Figure 4.
Figure 4. NMIIA is recruited to the secretory granules independently of F-actin. (A–F) Mice expressing either mTomato (mTom; A and B) or GFP-NMIIA (C–F) were exposed to 10 µM CytD for 10 min and injected SC with 0.03 mg/kg ISOP, as described in Materials and methods. (A and B) The SGs were explanted, processed for immunofluorescence, and labeled with Alexa Fluor 647 phalloidin and with antibodies directed against AQP5 (marker for the APM; A, middle), NKCC1 (marker for the basolateral plasma membrane; A, right), NMIIA (B, left), and NMIIB (B, right). (Left) Enlarged granules labeled with mTomato (red arrowheads). Bar, 3 µm. The series of small panels show high magnification of individual enlarged granules labeled for AQP5 but not NKCC1 (A, green arrowheads), phalloidin (A, blue arrowheads), and NMIIA and NMIIB (B, green arrowheads). Bars, 3 µm. (C–E) The SGs were explanted and labeled with Texas-Red phalloidin (red) and Hoechst (blue). (C, left) Maximal projection of a z stack. Bar, 5 µm. (Middle) The insets show a high magnification of the enlarged granules fused with the APM (dashed lines). Note that phalloidin staining is not detectable. (Right) The insets show fused granules under control conditions (arrowheads). Bars, 3 µm. (D) Maximal projection of a z stack in which phalloidin was detected (top, arrowheads). (Bottom) Isosurfaces generated as described in Materials and methods that show that GFP-NMIIA (green) does not overlap with the residual F-actin (red) on the enlarged granules. Bar, 3 µm. (E) Levels of GFP-NMIIA and phalloidin on the secretory granules after treatment with CytoD or LatA (top) and their colocalization (bottom). Error bars represent SD. ****, P < 0.0001; ANOVA. Ctrl, control; Fl. Int., fluorescence intensity. (F, left) ISMic of GFP-NMIIA recruitment onto the enlarged secretory granules (arrowheads) fused at the APM (red dashed line). Bar, 1 µm. (Right) Representative curves of the kinetics of recruitment of GFP-NMIIA in control (black curves) and CytoD-treated (red curves) mice. Curves are from one representative granule (5–10 granules per animal, total three animals). Norm., normalized.

NMIIA is recruited to the secretory granules independently of F-actin. (A–F) Mice expressing either mTomato (mTom; A and B) or GFP-NMIIA (C–F) were exposed to 10 µM CytD for 10 min and injected SC with 0.03 mg/kg ISOP, as described in Materials and methods. (A and B) The SGs were explanted, processed for immunofluorescence, and labeled with Alexa Fluor 647 phalloidin and with antibodies directed against AQP5 (marker for the APM; A, middle), NKCC1 (marker for the basolateral plasma membrane; A, right), NMIIA (B, left), and NMIIB (B, right). (Left) Enlarged granules labeled with mTomato (red arrowheads). Bar, 3 µm. The series of small panels show high magnification of individual enlarged granules labeled for AQP5 but not NKCC1 (A, green arrowheads), phalloidin (A, blue arrowheads), and NMIIA and NMIIB (B, green arrowheads). Bars, 3 µm. (C–E) The SGs were explanted and labeled with Texas-Red phalloidin (red) and Hoechst (blue). (C, left) Maximal projection of a z stack. Bar, 5 µm. (Middle) The insets show a high magnification of the enlarged granules fused with the APM (dashed lines). Note that phalloidin staining is not detectable. (Right) The insets show fused granules under control conditions (arrowheads). Bars, 3 µm. (D) Maximal projection of a z stack in which phalloidin was detected (top, arrowheads). (Bottom) Isosurfaces generated as described in Materials and methods that show that GFP-NMIIA (green) does not overlap with the residual F-actin (red) on the enlarged granules. Bar, 3 µm. (E) Levels of GFP-NMIIA and phalloidin on the secretory granules after treatment with CytoD or LatA (top) and their colocalization (bottom). Error bars represent SD. ****, P < 0.0001; ANOVA. Ctrl, control; Fl. Int., fluorescence intensity. (F, left) ISMic of GFP-NMIIA recruitment onto the enlarged secretory granules (arrowheads) fused at the APM (red dashed line). Bar, 1 µm. (Right) Representative curves of the kinetics of recruitment of GFP-NMIIA in control (black curves) and CytoD-treated (red curves) mice. Curves are from one representative granule (5–10 granules per animal, total three animals). Norm., normalized.

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