indicate the Standard Error of the Mean (SEM). The Energy Transfer Efficiency between mCerulean-Rab8a-Q67L and mVenus-Myosin Vb tail was 10.32 ± 3.8; between mCerulean-Rab8a-Q67L and mVenus-Myosin Va tail was -10.89 ± 3.1. This demonstrates that the FRET between Rab8a and Myosin Vb tail is specific. (B and C) The panels show representative examples of cells used for FRET measurements. mCerulean is pseudocolored with light blue and mVenus with yellow; overlap is represented by green. Unlike Myosin Vb tail, Myosin Va tail does not colocalize with Rab8a. The fluorescence intensity of a photobleached region of interest (ROI), indicated by a dashed-line circle on the example cells, was measured before (pre) and after (post) photobleaching and the average of pre-bleach images was compared to the average of the post-bleach images following background subtraction.
• Supplemental Figure S4 - Expressed Rab8a co-localizes with EHD1 and EHD3 tubules. (A) HeLa cells co-expressing myc-tagged EHD1 and mCherry-Rab8a were labeled with an anti-myc monoclonal antibody (9E10) and Alexa-488-labeled mouse secondary antibodies. Cofocal microscopy demonstrated that both expressed constructs co-localized to tubular structures. (B) A similar observation was made when myc-tagged EHD3 was co-expressed with mCherry-Rab8a. (C) Unlike endogenous Rab11a, expressed EGFP-Rab11a did show minor co-localization with myc-EHD1 on tubules in transiently transfected HeLa cells. (D) Similar to the pattern observed for myc-EHD1, EGFP-Rab11a partially co-localized with myc-EHD3 tubules in co-expressing HeLa cells. These results were similar to those in live cells co-expressing mCerulean-Rab8a and mVenus-Rab11a. Scale bars represent 10 µm.