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The Abl/enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons.

Kannan R, Kuzina I, Wincovitch S, Nowotarski SH, Giniger E - Mol. Biol. Cell (2014)

Bottom Line: The Abl effector, Enabled (Ena), selectively labels the cis-Golgi in developing PRs.Finally, we demonstrate that the effects of Abl signaling on Golgi are mediated via regulation of the actin cytoskeleton.Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion.

View Article: PubMed Central - PubMed

Affiliation: Axon Guidance and Neural Connectivity Unit, Basic Neuroscience Program, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

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Pharmacological disruption of actin structure is downstream of Abl signaling for Golgi distribution. (A–C) Confocal imaging of third-instar eye imaginal disks cultured in 20 mM cytochalasin B and mock-treated controls, stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). All sections were acquired at similar settings. Note the decrease in phalloidin staining at the cortex in CytoB-treated cultures compared with mock controls. A single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads highlight cis-Golgi cisternae. (A) WT. (B) dabmz. (C) Abl mutant (Df stJ7/abl4). Note that Golgi cisternae are concentrated in the basal cytoplasm in the mock-treated mutants but become randomized throughout the cell upon treatment with CytoB. (D, E) Quantification of CytoB-epistasis data. n for each experiment is reported on the bars. Error bars correspond to SEM. **p < 0.001 (ANOVA). (D) Quantification of cis-Golgi distribution after CytoB-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks. (E) Quantification of Golgi fragmentation phenotypes in CytoB-treated cultures of WT, dabmz, and Abl mutants (df stJ7/abl4). (F–H) Confocal micrographs of third-instar larval eye imaginal disks, acquired at similar settings, cultured in 10 mM LatD and DMSO-treated mock controls were stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). Note the decrease in phalloidin staining at the cortex in LatD-treated cultures compared with mock controls. Single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads represent cis-Golgi cisternae. (F) WT. (G) dabmz. (H) Abl mutant (Df stJ7/abl4). Note the random distribution of cis-Golgi structures in dabmz disks (G) and Abl mutant disks (H) compared with WT controls (F) treated with LatD. Scale bar, 5 μm. (I, J). Quantification of LatD epistasis data. n for each treatments is indicated on the bars. Error bars represent SEM. **p < 0.001 (estimated by ANOVA). Quantification of fragmentation (I) and basal redistribution (J) of cis-Golgi structures after LatD-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks.
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Figure 7: Pharmacological disruption of actin structure is downstream of Abl signaling for Golgi distribution. (A–C) Confocal imaging of third-instar eye imaginal disks cultured in 20 mM cytochalasin B and mock-treated controls, stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). All sections were acquired at similar settings. Note the decrease in phalloidin staining at the cortex in CytoB-treated cultures compared with mock controls. A single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads highlight cis-Golgi cisternae. (A) WT. (B) dabmz. (C) Abl mutant (Df stJ7/abl4). Note that Golgi cisternae are concentrated in the basal cytoplasm in the mock-treated mutants but become randomized throughout the cell upon treatment with CytoB. (D, E) Quantification of CytoB-epistasis data. n for each experiment is reported on the bars. Error bars correspond to SEM. **p < 0.001 (ANOVA). (D) Quantification of cis-Golgi distribution after CytoB-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks. (E) Quantification of Golgi fragmentation phenotypes in CytoB-treated cultures of WT, dabmz, and Abl mutants (df stJ7/abl4). (F–H) Confocal micrographs of third-instar larval eye imaginal disks, acquired at similar settings, cultured in 10 mM LatD and DMSO-treated mock controls were stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). Note the decrease in phalloidin staining at the cortex in LatD-treated cultures compared with mock controls. Single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads represent cis-Golgi cisternae. (F) WT. (G) dabmz. (H) Abl mutant (Df stJ7/abl4). Note the random distribution of cis-Golgi structures in dabmz disks (G) and Abl mutant disks (H) compared with WT controls (F) treated with LatD. Scale bar, 5 μm. (I, J). Quantification of LatD epistasis data. n for each treatments is indicated on the bars. Error bars represent SEM. **p < 0.001 (estimated by ANOVA). Quantification of fragmentation (I) and basal redistribution (J) of cis-Golgi structures after LatD-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks.

Mentions: Time-resolved pharmacological experiments further confirmed that Abl controls Golgi distribution through actin regulation (Figure 7, A– H). Third-instar larval eye disks were cultured for 1 h in media containing low concentrations of the actin depolymerizing agent cytochalasin B (CytoB; Figures 7, A– C) or latrunculin D (LatD; Figure 7, F– H) to select a concentration that reduced phalloidin staining but did not alter the overall morphology of PR. In WT, CytoB treatment fragments the cis-Golgi compartment without altering its apical-basal distribution (Figure 7A). These effects resemble Golgi phenotypes observed in Ena loss of function (Figure 2C). We then tested whether pharmacological disruption of actin structure modified the basal redistribution of cis-Golgi in dabmz (Figure 7B) and Abl mutants (Figure 7C). In mock-treated control cultures, we observed a high percentage of basally distributed cis-Golgi structures in dabmz (90.4% ± 6.1) and in Abl mutants (77.3% ± 5.7) compared with WT (60% ± 4.8; Figure 7D). Acute treatment with CytoB (20 μM) significantly restored the WT apical-basal distribution of cis-Golgi structures in dabmz (64% ± 6.4) and in Abl mutant (61.3% ± 4.9), respectively (Figure 7D). These data imply, first, that formation and maintenance of the basal redistribution of Golgi in Abl pathway mutants is an active process unleashed by derepression of Ena, and, second, that actin structure is functionally downstream of Abl/Ena activity for Golgi distribution. We also observed a synergistic increase in the mean number of cis-Golgi fragments in drug-treated dabmz (14.7 ± 1.8) and Abl mutant (18.9 ± 1.8) tissues compared with their respective mutant mock control alone (dabmz, 9.9 ± 1.4; Abl, 13.1 ± 1.3; Figure 7E). Comparable results were obtained with LatD-treated (10 mM) eye disk cultures (Figure 7, F– J). Taken together, these results suggest that the effects of the Abl/Ena pathway on Golgi localization, and probably on Golgi fragmentation, are mediated through regulation of actin dynamics. Consistent with this, in WT eye disks, we often detected enriched F-actin foci adjacent to cis-Golgi structures (Figure 6D).


The Abl/enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons.

Kannan R, Kuzina I, Wincovitch S, Nowotarski SH, Giniger E - Mol. Biol. Cell (2014)

Pharmacological disruption of actin structure is downstream of Abl signaling for Golgi distribution. (A–C) Confocal imaging of third-instar eye imaginal disks cultured in 20 mM cytochalasin B and mock-treated controls, stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). All sections were acquired at similar settings. Note the decrease in phalloidin staining at the cortex in CytoB-treated cultures compared with mock controls. A single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads highlight cis-Golgi cisternae. (A) WT. (B) dabmz. (C) Abl mutant (Df stJ7/abl4). Note that Golgi cisternae are concentrated in the basal cytoplasm in the mock-treated mutants but become randomized throughout the cell upon treatment with CytoB. (D, E) Quantification of CytoB-epistasis data. n for each experiment is reported on the bars. Error bars correspond to SEM. **p < 0.001 (ANOVA). (D) Quantification of cis-Golgi distribution after CytoB-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks. (E) Quantification of Golgi fragmentation phenotypes in CytoB-treated cultures of WT, dabmz, and Abl mutants (df stJ7/abl4). (F–H) Confocal micrographs of third-instar larval eye imaginal disks, acquired at similar settings, cultured in 10 mM LatD and DMSO-treated mock controls were stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). Note the decrease in phalloidin staining at the cortex in LatD-treated cultures compared with mock controls. Single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads represent cis-Golgi cisternae. (F) WT. (G) dabmz. (H) Abl mutant (Df stJ7/abl4). Note the random distribution of cis-Golgi structures in dabmz disks (G) and Abl mutant disks (H) compared with WT controls (F) treated with LatD. Scale bar, 5 μm. (I, J). Quantification of LatD epistasis data. n for each treatments is indicated on the bars. Error bars represent SEM. **p < 0.001 (estimated by ANOVA). Quantification of fragmentation (I) and basal redistribution (J) of cis-Golgi structures after LatD-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks.
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Figure 7: Pharmacological disruption of actin structure is downstream of Abl signaling for Golgi distribution. (A–C) Confocal imaging of third-instar eye imaginal disks cultured in 20 mM cytochalasin B and mock-treated controls, stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). All sections were acquired at similar settings. Note the decrease in phalloidin staining at the cortex in CytoB-treated cultures compared with mock controls. A single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads highlight cis-Golgi cisternae. (A) WT. (B) dabmz. (C) Abl mutant (Df stJ7/abl4). Note that Golgi cisternae are concentrated in the basal cytoplasm in the mock-treated mutants but become randomized throughout the cell upon treatment with CytoB. (D, E) Quantification of CytoB-epistasis data. n for each experiment is reported on the bars. Error bars correspond to SEM. **p < 0.001 (ANOVA). (D) Quantification of cis-Golgi distribution after CytoB-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks. (E) Quantification of Golgi fragmentation phenotypes in CytoB-treated cultures of WT, dabmz, and Abl mutants (df stJ7/abl4). (F–H) Confocal micrographs of third-instar larval eye imaginal disks, acquired at similar settings, cultured in 10 mM LatD and DMSO-treated mock controls were stained for phalloidin (green), cis-Golgi (anti-GM130; red), and anti-Elav (blue). Note the decrease in phalloidin staining at the cortex in LatD-treated cultures compared with mock controls. Single PR cell body is outlined in enlarged panels for the various conditions. Arrowheads represent cis-Golgi cisternae. (F) WT. (G) dabmz. (H) Abl mutant (Df stJ7/abl4). Note the random distribution of cis-Golgi structures in dabmz disks (G) and Abl mutant disks (H) compared with WT controls (F) treated with LatD. Scale bar, 5 μm. (I, J). Quantification of LatD epistasis data. n for each treatments is indicated on the bars. Error bars represent SEM. **p < 0.001 (estimated by ANOVA). Quantification of fragmentation (I) and basal redistribution (J) of cis-Golgi structures after LatD-mediated disruption of actin polymerization in WT, dabmz, and Abl mutant (Df stJ7/abl4) eye disks.
Mentions: Time-resolved pharmacological experiments further confirmed that Abl controls Golgi distribution through actin regulation (Figure 7, A– H). Third-instar larval eye disks were cultured for 1 h in media containing low concentrations of the actin depolymerizing agent cytochalasin B (CytoB; Figures 7, A– C) or latrunculin D (LatD; Figure 7, F– H) to select a concentration that reduced phalloidin staining but did not alter the overall morphology of PR. In WT, CytoB treatment fragments the cis-Golgi compartment without altering its apical-basal distribution (Figure 7A). These effects resemble Golgi phenotypes observed in Ena loss of function (Figure 2C). We then tested whether pharmacological disruption of actin structure modified the basal redistribution of cis-Golgi in dabmz (Figure 7B) and Abl mutants (Figure 7C). In mock-treated control cultures, we observed a high percentage of basally distributed cis-Golgi structures in dabmz (90.4% ± 6.1) and in Abl mutants (77.3% ± 5.7) compared with WT (60% ± 4.8; Figure 7D). Acute treatment with CytoB (20 μM) significantly restored the WT apical-basal distribution of cis-Golgi structures in dabmz (64% ± 6.4) and in Abl mutant (61.3% ± 4.9), respectively (Figure 7D). These data imply, first, that formation and maintenance of the basal redistribution of Golgi in Abl pathway mutants is an active process unleashed by derepression of Ena, and, second, that actin structure is functionally downstream of Abl/Ena activity for Golgi distribution. We also observed a synergistic increase in the mean number of cis-Golgi fragments in drug-treated dabmz (14.7 ± 1.8) and Abl mutant (18.9 ± 1.8) tissues compared with their respective mutant mock control alone (dabmz, 9.9 ± 1.4; Abl, 13.1 ± 1.3; Figure 7E). Comparable results were obtained with LatD-treated (10 mM) eye disk cultures (Figure 7, F– J). Taken together, these results suggest that the effects of the Abl/Ena pathway on Golgi localization, and probably on Golgi fragmentation, are mediated through regulation of actin dynamics. Consistent with this, in WT eye disks, we often detected enriched F-actin foci adjacent to cis-Golgi structures (Figure 6D).

Bottom Line: The Abl effector, Enabled (Ena), selectively labels the cis-Golgi in developing PRs.Finally, we demonstrate that the effects of Abl signaling on Golgi are mediated via regulation of the actin cytoskeleton.Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion.

View Article: PubMed Central - PubMed

Affiliation: Axon Guidance and Neural Connectivity Unit, Basic Neuroscience Program, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

Show MeSH
Related in: MedlinePlus