Figure S6.
Axonal transport and neuronal distribution of DCVs in LRRK null larvae, decreased peripheral nerve DCV content under RUFY RNAi knockdown, and decreased DCV content in syd mutant NMJs. (A) Representative kymographs showing transport of ILP2-GFP–positive DCVs in motor axons of control and LRRK/Df larvae. Scale bar: 10 µm. (B) Left, directional distributions derived from A. Right, the logarithmic ratio of retrograde to anterograde peak amplitude for the directional distributions at the left. (C) Left: Representative confocal images showing the pre-bleach ILP2-GFP intensity in A7 nerves of control and LRRK/Df larvae. Scale bar: 10 µm. Right: Quantification of the ILP2-GFP signal intensity. Number of larvae analyzed in B and C: control 4, LRRK/Df 10. (D) Confocal images (sum projection of z-stacks) showing the ILP2-GFP signal in VNCs and peripheral nerves of third instar control and LRRK/Df larvae. Images are shown at both low contrast settings at which the VNC cell body signal is not saturated (left) and high contrast settings at which the axonal signal in peripheral nerves is visible (right). Scale bar: 100 µm. (E) Quantification of ILP2-GFP fluorescence in D from VNC cell bodies (left) and A7 peripheral nerves (middle), as well as the ratio between nerve and cell body fluorescence (right). Number of larvae analyzed: control 11, LRRK/Df 11. (F) Left: Pre-bleach ILP2-GFP intensity in A7 nerves of control larvae and larvae subjected to motor neuron-targeted knockdown of RUFY (KK or VALIUM20 UAS-RNAi lines, cf. Fig. 5). Scale bar: 10 µm. Right: Quantification of the ILP2-GFP signal intensity. Number of larvae analyzed: control for RUFY-KD (KK) 9, RUFY-KD (KK) 7; control for RUFY-KD (VAL20) 10, RUFY-KD (VAL20) 12. (G) Confocal images showing the intensity of the presynaptic ILP2-GFP signal in the neuromuscular junction of muscle fiber 6/7 in control and sydz4/Df larvae. The sydz4/Df micrograph is shown with brightness and contrast settings matching those of the control (middle), as well as with the contrast digitally enhanced for better visibility (right). Scale bar: 10 µm. (H) Quantification of the presynaptic ILP2-GFP signal intensity in G. Number of larvae analyzed: control 8, sydz4/Df 8. Data in all panels are from third instar larvae. Bar graphs in B, C, E, F, G, and H represent mean + SEM and were analyzed using Student’s t test.

Axonal transport and neuronal distribution of DCVs in LRRK null larvae, decreased peripheral nerve DCV content under RUFY RNAi knockdown, and decreased DCV content in syd mutant NMJs. (A) Representative kymographs showing transport of ILP2-GFP–positive DCVs in motor axons of control and LRRK/Df larvae. Scale bar: 10 µm. (B) Left, directional distributions derived from A. Right, the logarithmic ratio of retrograde to anterograde peak amplitude for the directional distributions at the left. (C) Left: Representative confocal images showing the pre-bleach ILP2-GFP intensity in A7 nerves of control and LRRK/Df larvae. Scale bar: 10 µm. Right: Quantification of the ILP2-GFP signal intensity. Number of larvae analyzed in B and C: control 4, LRRK/Df 10. (D) Confocal images (sum projection of z-stacks) showing the ILP2-GFP signal in VNCs and peripheral nerves of third instar control and LRRK/Df larvae. Images are shown at both low contrast settings at which the VNC cell body signal is not saturated (left) and high contrast settings at which the axonal signal in peripheral nerves is visible (right). Scale bar: 100 µm. (E) Quantification of ILP2-GFP fluorescence in D from VNC cell bodies (left) and A7 peripheral nerves (middle), as well as the ratio between nerve and cell body fluorescence (right). Number of larvae analyzed: control 11, LRRK/Df 11. (F) Left: Pre-bleach ILP2-GFP intensity in A7 nerves of control larvae and larvae subjected to motor neuron-targeted knockdown of RUFY (KK or VALIUM20 UAS-RNAi lines, cf. Fig. 5). Scale bar: 10 µm. Right: Quantification of the ILP2-GFP signal intensity. Number of larvae analyzed: control for RUFY-KD (KK) 9, RUFY-KD (KK) 7; control for RUFY-KD (VAL20) 10, RUFY-KD (VAL20) 12. (G) Confocal images showing the intensity of the presynaptic ILP2-GFP signal in the neuromuscular junction of muscle fiber 6/7 in control and sydz4/Df larvae. The sydz4/Df micrograph is shown with brightness and contrast settings matching those of the control (middle), as well as with the contrast digitally enhanced for better visibility (right). Scale bar: 10 µm. (H) Quantification of the presynaptic ILP2-GFP signal intensity in G. Number of larvae analyzed: control 8, sydz4/Df 8. Data in all panels are from third instar larvae. Bar graphs in B, C, E, F, G, and H represent mean + SEM and were analyzed using Student’s t test.

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