Figure S7.
Trafficking defects of HA-VMAT and SYTα-mCherry in motor neuron somata and axons of Rab2 null larvae evaluated by STED microscopy. (A) Spatial relationship between ILP2-GFP and HA-VMATY600A in cell bodies. Representative STED images showing the distribution of ILP2-GFP and HA-tagged VMATY600A in motor neuron cell bodies in VNCs of control and Rab2 third instar larvae. Scale bars (left to right): 2 µm, 200 nm (inset), 2 µm, and 200 nm (inset). (B) Quantification of A. Pearson’s correlation coefficient (PCC) between the ILP2-GFP and HA-VMATY600A signals is shown for two control and five Rab2 larvae. Counts of HA-VMATY600A aggregates are also given (numbers below graph). (C) Spatial relationship between ILP2-GFP and Sytα-mCherry in cell bodies. Representative STED images showing the distribution of ILP2-GFP and mCherry-tagged Sytα in motor neuron cell bodies in VNCs of control and Rab2 third instar larvae. Examples of diffuse (middle) and dense (right) Sytα-mCherry aggregates in Rab2 mutants are shown. Scale bars (left to right): 2 µm, 200 nm (inset), 2 µm, 400 nm (inset), 2 µm, and 500 nm (inset). (D) The PCC between ILP2-GFP and Sytα-mCherry is shown for three control and three Rab2 larvae. Counts of diffuse and dense Sytα-mCherry aggregates are also given (numbers below graph). (E) Size distribution of VMAT aggregates (n = 262) in Rab2 cell bodies (Fig. 7 A). Q1 and Q3, first and third quartile. Diameters were calculated from the areas of the aggregates, assuming a circular shape. A lower diameter cutoff of 178.4 nm (area 0.025 µ2) was applied. (F) Representative STED image showing the distribution of ILP2-GFP and HA-VMAT in a VNC motor neuron cell body from third instar larva subjected to motor neuron-targeted knockdown of RUFY (OK6 > ILP2-GFP, HA-VMAT, RUFY-RNAiVAL20). Scale bars: 2 µm, 200 nm (inset). (G) Spatial relationship between ILP2-GFP and HA-tagged WT VMAT in motor axons. The multiple ROIs defined by yellow outlines mark the DCV-associated ILP2-GFP2 signal superimposed on the VMAT image. In the bottom row, the VMAT signal outside the DCV-associated ROIs has been digitally erased. Scale bar: 500 nm. (H) Quantification of E, showing the percentage of VMAT signal located outside the DCV-associated ROIs (top) and the VMAT signal intensity inside the ROIs (bottom) for eight control and eight Rab2 larvae. Bar graphs in B, D, and H represent mean + SEM. Student’s t test (B, D, and H).

Trafficking defects of HA-VMAT and SYTα-mCherry in motor neuron somata and axons of Rab2 null larvae evaluated by STED microscopy. (A) Spatial relationship between ILP2-GFP and HA-VMATY600A in cell bodies. Representative STED images showing the distribution of ILP2-GFP and HA-tagged VMATY600A in motor neuron cell bodies in VNCs of control and Rab2 third instar larvae. Scale bars (left to right): 2 µm, 200 nm (inset), 2 µm, and 200 nm (inset). (B) Quantification of A. Pearson’s correlation coefficient (PCC) between the ILP2-GFP and HA-VMATY600A signals is shown for two control and five Rab2 larvae. Counts of HA-VMATY600A aggregates are also given (numbers below graph). (C) Spatial relationship between ILP2-GFP and Sytα-mCherry in cell bodies. Representative STED images showing the distribution of ILP2-GFP and mCherry-tagged Sytα in motor neuron cell bodies in VNCs of control and Rab2 third instar larvae. Examples of diffuse (middle) and dense (right) Sytα-mCherry aggregates in Rab2 mutants are shown. Scale bars (left to right): 2 µm, 200 nm (inset), 2 µm, 400 nm (inset), 2 µm, and 500 nm (inset). (D) The PCC between ILP2-GFP and Sytα-mCherry is shown for three control and three Rab2 larvae. Counts of diffuse and dense Sytα-mCherry aggregates are also given (numbers below graph). (E) Size distribution of VMAT aggregates (n = 262) in Rab2 cell bodies (Fig. 7 A). Q1 and Q3, first and third quartile. Diameters were calculated from the areas of the aggregates, assuming a circular shape. A lower diameter cutoff of 178.4 nm (area 0.025 µ2) was applied. (F) Representative STED image showing the distribution of ILP2-GFP and HA-VMAT in a VNC motor neuron cell body from third instar larva subjected to motor neuron-targeted knockdown of RUFY (OK6 > ILP2-GFP, HA-VMAT, RUFY-RNAiVAL20). Scale bars: 2 µm, 200 nm (inset). (G) Spatial relationship between ILP2-GFP and HA-tagged WT VMAT in motor axons. The multiple ROIs defined by yellow outlines mark the DCV-associated ILP2-GFP2 signal superimposed on the VMAT image. In the bottom row, the VMAT signal outside the DCV-associated ROIs has been digitally erased. Scale bar: 500 nm. (H) Quantification of E, showing the percentage of VMAT signal located outside the DCV-associated ROIs (top) and the VMAT signal intensity inside the ROIs (bottom) for eight control and eight Rab2 larvae. Bar graphs in B, D, and H represent mean + SEM. Student’s t test (B, D, and H).

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