Fig 1.
Quantitative co-localization analysis of different intracellular transport markers in control (-BFA) and BFA-treated cells (+BFA).
A-D) Confocal microscopy images of HeLa cells. Anti-GFP antibody and antibody against the indicated endogenous marker were used. A) Positive control with COPII markers Sec31 and YFP-Sec24wt. B) Negative control using an early endosome marker (EEA-1) and the COPII marker Sec31. C and D) Co-localization of the Golgi markers GalNAcT2 or GM130 with GFP-Rab1b, respectively. Scale bars represent 10 μm. E) Bar graphs depicting the Pearson correlation coefficient (PCC) of co-localization between the different markers (8 to 10 cells per condition, from 2–3 independent experiments). Data are expressed as mean ± SD (** = p<0.001).
Fig 2.
Quantitative co-localization analysis of different intracellular transport markers in control (-BFA) and BFA-treated cells (5 ug/mL for 2h).
(A-D) Confocal microscopy images of transfected HeLa cells (A, C and D). Immunofluorescence assays were performed with anti-GFP antibody (to detect GFP-Rab1bwt or GFP-p115) and an antibody against the indicated endogenous marker (ERGIC53 or Sec31). (B) Direct fluorescence detection of the mCherry or GFP indicated fusion proteins. Scale bars represent 10 μm. (E) Bar graphs depicting the Pearson correlation coefficient (PCC) of co-localization between the different markers (8 to 10 cells per condition, from 2–3 independent experiments). Data are expressed as mean ± SD (*** = p<0.0001).
Fig 3.
Representative time-lapse imaging of HeLa cells co-transfected with mCherry-Rab1bwt and YFP-Sec24wt (S1 Movie).
(A) Confocal images showing individual Sec24 and Rab1b signals and their overlap (B 1–2) Magnification of different ROIs shown in A at the indicated time points: 1) Rab1b-labeled structures (arrow) moving between two Sec24-labeled stable structures (arrowheads). 2) Rab1b—labeled punctate structures moving towards the Golgi complex (arrow) and passing through a stable Sec24-labeled structure (arrowhead 185 sec). Data represent at least three independent experiments. Scale bars represent 10 μm (A) and 3 μm (B).
Fig 4.
Representative time-lapse imaging of HeLa cells co-transfected with mCherry-Rab1bwt and GFP-p115wt (S2 Movie).
(A) Confocal images showing individual p115 and Rab1b signals and their overlap (B 1–2) Magnification of different ROIs shown in A at the indicated time points: 1) Punctate structures labeled with both Rab1b and p115 (B1, arrows) which remained stable throughout the recorded time (more than 6 minutes, 392sec). It was also possible to detect p115-labeled structures that did not co-localize with Rab1b over the same period of time (B1, arrowhead); 2) tubular structure labeled with both p115 and Rab1b originated from the Golgi and rapidly moving towards the cell periphery until it vanished (B3, arrow). Data represent at least three independent experiments. Scale bars represent 10 μm (A) and 3 μm (B).
Fig 5.
Representative time-lapse imaging of HeLa cells co-transfected with mCherry-Rab1b, YFP-Sec24 and SialT2-CFP during BFA washout.
After 24h of transfection, cells were incubated with BFA (1 μg/mL for 2h), then BFA was removed (washout) and in vivo time-lapse assays were performed. (A) Confocal image showing the overlap of YFP-Sec24 and mCherry-Rab1b and their individual signals. (B) Magnification of ROIs indicated in A, showing panels of the individual YFP-Sec24 and mCherry-Rab1b signals and their merge at the indicated times. Asterisks in Sec24 panel indicate five structures (labeled from 1 to 5, at the first indicated time, and with green circles at all time points) that remained stable at the indicated times. Asterisk in Rab1b panel indicates the first time that Rab1b associated to one of the Sec24 labeled structures. Arrows in the merge panel indicate structures where Sec24 and Rab1b are associated at the indicated times. (C) Magnification of same ROIs and times indicated in A and B respectively. Panels show individual SialT2-CFP signal and the merge including YFP-Sec24, mCherry-Rab1b and SialT2-CFP, and green circles specify the location of the same YFP-Sec24 structures indicated in B. Arrows in SialT2-CFP panel indicate the SialT2 concentration/sorting. Asterisk and circle in the merge panel indicate the Sec24 stable structures (#3), where the SialT2-CFP signal is concentrated at the indicated times (arrows). These arrows also show YFP-Sec24, mCherry-Rab1b and SialT2-CFP co-localization. Scale bars represent 10 μm (A) and 3 μm (B and C).
Fig 6.
Representative time-lapse imaging of HeLa cells co-transfected with mCherry-Rab1b, YFP-Sec24 and SialT2-CFP during BFA washout.
After 24h of transfection, cells were incubated with BFA (1 μg/mL for 2 h), then the BFA was removed (washout) and the time-lapse assays were performed. (A) Confocal image showing the individual YFP-Sec24, mCherry-Rab1b and SialT2-CFP signals and their merge at the indicated time points. Scale bars represent 3 μm. (B) Time-dependent changes in relative PFIVar of Rab1b or SialT2, at the region of interest depicted in A, calculated as described in Materials and Methods. (C) Bar graph depicting the percentage of the SialT2-CFP PFIVar peak behaviors (n = 62 events) clustered into three different groups: The first group containing SialT2 peaks coinciding with high Rab1b levels (named SialT2 coinciding with Rab1b), with this group including the SialT2 PFIVar peaks occurring simultaneously, 30 seconds before or after the Rab1b PFIVar peaks. In the second group were included the SialT2 PFIVar peaks which occurred completely before a Rab1b peak (named SialT2 before Rab1b), and finally, the third group collected all the SialT2 PFIVar peaks in the absence of Rab1 (named SialT2 without Rab1b).
Fig 7.
Co-localization time of Rab1b with ERES structures. Dual color time-lapse assays were performed in cells expressing YFP-Sec24 and mCherry-Rab1bwt (S3 Movie).
(A) Representative images of the time-lapse assays performed in HeLa cells for three different conditions: Control (-BFA), BFA treated cells (+BFA, 5 μg/mL for 2h), and in cells also co-transfected with GBF1-E794K-myc. (B, panels 1–3) Magnification of the different ROIs shown in A at the indicated time points of the time-lapse assay. Arrows indicate the Rab1b- labeled structure analyzed in the example. Asterisks in 1 indicate the times that the Rab1b-labeled structure is no longer associated to the Sec24-labeled structure. Scale bars represent 10 μm (A) and 3 μm (B). (C) Scatter plot of the co-localization time for cells at the indicated conditions. Sec24 and Rab1b-labeled structures were randomly selected for the analysis for each condition (-BFA: 40, +BFA: 45, GBF-E794K: 51 structures, n = 3 cells per condition).
Fig 8.
Rab1b and GBF1 co-localization and dynamic analysis.
(A and B) Confocal microscopy and co-localization analysis of mCherry-Rab1b and GBF1 or mCherry-Rab1b and GBF1-E794K-myc. Data are expressed as mean ± SD (* = p<0.05). (C-E) Kinetics of mCherry-Rab1b binding to and dissociation from ERES structures in HeLa cells treated with Nocodazole (2,5 μg/mL for 2h at 37°C), BFA (5 μg/mL for 2h) or expressing GBF1-E794K-myc. (C) Representative FRAP experiment in Hela cells, expressing mCherry-Rab1b and YFP-Sec24 and treated with Nocodazole. (D) Magnification of the ROI indicated in C showing a Rab1b-labeled structure selected for photobleaching (white circle, Rab1b panel) that localizes with a YFP-Sec24 labeled structure (white circle, Sec 24 panel). (E) Quantification of the mCherry-Rab1b intensity of a punctate structure localizing with an ERES (labeled with YFP-Sec24) in Nocodazole treated cells (n = 5), cells incubated with BFA (n = 8), or co-transfected with GBF1-E794K (n = 8); error bars, SD. Scale bars represent 10 μm (A and C) and 3 μm (D).