American Society of Hematology

Figure 7

$Rab32 or Rab38 knock-down impairs normal fusion of vesicles containing dense granule proteins with the organelle. (A-F) MEG-01 cells were cotransfected with LAMP2-Cherry as a DG reporter and either control siRNA shown in panels A and B, Rab32 siRNA in panels C and D, or Rab38 siRNA in panels E and F (see supplemental Figure 11 for Rab32/38 siRNA knockdown confirmation by immunoblotting). The kymographs presented in panels B, D, and F were made by aligning on a time axis the pieces of images indicated with a white rectangle in panels A, C, and E, respectively, from each of the 60 frames of the corresponding movies (1 frame/second). (A) Control siRNA cells present diffraction-limited LAMP2 structures consistent in size with organelles. (B) The LAMP2 structures in Control siRNA cells present a limited range of motion. (C,E) Both Rab32 and Rab38 siRNA cells present LAMP2 structures that are more consistent in size with vesicles or small organelles. (D,F) The smaller LAMP2 structures in Rab32 and Rab38 siRNA cells are more dynamic and move faster than structures in Control siRNA cells. (G) The diameter of LAMP2 structures present in the representative cells shown in panels A, C, and E was measured using the Ruler function in Slidebook. (H) The average speed of LAMP2 structures present in the representative cells shown in panels A, C, and E was measured using the Manual Particle Tracking function in Slidebook. (I) Extracts from control, Rab32, and Rab38 siRNA-treated cells were fractionated in sucrose gradients. For each treatment, the amount of LAMP2 in the first fraction of the gradient, which corresponds to the vesicular LAMP2, was analyzed by immunoblotting. The levels of tubulin in the same blot were used to confirm equal loading. Bars represent 5 μm (*P < .05; **P < .001). (J) DG membrane proteins are sorted in early endosomal compartments by adaptor protein complexes, such as AP-3, which recognize sorting signals present in their cytosolic tails. Rab32 and Rab38 are recruited to the nascent clathrin-coated vesicle and through interactions with so far unknown effectors target the vesicle to the maturing DG. The DG precursor is a MVB that on receiving Rab32/Rab38 vesicles containing DG proteins, such as the ADP transporter MRP4 or the serotonin transporter VMAT2, matures into a DG.$

Rab32 or Rab38 knock-down impairs normal fusion of vesicles containing dense granule proteins with the organelle. (A-F) MEG-01 cells were cotransfected with LAMP2-Cherry as a DG reporter and either control siRNA shown in panels A and B, Rab32 siRNA in panels C and D, or Rab38 siRNA in panels E and F (see supplemental Figure 11 for Rab32/38 siRNA knockdown confirmation by immunoblotting). The kymographs presented in panels B, D, and F were made by aligning on a time axis the pieces of images indicated with a white rectangle in panels A, C, and E, respectively, from each of the 60 frames of the corresponding movies (1 frame/second). (A) Control siRNA cells present diffraction-limited LAMP2 structures consistent in size with organelles. (B) The LAMP2 structures in Control siRNA cells present a limited range of motion. (C,E) Both Rab32 and Rab38 siRNA cells present LAMP2 structures that are more consistent in size with vesicles or small organelles. (D,F) The smaller LAMP2 structures in Rab32 and Rab38 siRNA cells are more dynamic and move faster than structures in Control siRNA cells. (G) The diameter of LAMP2 structures present in the representative cells shown in panels A, C, and E was measured using the Ruler function in Slidebook. (H) The average speed of LAMP2 structures present in the representative cells shown in panels A, C, and E was measured using the Manual Particle Tracking function in Slidebook. (I) Extracts from control, Rab32, and Rab38 siRNA-treated cells were fractionated in sucrose gradients. For each treatment, the amount of LAMP2 in the first fraction of the gradient, which corresponds to the vesicular LAMP2, was analyzed by immunoblotting. The levels of tubulin in the same blot were used to confirm equal loading. Bars represent 5 μm (*P < .05; **P < .001). (J) DG membrane proteins are sorted in early endosomal compartments by adaptor protein complexes, such as AP-3, which recognize sorting signals present in their cytosolic tails. Rab32 and Rab38 are recruited to the nascent clathrin-coated vesicle and through interactions with so far unknown effectors target the vesicle to the maturing DG. The DG precursor is a MVB that on receiving Rab32/Rab38 vesicles containing DG proteins, such as the ADP transporter MRP4 or the serotonin transporter VMAT2, matures into a DG.

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