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First published online 25 September 2007
doi: 10.1242/jcs.006296

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Rho/ROCK and myosin II control the polarized distribution of endocytic clathrin structures at the uropod of moving T lymphocytes

¹ Unidad de Microscopía Confocal, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain
² Servicio de Inmunología, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain

View larger version (63K): [in this window] [in a new window]   Fig. 1. CME components are polarized toward the uropod. (A) In vivo confocal sectioning of a representative T cell transfected with GFP-clathrin light chain (ClLC) and pulsed with Tfn-Tx (10 µg/ml). The z projection of 19 confocal sections taken each 300 nm, the lateral reconstruction, and the basal and mid-uropod planes, are shown. The selected middle section avoids most of the cytoplasmic TGN-labeling, and several CSs located at the cell periphery are enlarged below. A number of Tfn spots are observed in some CSs (arrows), probably corresponding to CCPs or budding-CCV. The double labeling can be viewed section by section in Movie 1 (see supplementary material). (B) Polarized distribution of endogenous AP-2 (-adaptin) in a T lymphoblast. The z projection, a confocal section and the DIC image are shown. (C) AP-2 (-adaptin, red) and GFP-ClLC (green) double labeling. Several co-localizing large CSs distributed at the tip and the uropod neck (arrows) are observed in the mid-uropod confocal section. The single AP-2 labeling and the merged image are shown; the enlarged area appears outlined in blue. (D) Intensity profile of clathrin staining along the cell contour (yellow line). This is the same cell shown in A. (E) Schematic representation of membrane CS distribution (green circles) in a polarized T cell. Cells were virtually divided in uropod, uropod-neck and front. However, generally, both the uropod and its neck are overall similarly rich in CSs/µm, in contrast to the front cell pole. (F) Histograms summarizing the frequency of membrane CSs/µm at the uropod, uropod-neck and the front part of the cell (check all three domains in E). Over 1000 µm of membrane were carefully checked in highly magnified images for 200-800 µm CSs co-localizing with Tfn or AP-2. Bars, 5 µm, or as indicated.

View larger version (78K): [in this window] [in a new window]   Fig. 2. Spatio-temporal analysis of endocytosis in polarized T cells. (A) In vivo imaging of a steady membrane CS detected with GFP-ClLC (green) 3 minutes after a Tfn-Tx pulse (red). Rows show several Tfn/GFP-ClLC overlaid images (upper), and the single Tfn staining (bottom), at 30 second intervals (arrowheads point Tfn spots). The whole uropod image corresponding to the last frame is shown (120 seconds). Multiple co-localization events occur during consecutive frames. Occasionally, Tfn-rich endosomes steadily located very close to the plasma membrane were observed (arrows), even under the CS. (B) In vivo imaging of a steady membrane CS detected with GFP--adaptin (green) 3 minutes after a Tfn-A633 pulse (red). As in A, multiple co-localization events occurred during consecutive frames (arrowheads). Rows show several Tfn/AP-2 overlaid images (upper), and the single Tfn staining (bottom), at 20 second intervals. (C) In vivo imaging of a T cell over longer periods of time. Time/space integrated images over 5-10 and 10-15 minute observation intervals, corresponding to three confocal sections taken every 30 seconds, are shown. Co-localization pixels are depicted in white, and the ROIs used for the analysis are shown in the corresponding fluorograms, as indicated in Materials and Methods. After 5 minutes, most of the Tfn has already internalized and co-localizes with clathrin around the endosomes (see details in D), then co-localization decays (13 minutes). This observation suggests that the traffic of cytoplasmic CCVs has diminished, and also serves as internal control of the co-localization analysis. Enlarged inserts show several red/green-merged images at discrete time/space points (7 minutes). (D) In vivo imaging of a cytoplasmic endosome where Tfn has accumulated during 5 minutes (red). From the 3rd to approximately the 13th minute after the Tfn pulse, an intense dynamic co-localization with cytoplasmic clathrin (green) occurs exclusively inside and around the endosomes within the uropod, including a subset of very small clathrin spots (arrowheads). The z-projection and the DIC image, both taken around the 11th minute, are shown. Bars, 5 µm, or as indicated.

View larger version (78K): [in this window] [in a new window]   Fig. 3. Kinetics of transferrin binding to its receptor and internalization. (A) Imaging of a T cell migrating on ICAM-1, 2 minutes after Tfn-Tx addition, at 60 second intervals. DIC images of the first and last frame are shown. Arrowheads point to the trailing uropod. (B,C) Kinetics of Tfn-A488 distribution 1 and 4 minutes after addition, respectively, and co-localization analysis with membrane-TfnR in non-permeabilized T cells. In this analysis, Tfn/TfnR co-localizing pixels correspond to surface-associated Tfn. Scatter plots display the intensity distribution and degree of co-localization corresponding to the entire cell, which is shown next to it. The enclosed areas (yellow rectangles) determine the co-localizing pixels, overlaid in white on the merged images. Magnification of Tfn/TfnR at the uropod membrane is shown at 4 minutes (the enlarged area is depicted in blue). Images at the 4th (20/20) and the 1st minute (16/20) are totally and widely representative, respectively. In the last case, 20% of the cells already showed a polarized distribution of membrane-TfnR/Tfn after 1 minute. The evolution of Tfn/TfnR distribution can also be observed in intensity profiles along membrane vectors from the uropod to the front pole (yellow lines). Bars, 5 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 4. In vivo analysis of Tfn plasma membrane flow and internalization. (A) For analysis of Tfn lateral diffusion on the plasma membrane, ice-chilled cells were incubated with Tfn-A488 for 5 minutes, washed and bleached at their rear halve membranes. Incubation at 37°C of the representative cell quickly restored the membrane diffusion, and fluorescence intensity recovered 50% within 300 seconds at the uropod membrane (100% is the pre-bleaching intensity). Similar analyses were repeated in other five cells and the average recovery ±s.d. was 43±18%. The uptake curve is also shown as the mean intensity measured within the uropod over time (see D). (B) Recovery ratios of Tfn-A488 or anti-CD45-FITC (control). Recovery ratio at one bleached pole corresponds to the mean intensity of the bleached membrane, divided by the intensity of the opposite unbleached membrane. (C) Similar analyses to A were performed bleaching the front membrane. In this way, the membrane fluorescence recovery was minimal (5±2%, n=5), while Tfn uptake at the uropod was comparable with that of the neighbor cells during the 300 second assays (91±4% of control cells, n=5). (D) Tfn internalization in the rear-bleached cell was almost undetectable in the 300 seconds of the assay (arrow), while uptake in control neighbor unbleached cells was as usual (normalized to 100%). The average uptake in these conditions compared with control cells was 4±6% (n=6). Projection of a complete z scanning – taken at time 350 seconds – is shown to allow the identification of any out-of-focus intracellular Tfn. Simultaneous membrane recovery and uptake can be checked in Movie 3 (see supplementary material). Bar, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 5. Actomyosin disruption inhibits CME. (A) T cells were pre-treated with Y-27632 (20 µM, 2 hours), blebbistatin (30 µM, 20 minutes), cytochalasin D (2.5 µg/ml, 2 hours), latrunculin B (0.6 µg/ml, 5 minutes) or colchicine (1 µg/ml, 2 hours) were evaluated for Tfn uptake for 5 and 10 minutes. For the rescue assays the Tfn uptake was re-evaluated after a 5-10 minute wash in fresh medium (n=50 cells for each condition). Similarly, C3-GFP- and the dominant-negative RhoAN19-GFP-transfected cells were evaluated for Tfn uptake for 5 and 10 min (n=10 cells for each condition). The internal pool of Tfn was evaluated as described in Materials and Methods and supplementary material Fig. S3. Values are compared with the control. (B) T cells plated on ICAM-1 were checked for Tfn internalization (10 minute pulse of Tfn-A488, or Tfn-A633 in C3-GFP-transfected cell) and surface TfnR distribution in control, C3-GFP-transfected cells (pseudo-colored in cyan), and pre-treated with Y-27632 and blebbistatin as in A. Bar, 5 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 6. ROCK and actomyosin inhibitors disrupt the polarized distribution of clathrin at the uropod. (A) The effects of Rho/ROCK and myosin II inhibitors on the distribution of membrane CSs (green circles) and on the cell morphology during integrin-mediated cell adhesion. (B) Density of CSs/µm2 – labeled with GFP-ClLC or endogenous -adaptin – at the basal plasma membrane (adhesion plane) of cells pre-incubated with Y-27632 (1 hour), blebbistatin (30 minutes) or cytochalasin D (1 hours). More than 1000 CSs from 10 representative cells were visualized for each inhibitor and labeling, except the control cells, in highly magnified images. Frequency of CSs at the basal membrane of control cells was minimal. The distribution of AP-2 is shown in supplementary material Fig. S2E. (C) Distribution of GFP-ClLC (green) in a representative control cell. The adhesion plane is pointed at the lateral view (arrow). Basal membrane was simultaneously stained with the membrane marker FM 4-64 (red); note how the frequency of CSs is very low in control cells compared with the plasma membrane red stain. (D) Distribution of CSs in cells plated on fibronectin and pre-treated with C3 (14 hours), Y-27632 (1 hour), or blebbistatin (30 minutes). Panels show the endogenous clathrin heavy chain (ClHC) (C3) and GFP-ClLC (Y-27632, blebbistatin) labeling at the basal membrane. Enlarged areas are indicated by blue boxes, and DIC images merged with nuclear DAPI (blue) are shown. Bars, 5 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Specific disruption of CME inhibits chemotaxis. (A) Tfn uptake in cells transfected with GFP, D32-GFP and DIII-GFP, separating the last condition into mock (–) and transfected (+) populations. For quantification, CME was considered totally inhibited when the cell lacked any cytoplasmic signal 20 minutes after Tfn addition. Averages ±s.d. are shown (n=100 cells by condition). (B) Cells transfected with DIII-GFP; and D32-GFP or GFP as controls, were allowed to migrate for 45 minutes in response to 100 ng/ml CXCL12 through transwell membranes. Chemotactic migration is analyzed as the ratio between migrated and non-migrated cells as a function of the fluorescence intensity levels, where region R0 corresponds to the non-transfected cells, and R1, R2, R3 and R4 correspond to increasing intensities. Migration ratios averages ±s.d. obtained from three (GFP), five (D32-GFP) and six (DIII-GFP) independent experiments are shown. Chemotactic analysis was performed only when CME inhibition was affecting >75% of DIII-GFP-transfected cells. The migration ratio of DIII-GFP significantly decreased as the fluorescence intensity (CME inhibition) increased (P<0.05). (C) Immunoblot of T cells transfected with control siRNA (siC) and siRNAs pre-designed against the clathrin heavy chain (si1, si2, si3) to calculate the efficiency relative to control. Note that only si2 and si3 significantly inhibited clathrin expression. (D) Tfn uptake quantification in siRNA-transfected cells. Note that CME is severely reduced but not completely abolished (n=50 cells by condition). (E,F) T cells transfected with siRNAs were allowed to migrate for 45 minutes in response to 100 ng/ml CXCL12 and for 1 hour in basal conditions through transwell membranes, respectively. The total number of migrated cells was quantified by flow-cytometry from three independent experiments, showing that the migration rates of si2 and si3 were significantly decreased compared with siC and si1 controls (P<0.05).

View larger version (40K): [in this window] [in a new window]   Fig. 8. Tfn does not accumulate at the uropod membrane in AP-2 blocked cells. (A) ICAM-3 spontaneously clusters at the uropod tip (green) of both a DIII+ (cyan) and a non-transfected cell. Internalized Tfn only appears in the non-transfected cell (red). The image is a z projection, and the DIC image merged with ICAM-3 labeling is inserted. (B) Time-lapse projections of Tfn-Tx dynamics in a DIII+ (arrow) and a control cell, as indicated. Both cells are polarized (F, front; U, uropod) and representative (10/10). Note that Tfn only enters the control cell, and that it remains evenly distributed on the membrane of the DIII+ cell, with no apparent redistribution towards the rear part along time (see C). (C) Intensity profiles from the front to the rear membrane (blue lines) at different Tfn-chase times (1, 2, 5 min). Note that Tfn is homogenously distributed at the first minute in both cells, and eventually redistributes towards the uropod neck of the control cell (arrowheads). This usual accumulation was never observed in AP-2-blocked cells (DIII+). DIC images merged to Tfn (red) or DIII-GFP (green), are shown at 5 minutes.