Figure 3
Figure 3. INF2 knockdown blocks the formation of exocytic transport vesicles containing Lck and decreases the expression of Lck at the plasma membrane. (A) The subcellular distribution of endogenous Lck was analyzed in Jurkat cells coexpressing GFP and shControl, shINF2a, or shINF2b using anti-Lck antibodies followed by secondary antibodies coupled to Alexa-647 antibodies. Lck staining intensity was measured by densitometric analysis along rectilinear lines as described for MAL in Figure 2C. The histogram shows the mean percentage ± SEM of cells with low levels of peripheral Lck or TCR/CD3. (B-C) Jurkat cells transiently expressing Lck-Cherry (B) or p75-GFP (C) were cotransfected with a construct coexpressing GFP and shControl or shINF2a (B) or Cherry and shControl or shINF2a (C) for 48 hours, the activity being captured with time-lapse videomicroscopy. Transfected cells were identified by expression of GFP (B) or Cherry (C). The processes occurring in the cell within the boxed regions are shown at higher magnification in the right panels. Arrowheads indicate 2 vesicles transporting Lck to the plasma membrane. Numbers indicate time in seconds. (D) Jurkat cells transiently expressing GFP fusions of INF2-1 or Cterm1 were stained for endogenous Lck using anti-Lck antibodies and secondary antibodies coupled to Alexa-647 (left). The histogram shows the mean percentage ± SEM of cells with low levels of peripheral Lck. (E) Jurkat cells transiently expressing GFP fused to Cterm1 were stained for endogenous INF2 using anti-INF2 antibodies and secondary antibodies coupled to Alexa-594 and with TRITC-phalloidin. The staining of F-actin is presented in gray. (F) Jurkat cells coexpressing GFP (top) and the indicated shRNA or a trasmembrane CD4/Lck chimera (bottom) were conjugated to staphylococcal enterotoxin E–pulsed antigen presenting cells (APCs) for 15 minutes. After cell fixation, the distribution of TCR/CD3 and F-actin was analyzed with anti-CD3 antibodies and secondary antibodies coupled to Alexa-647 and with TRITC-phalloidin, respectively. The staining of TCR/CD3 was pseudocolored in green and that of GFP in gray to help visualization of the distribution of TCR/CD3. The cells expressing CD4/Lck were identified with anti–mouse CD4 mAb and secondary antibodies coupled to Alexa-488 (bottom). (G) The histogram represents the mean percentage of transfected Jurkat cells in T-cell–APC conjugates displaying polarized distribution of F-actin or TCR/CD3 to the IS. Three independent experiments were performed in panels A, D, G; n > 50 T cells/experiment (**P < .01). Scale bars correspond to 5 μm unless other value is indicated.

INF2 knockdown blocks the formation of exocytic transport vesicles containing Lck and decreases the expression of Lck at the plasma membrane. (A) The subcellular distribution of endogenous Lck was analyzed in Jurkat cells coexpressing GFP and shControl, shINF2a, or shINF2b using anti-Lck antibodies followed by secondary antibodies coupled to Alexa-647 antibodies. Lck staining intensity was measured by densitometric analysis along rectilinear lines as described for MAL in Figure 2C. The histogram shows the mean percentage ± SEM of cells with low levels of peripheral Lck or TCR/CD3. (B-C) Jurkat cells transiently expressing Lck-Cherry (B) or p75-GFP (C) were cotransfected with a construct coexpressing GFP and shControl or shINF2a (B) or Cherry and shControl or shINF2a (C) for 48 hours, the activity being captured with time-lapse videomicroscopy. Transfected cells were identified by expression of GFP (B) or Cherry (C). The processes occurring in the cell within the boxed regions are shown at higher magnification in the right panels. Arrowheads indicate 2 vesicles transporting Lck to the plasma membrane. Numbers indicate time in seconds. (D) Jurkat cells transiently expressing GFP fusions of INF2-1 or Cterm1 were stained for endogenous Lck using anti-Lck antibodies and secondary antibodies coupled to Alexa-647 (left). The histogram shows the mean percentage ± SEM of cells with low levels of peripheral Lck. (E) Jurkat cells transiently expressing GFP fused to Cterm1 were stained for endogenous INF2 using anti-INF2 antibodies and secondary antibodies coupled to Alexa-594 and with TRITC-phalloidin. The staining of F-actin is presented in gray. (F) Jurkat cells coexpressing GFP (top) and the indicated shRNA or a trasmembrane CD4/Lck chimera (bottom) were conjugated to staphylococcal enterotoxin E–pulsed antigen presenting cells (APCs) for 15 minutes. After cell fixation, the distribution of TCR/CD3 and F-actin was analyzed with anti-CD3 antibodies and secondary antibodies coupled to Alexa-647 and with TRITC-phalloidin, respectively. The staining of TCR/CD3 was pseudocolored in green and that of GFP in gray to help visualization of the distribution of TCR/CD3. The cells expressing CD4/Lck were identified with anti–mouse CD4 mAb and secondary antibodies coupled to Alexa-488 (bottom). (G) The histogram represents the mean percentage of transfected Jurkat cells in T-cell–APC conjugates displaying polarized distribution of F-actin or TCR/CD3 to the IS. Three independent experiments were performed in panels A, D, G; n > 50 T cells/experiment (**P < .01). Scale bars correspond to 5 μm unless other value is indicated.

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