Figure 7.
Missorting of HA-VMAT to ectopic phase-separated vesicle aggregates and reduction of DCV numbers in cell bodies of Rab2 mutants. (A) Representative STED images showing the distribution of ILP2-GFP and HA-tagged WT VMAT in motor neuron cell bodies in VNCs of third instar larvae. KD, motor neuron-specific knockdown. Scale bar: 2 µm, 500 nm (insets). (B) Left, the co-localization of ILP2-GFP and HA-VMAT in A quantified using Pearson’s correlation coefficient (PCC). P values in red and blue result from comparison with the control and Rab2 group, respectively. Right, the correlation between ILP2-GFP and HA-VMAT in motor neuron cell bodies of RUFY-KD and control larvae. Counts of VMAT aggregates made in confocal images are also shown (numbers below the graphs). (C) Left, ILP2-GFP-positive vesicle densities in cell bodies for the larval genotypes in A, and in RUFY-KD larvae with controls (right). Number of larvae analyzed in B and C: control 26, Rab2 10, sydz4/Df 9, Dhc-KD 9, Arl8/Df; control for RUFY-KD 7, RUFY-KD 8. (D) Representative confocal and STED (insets) images of WT HA-VMAT in motor neuron cell bodies from untreated Rab2 third instar VNCs (top) or Rab2 VNCs treated with 10% 1,6 hexanediol (1,6-HD) prior to fixation (bottom). Scale bar: 10 µm. (E) Density and relative area of large droplet-shaped VMAT-positive aggregates in motor neuron cell bodies in D. Number of larvae analyzed: control 8, 1,6-HD 7. (F) Representative STED images showing the distribution of ILP2-GFP and WT HA-VMAT or HA-VMATDE/LI/Y>A (HA-VMATD584A, E585A, L589A, I590A, Y600A) in motor neuron cell bodies from control (top) and Rab2 larvae (bottom). Scale bar: 2 µm, 500 nm (insets). (G) Left, quantification using PCC of the co-localization of ILP2-GFP with HA-VMAT or HA-VMATDE/LI/Y>A in control and Rab2 larvae. Right, density and relative area of large droplet-shaped VMAT-positive aggregates in Rab2 motor neuron cell bodies. Number of larvae analyzed in G, left: control, HA-VMATWT 7; control, HA-VMATDE/LI/Y>A 6; Rab2, HA-VMATWT 6; Rab2, HA-VMATDE/LI/Y>A 8. Number of larvae analyzed in G, right: Rab2, HA-VMATWT 9; Rab2, HA-VMATDE/LI/Y>A 10. Bar graphs in B, C, E, G represent mean + SEM. ANOVA followed by Tukey’s test (B, left; G, left), Steel with control test (C, left), Student's t test (B, right; C, right; E; G, right).

Missorting of HA-VMAT to ectopic phase-separated vesicle aggregates and reduction of DCV numbers in cell bodies of Rab2 mutants. (A) Representative STED images showing the distribution of ILP2-GFP and HA-tagged WT VMAT in motor neuron cell bodies in VNCs of third instar larvae. KD, motor neuron-specific knockdown. Scale bar: 2 µm, 500 nm (insets). (B) Left, the co-localization of ILP2-GFP and HA-VMAT in A quantified using Pearson’s correlation coefficient (PCC). P values in red and blue result from comparison with the control and Rab2 group, respectively. Right, the correlation between ILP2-GFP and HA-VMAT in motor neuron cell bodies of RUFY-KD and control larvae. Counts of VMAT aggregates made in confocal images are also shown (numbers below the graphs). (C) Left, ILP2-GFP-positive vesicle densities in cell bodies for the larval genotypes in A, and in RUFY-KD larvae with controls (right). Number of larvae analyzed in B and C: control 26, Rab2 10, sydz4/Df 9, Dhc-KD 9, Arl8/Df; control for RUFY-KD 7, RUFY-KD 8. (D) Representative confocal and STED (insets) images of WT HA-VMAT in motor neuron cell bodies from untreated Rab2 third instar VNCs (top) or Rab2 VNCs treated with 10% 1,6 hexanediol (1,6-HD) prior to fixation (bottom). Scale bar: 10 µm. (E) Density and relative area of large droplet-shaped VMAT-positive aggregates in motor neuron cell bodies in D. Number of larvae analyzed: control 8, 1,6-HD 7. (F) Representative STED images showing the distribution of ILP2-GFP and WT HA-VMAT or HA-VMATDE/LI/Y>A (HA-VMATD584A, E585A, L589A, I590A, Y600A) in motor neuron cell bodies from control (top) and Rab2 larvae (bottom). Scale bar: 2 µm, 500 nm (insets). (G) Left, quantification using PCC of the co-localization of ILP2-GFP with HA-VMAT or HA-VMATDE/LI/Y>A in control and Rab2 larvae. Right, density and relative area of large droplet-shaped VMAT-positive aggregates in Rab2 motor neuron cell bodies. Number of larvae analyzed in G, left: control, HA-VMATWT 7; control, HA-VMATDE/LI/Y>A 6; Rab2, HA-VMATWT 6; Rab2, HA-VMATDE/LI/Y>A 8. Number of larvae analyzed in G, right: Rab2, HA-VMATWT 9; Rab2, HA-VMATDE/LI/Y>A 10. Bar graphs in B, C, E, G represent mean + SEM. ANOVA followed by Tukey’s test (B, left; G, left), Steel with control test (C, left), Student's t test (B, right; C, right; E; G, right).

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