|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
| ||||||||||||||||||||
Files in this Data Supplement:
Fig. S1. Live cell imaging of retrograde STxB sorting on Tf-containing EE/RE. (A) HeLa cells were incubated with Alexa-488-labeled STxB (green) and Alexa-568-labeled human Tf (red), for 60 minutes at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy. A cell with STxB and Tf accumulated in early endosomes is shown. Merged image shows overlapping localization (yellow). The right panels are magnified views marked with a dashed box in the indicated channels on the left. The series shows a Tf-containing tubule moving away from an early endosome (red arrows), wheras STxB remains associated only with the endosome (green arrows). Time after the start of imaging is shown in seconds. Bar, 10 μm.
Fig. S2. Endosmal STxB accumulation in SNX1 RNAi cells. Cy3-STxB (red) was incubated with SNX1-depleted cells for 45 minutes at 37°. After fixation, cells were labeled for TfR (green). (A) Low STxB accumulation in perinuclear Golgi membranes was use as surrogated marker for the identification of well-transfected cells. In A, an untransfected cell has been marked (*). Bar, 10 μm. (B) represents a high magnification view of the boxed area in A. Additional magnification views are shown as insets in B. Like in Vps26-depleted cells, STxB and TfR structures are juxtaposed but not superposed in SNX1-depleted cells. Bar, 5 μm.
Movie 1. Live cell imaging of retrograde STxB sorting on Tf-containing EE/RE. HeLa cells were incubated with Alexa-488-labeled STxB (green) and Alexa-568-labeled human transferrin (red), for 60 minutes at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy at ∼0.59-second intervals. A cell with STxB and Tf accumulated in early endosomes is shown; yellow indicates overlapping localization. The movie shows STxB and Tf colocalizing in a large population of vesicles. Several STxB-containing tubules moving away from early endosomes are also observed, while transferrin remains associated only with the endosomes.
Movie 2. Live cell imaging of retrograde STxB sorting on Tf-containing EE/RE. HeLa cells were incubated with Alexa-488-labeled STxB (green) and Alexa-568-labeled human transferrin (red), for 60 min at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy at ∼0.59 seconds intervals. Accumulated STxB and Tf in early endosomes is shown; yellow indicates overlapping localization. The movie shows STxB and Tf colocalizing in several vesicles. STxB-containing tubules moving away from early endosomes are also observed, whereas Tf remains associated only with the endosomes.
Movie 3. Live cell imaging of Tf sorting on STxB-containing EE/RE. HeLa cells were incubated with Alexa-488-labeled STxB (green) and Alexa-568-labeled human transferrin (red), for 60 minutes at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy at ∼0.55 seconds intervals. Accumulated STxB and Tf in early endosomes is shown; yellow indicates overlapping localization. The movie shows STxB and Tf colocalizing in several vesicles. A transferrin-containing tubule moving away from an early endosome is also observed, whereas STxB remains associated with the endosome.
Movie 4. Live cell imaging of STxB and Vps29. HeLa cells transiently expressing Vps29-GFP (green), were incubated with Cy3-STxB (red), for 60 min at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy at ∼1.58-second intervals. Merged movie shows overlapping localization (yellow). The movie shows accumulated Cy3-STxB in early endosomes where colocalizes with Vps29-GFP. Tubules moving away from endosomes containing both proteins are also observed.
Movie 5. Live cell imaging of STxB and Vps29. HeLa cells transiently expressing Vps29-GFP (green), were incubated with Cy3-STxB (red), for 60 min at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy at ∼1.58-second intervals. Merged movie shows overlapping localization (yellow). The movie shows very dynamic tubules containing both proteins moving away from endosomes.
Movie 6. Live cell imaging of STxB in Vps26-depleted cells. Mock- or Vps26-depleted cells were incubated with Alexa-488-labeled STxB for 45 min at 37°C. After incubation cells were immediately imaged at ∼0.25-second intervals. In mock-depleted cells, STxB was correctly targeted to the TGN/Golgi. In Vps26-depleted cells, STxB was present in peripheral vesicles/endosomes and also in long tubular structures.
Movie 7. Live cell imaging of STxB and Tf in Vps26-depleted cells. Vps26-depleted cells were incubated with Alexa-488-labeled STxB (green) and Alexa-568-labeled human Tf (red), for 45 min at 37°C. After incubation cells were immediately imaged at ∼1.16-second intervals. Merged movie shows overlapping localization (yellow).The movie shows accumulated STxB colocalizing with transferrin in several endosomes. It also shows regions on the same endosomes where STxB and transferrin are stably segregated.
Movie 8. Live cell imaging of STxB and Tf in Vps26-depleted cells. Vps26-depleted cells were incubated with Alexa-488-labeled STxB (green) and Alexa-568-labeled human Tf (red), for 45 min at 37°C. After incubation cells were immediately imaged at ∼1.16-second intervals. Merged movie shows overlapping localization (yellow). The movie shows accumulated STxB colocalizing with transferrin in several endosomes. It also shows regions on the same endosomes where STxB and transferrin are stably segregated.
| ||||||||||||||||||||
