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Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes.

Racchetti G, D'Alessandro R, Meldolesi J - Glia (2011)

Bottom Line: Contrary to previous reports, we found stellation to be governed by the small G protein Rac1, up to disappearance of the process when Rac1 was downregulated or blocked by a specific drug.The surface expansion concomitant to cytoskeleton restructuring, also dependent on Rac1, was found to be at least partially sustained by the exocytosis of enlargeosomes, small vesicles distinct from classical cell organelles, which are abundant in astrocytes.Exhaustion of stellation induced by repeated administrations of Y27632 correlated with the decrease of the enlargeosome pool.

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Affiliation: Scientific Institute San Raffaele, Division of Neuroscience and IIT Network, Research Unit of Molecular Neuroscience, via Olgettina 58, Milan, Italy.

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Enlargeosomes participate in the surface enlargement of stellation. (A) Western blot results showing that astrocytes are rich of enlargeosomes (marker Ahnak, expressed in the range of enlargeosome-rich neural cells, PC12-27 and SH-SY5Y). (B) Astrocytes before (left) and after (right) stellation, immunolabeled for GFAP (green) and Ahnak (red). (C) Flat cultured astrocytes dually labeled for Ahnak (red) and markers of cytoplasmic organelles (green): calnexin for endoplasmic reticulum (left panel), 58 K for Golgi complex (arrows, middle panel), TGN38 for transGolgi network (arrows, right panel). (D, E) astrocytes before (left panels) and after Y27632-induced (25 μM, 60 min) stellation (middle panels) dually immuno-labeled for Ahnak (red) and the vesicular glutamate transporters vGLUT1 (D) and vGLUT2 (E) (green). The right panels of (D, E) are higher magnification, deconvolved images of the corresponding middle panels. Bars in (B) (valid in C and left/middle panels of D, E), and in (D, E) right panels, are 10 μm.
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fig04: Enlargeosomes participate in the surface enlargement of stellation. (A) Western blot results showing that astrocytes are rich of enlargeosomes (marker Ahnak, expressed in the range of enlargeosome-rich neural cells, PC12-27 and SH-SY5Y). (B) Astrocytes before (left) and after (right) stellation, immunolabeled for GFAP (green) and Ahnak (red). (C) Flat cultured astrocytes dually labeled for Ahnak (red) and markers of cytoplasmic organelles (green): calnexin for endoplasmic reticulum (left panel), 58 K for Golgi complex (arrows, middle panel), TGN38 for transGolgi network (arrows, right panel). (D, E) astrocytes before (left panels) and after Y27632-induced (25 μM, 60 min) stellation (middle panels) dually immuno-labeled for Ahnak (red) and the vesicular glutamate transporters vGLUT1 (D) and vGLUT2 (E) (green). The right panels of (D, E) are higher magnification, deconvolved images of the corresponding middle panels. Bars in (B) (valid in C and left/middle panels of D, E), and in (D, E) right panels, are 10 μm.

Mentions: Western blot results in cultured, flat astrocytes revealed the expression of the enlargeosome marker, Ahnak, to be much higher than in wtPC12 and in the same range as other cells rich of enlargeosomes, PC12-27 and SH-SY5Y (Fig. 4A; see Prada et al., 2011). Ahnak immunolabeling showed the marker concentrated in numerous small puncta spread throughout the cytoplasm (Fig. 4B, left panel), that upon stellation redistributed also to the processes (Fig. 4B, right panel). As in many other cell types, the Ahnak-positive puncta did not colocalize with markers of classical organelles such as the ER, Golgi complex, and transGolgi network (calnexin, 58K, TGN38, respectively, Fig. 4C), concentrated in discrete areas of the cytoplasm. Likewise, Ahnak did not colocalize with various glutamate transporters, neither the clear vesicle vGLUT1 (Fig. 4D) nor the plasma membrane transporters GLAST and EAA2, which in cultured astrocytes exhibited a widespread cytoplasmic distribution (not shown). A partial colocalization of Ahnak was observed only with the vesicular glutamate transporter vGLUT2. Deconvolution analyses of the samples (Fig. 4E) revealed it to occur in discrete puncta distributed in the cell body and the processes, especially proximal to the surface. The relevance of this colocalization remains to be investigated.


Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes.

Racchetti G, D'Alessandro R, Meldolesi J - Glia (2011)

Enlargeosomes participate in the surface enlargement of stellation. (A) Western blot results showing that astrocytes are rich of enlargeosomes (marker Ahnak, expressed in the range of enlargeosome-rich neural cells, PC12-27 and SH-SY5Y). (B) Astrocytes before (left) and after (right) stellation, immunolabeled for GFAP (green) and Ahnak (red). (C) Flat cultured astrocytes dually labeled for Ahnak (red) and markers of cytoplasmic organelles (green): calnexin for endoplasmic reticulum (left panel), 58 K for Golgi complex (arrows, middle panel), TGN38 for transGolgi network (arrows, right panel). (D, E) astrocytes before (left panels) and after Y27632-induced (25 μM, 60 min) stellation (middle panels) dually immuno-labeled for Ahnak (red) and the vesicular glutamate transporters vGLUT1 (D) and vGLUT2 (E) (green). The right panels of (D, E) are higher magnification, deconvolved images of the corresponding middle panels. Bars in (B) (valid in C and left/middle panels of D, E), and in (D, E) right panels, are 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Enlargeosomes participate in the surface enlargement of stellation. (A) Western blot results showing that astrocytes are rich of enlargeosomes (marker Ahnak, expressed in the range of enlargeosome-rich neural cells, PC12-27 and SH-SY5Y). (B) Astrocytes before (left) and after (right) stellation, immunolabeled for GFAP (green) and Ahnak (red). (C) Flat cultured astrocytes dually labeled for Ahnak (red) and markers of cytoplasmic organelles (green): calnexin for endoplasmic reticulum (left panel), 58 K for Golgi complex (arrows, middle panel), TGN38 for transGolgi network (arrows, right panel). (D, E) astrocytes before (left panels) and after Y27632-induced (25 μM, 60 min) stellation (middle panels) dually immuno-labeled for Ahnak (red) and the vesicular glutamate transporters vGLUT1 (D) and vGLUT2 (E) (green). The right panels of (D, E) are higher magnification, deconvolved images of the corresponding middle panels. Bars in (B) (valid in C and left/middle panels of D, E), and in (D, E) right panels, are 10 μm.
Mentions: Western blot results in cultured, flat astrocytes revealed the expression of the enlargeosome marker, Ahnak, to be much higher than in wtPC12 and in the same range as other cells rich of enlargeosomes, PC12-27 and SH-SY5Y (Fig. 4A; see Prada et al., 2011). Ahnak immunolabeling showed the marker concentrated in numerous small puncta spread throughout the cytoplasm (Fig. 4B, left panel), that upon stellation redistributed also to the processes (Fig. 4B, right panel). As in many other cell types, the Ahnak-positive puncta did not colocalize with markers of classical organelles such as the ER, Golgi complex, and transGolgi network (calnexin, 58K, TGN38, respectively, Fig. 4C), concentrated in discrete areas of the cytoplasm. Likewise, Ahnak did not colocalize with various glutamate transporters, neither the clear vesicle vGLUT1 (Fig. 4D) nor the plasma membrane transporters GLAST and EAA2, which in cultured astrocytes exhibited a widespread cytoplasmic distribution (not shown). A partial colocalization of Ahnak was observed only with the vesicular glutamate transporter vGLUT2. Deconvolution analyses of the samples (Fig. 4E) revealed it to occur in discrete puncta distributed in the cell body and the processes, especially proximal to the surface. The relevance of this colocalization remains to be investigated.

Bottom Line: Contrary to previous reports, we found stellation to be governed by the small G protein Rac1, up to disappearance of the process when Rac1 was downregulated or blocked by a specific drug.The surface expansion concomitant to cytoskeleton restructuring, also dependent on Rac1, was found to be at least partially sustained by the exocytosis of enlargeosomes, small vesicles distinct from classical cell organelles, which are abundant in astrocytes.Exhaustion of stellation induced by repeated administrations of Y27632 correlated with the decrease of the enlargeosome pool.

View Article: PubMed Central - PubMed

Affiliation: Scientific Institute San Raffaele, Division of Neuroscience and IIT Network, Research Unit of Molecular Neuroscience, via Olgettina 58, Milan, Italy.

Show MeSH
Related in: MedlinePlus