Figure 5
Figure 5. TEM of cytoplasmic and membrane remodeling in erythroblasts and reticulocytes and detection of GPA-positive vesicles by confocal microscopy. (A) Representative TEM images of day 23 filtered reticulocytes containing large autophagic compartments (autophagosomes and amphisomes). (i-ii) Fields of R2 reticulocytes, some retaining large autophagic vacuoles (arrowheads) containing poorly degraded organelles; identical compartments were also observed undergoing exocytosis (arrow). (iii) Higher magnification image of the autophagic vacuole outlined in panel ii. (iv-v) Examples of exocytic events in reticulocytes. (B) Evidence for plasma membrane blebbing during human in vitro erythropoiesis. (i-ii) Plasma membrane blebs on the surface of erythroblasts. (iii-v) Plasma membrane blebs showing electron-dense constrictions at their bases (arrowheads). (vi) Example field of a reticulocyte culture containing cellular fragments (arrows) of comparable size to the plasma membrane blebs observed in erythroblasts. (C) Analysis of reticulocytes isolated after 7 days in final stage medium and analyzed at days 7, 11, and 14 (numbers of cells analyzed: 54 at day 7, 37 at day 11, and 43 at day 14). Black bars represent the mean. *P ≤ .05; **P ≤ .01; ***P ≤ .001. (D) GPA-stained cells before and after filtration (left and right panels, respectively) show the presence of vesicles (arrows; i). (ii-iv) GPA (red) dual-stained aquaporin-1 (Aq-1), Glut-1, and band 3 (all green), respectively. (E) Filtered reticulocytes were dual stained with GPA (green) and Abs to organelle markers (red) for autophagy (LC3; i), ER (calreticulin; ii), Golgi (giantin; iii), lysosomes (LAMP1; iv), and mitochondria (MitoTracker; v). Arrows highlight vesicles fusing with the plasma membrane. All cells were cultured following protocol A. (F) Presence of GPA-positive vesicles in vivo. GPA-positive vesicles (red) containing organelle markers (green) for autophagy (LC3; i) and Golgi (giantin; ii) and GPA-positive vesicles (green) with mitochondrial probe MitoTracker (red) found in cells from peripheral blood (iii). Arrows highlight colocalization. Scale bars indicate 5 μm.

TEM of cytoplasmic and membrane remodeling in erythroblasts and reticulocytes and detection of GPA-positive vesicles by confocal microscopy. (A) Representative TEM images of day 23 filtered reticulocytes containing large autophagic compartments (autophagosomes and amphisomes). (i-ii) Fields of R2 reticulocytes, some retaining large autophagic vacuoles (arrowheads) containing poorly degraded organelles; identical compartments were also observed undergoing exocytosis (arrow). (iii) Higher magnification image of the autophagic vacuole outlined in panel ii. (iv-v) Examples of exocytic events in reticulocytes. (B) Evidence for plasma membrane blebbing during human in vitro erythropoiesis. (i-ii) Plasma membrane blebs on the surface of erythroblasts. (iii-v) Plasma membrane blebs showing electron-dense constrictions at their bases (arrowheads). (vi) Example field of a reticulocyte culture containing cellular fragments (arrows) of comparable size to the plasma membrane blebs observed in erythroblasts. (C) Analysis of reticulocytes isolated after 7 days in final stage medium and analyzed at days 7, 11, and 14 (numbers of cells analyzed: 54 at day 7, 37 at day 11, and 43 at day 14). Black bars represent the mean. *P ≤ .05; **P ≤ .01; ***P ≤ .001. (D) GPA-stained cells before and after filtration (left and right panels, respectively) show the presence of vesicles (arrows; i). (ii-iv) GPA (red) dual-stained aquaporin-1 (Aq-1), Glut-1, and band 3 (all green), respectively. (E) Filtered reticulocytes were dual stained with GPA (green) and Abs to organelle markers (red) for autophagy (LC3; i), ER (calreticulin; ii), Golgi (giantin; iii), lysosomes (LAMP1; iv), and mitochondria (MitoTracker; v). Arrows highlight vesicles fusing with the plasma membrane. All cells were cultured following protocol A. (F) Presence of GPA-positive vesicles in vivo. GPA-positive vesicles (red) containing organelle markers (green) for autophagy (LC3; i) and Golgi (giantin; ii) and GPA-positive vesicles (green) with mitochondrial probe MitoTracker (red) found in cells from peripheral blood (iii). Arrows highlight colocalization. Scale bars indicate 5 μm.

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