Figure 4.
Endogenous localization of RIC-7. (A) Labeling scheme of endogenous RIC-7. (B–D) Representative images showing RIC-7::7xSplitGFP puncta associated with mitochondria both in the processes and in the cell body. Note that RIC-7 is also labeled with 7xSplitGFP in VA12, the axon of which is visible next to the dendrite of DA9. Arrowheads indicate RIC-7::7xSplitGFP puncta in DA9. Scale bars = 5 μm. (E) Percentage of mitochondria associated with RIC-7::7xSplitGFP puncta in the axon and the dendrite was quantified from still images. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from three independent experiments. Number of animals = 14. ****P < 0.0001 (Fisher’s exact test). (F) Percentage of mitochondria associated with RIC-7::7xSplitGFP based on its polarization status from the same dataset as shown in E. If one or more RIC-7 puncta are localized to the distal or proximal half of the mitochondria, it is regarded as “Distal” or “Proximal.” If multiple RIC-7 puncta are spread out on the mitochondria, or if RIC-7 is diffuse and relatively homogenous on the mitochondria, it is regarded as “Non-polarized.” Error bars represent 95% confidence intervals. (G) Percentage of moving mitochondria associated with RIC-7::7xSplitGFP puncta in the axon. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from 13 independent experiments. Number of animals = 47. ****P < 0.0001 (Fisher’s exact test). (H) Percentage of moving mitochondria associated with RIC-7::7xSplitGFP puncta in the dendrite. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from 13 independent experiments. Number of animals = 38. P value = 0.12 (Fisher’s exact test). (I) Representative images of anterogradely trafficking mitochondria in the axon at two different timepoints, with RIC-7::7xSplitGFP accumulating at the leading end, which is indicated by the arrowhead. Scale bar = 2 μm. See Video 4. (J) Normalized intensity of RIC-7::7xSplitGFP averaged across different timepoints along the length of mitochondrion shown in Video 4. Leading end is on the left and trailing end on the right. Error bars represent SEM. (K) Representative images of mitochondria entering the axon, with RIC-7::7xSplitGFP accumulating at the leading end, which is indicated by the arrowhead. Scale bar = 5 μm. See Video 5. (L) Percentage of mitochondria exiting the cell body with RIC-7::7xSplitGFP accumulation at the tip. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from five independent experiments. Number of animals = 15. ***P < 0.001 (Fisher’s exact test).

Endogenous localization of RIC-7. (A) Labeling scheme of endogenous RIC-7. (B–D) Representative images showing RIC-7::7xSplitGFP puncta associated with mitochondria both in the processes and in the cell body. Note that RIC-7 is also labeled with 7xSplitGFP in VA12, the axon of which is visible next to the dendrite of DA9. Arrowheads indicate RIC-7::7xSplitGFP puncta in DA9. Scale bars = 5 μm. (E) Percentage of mitochondria associated with RIC-7::7xSplitGFP puncta in the axon and the dendrite was quantified from still images. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from three independent experiments. Number of animals = 14. ****P < 0.0001 (Fisher’s exact test). (F) Percentage of mitochondria associated with RIC-7::7xSplitGFP based on its polarization status from the same dataset as shown in E. If one or more RIC-7 puncta are localized to the distal or proximal half of the mitochondria, it is regarded as “Distal” or “Proximal.” If multiple RIC-7 puncta are spread out on the mitochondria, or if RIC-7 is diffuse and relatively homogenous on the mitochondria, it is regarded as “Non-polarized.” Error bars represent 95% confidence intervals. (G) Percentage of moving mitochondria associated with RIC-7::7xSplitGFP puncta in the axon. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from 13 independent experiments. Number of animals = 47. ****P < 0.0001 (Fisher’s exact test). (H) Percentage of moving mitochondria associated with RIC-7::7xSplitGFP puncta in the dendrite. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from 13 independent experiments. Number of animals = 38. P value = 0.12 (Fisher’s exact test). (I) Representative images of anterogradely trafficking mitochondria in the axon at two different timepoints, with RIC-7::7xSplitGFP accumulating at the leading end, which is indicated by the arrowhead. Scale bar = 2 μm. See Video 4. (J) Normalized intensity of RIC-7::7xSplitGFP averaged across different timepoints along the length of mitochondrion shown in Video 4. Leading end is on the left and trailing end on the right. Error bars represent SEM. (K) Representative images of mitochondria entering the axon, with RIC-7::7xSplitGFP accumulating at the leading end, which is indicated by the arrowhead. Scale bar = 5 μm. See Video 5. (L) Percentage of mitochondria exiting the cell body with RIC-7::7xSplitGFP accumulation at the tip. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Data are pooled from five independent experiments. Number of animals = 15. ***P < 0.001 (Fisher’s exact test).

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