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Volume 7 issue 5 May 2006
Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells
Marcel Mettlen, Anna Platek, Patrick Van Der Smissen, Sarah Carpentier, Mustapha Amyere, Letizia Lanzetti, Philippe de Diesbach, Donatienne Tyteca, and Pierre J. Courtoy
Sup. Figure Legends
Sup. Fig. 1 . Src association with apical plasma membrane and endocytic structures. Polarized cells were kept at 40°C (A-C), or transferred to 34°C for 6 hours (D-F). F-actin (Alexa 568-phalloidin, red) and v-Src protein (green) are shown in single horizontal sections of the apical pole, around the level of tight junctions. Arrowheads, membrane recruitment of Src; thin arrows, macropinosomes; thick arrow, large endocytic vacuole. Bars, 5 µm.
Figure 1 (.jpg)
Sup. Fig. 2 . Selective inhibition of apical membrane ruffling by latrunculin B. Polarized cells were kept at 40°C (A-C), or transferred to 34°C for 6 hours (D-F) and left untreated (A, B, D and E), or treated for 1 hour with 100 nM latrunculin B (C, F), then F-actin was labeled by Alexa 568-phalloidin (red). Representative images from single apical or basolateral horizontal sections. Arrows, position of central sensory cilium; arrowheads, actin-based ruffles. Bars, 5 µm.
Figure 2 (.jpg)
Sup. Fig. 3 . Recruitment of cortactin into v-Src-induced apical membrane ruffles. Polarized cells were kept at 40°C (A-C), or transferred to 34°C for 6 hours (D-F), then F-actin and cortactin were labeled. Representative images from single horizontal sections. Arrows, cortactin labeling of actin-rich ruffles. Bars, 10 µm. Inserts D-F: a circular apical membrane ruffle at higher magnification. Notice the dotty appearance of cortactin labeling. Bars, 5 µm.
Figure 3 (.jpg)
Sup. Fig. 4. Spatial segregation of macropinocytosis and micropinocytosis. Polarized cells were transferred to 34°C for 6 hours, then allowed to apically endocytose Alexa 568-dextran for 5 minutes, extensively washed, fixed and examined by confocal microscopy. Merge of transmission image (gray) and fluorescent apical section colored in red (A); or merge of a similar fluorescent apical section, in red and a nearby lower fluorescent section at the tight junction level, pseudo-colored in green (B). Thick arrows, large endocytic vacuoles. Bars, 5 µm.
Figure 4 (.jpg)
Sup. Fig. 5 . Tubulation of the large apical endocytic vacuole. Cells polarized in LabTek chambers were transferred to 34°C for 6 hours. After uptake of fluorescent dextran for 3 minutes, living cells were rapidly washed and observed at the confocal microscope. Tubulation of the large apical endocytic vacuole was followed by time-lapse recording at 5-s intervals. Representative images are shown at the indicated intervals. Arrows point to visible tips of tubules emitted by the large vacuole. Bar, 5 µm.
Figure 5 (.jpg)
Sup. Fig. 6 . Preferential localization of RN-tre, a Rab5-GAP, at the basolateral membrane and absence of apical ruffling in transfected cells. Polarized cells were transiently transfected for GFP-RN-tre and either kept at 40°C (A, C, E), or transferred to 34°C for 6 hours (B, D, F). Position of GFP-RN-tre (green) is shown by reference to gp135/podocalixin (red, A) or F-actin (red, B-F) in confocal vertical sections (A, B) or horizontal sections (C-F). The small bracket at left indicates the filter position. Arrows in F point to ruffles. RN-tre transfected and v-Src-transformed cells never showed membrane ruffles. Bars, 10 µm.
Figure 6 (.jpg)
Supplemental Movie 1: View Movie 1 (.mov)
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