Figure 4.
Figure 4. Patronin is required for normal microtubule polarity in ddaE and ddaC neurons. (A) Schematic representation of the Patronin gene region (based on RA isoform). Target sites for RNAi lines as well as transposon insertion sites are indicated. The two VDRC lines generate long hairpin RNAs, while the Bloomington (BL) line generates a small hairpin. (B and C) Quantification of dendrite microtubule polarity in ddaE neurons expressing EB1-GFP in genetic backgrounds that reduce Patronin. Numbers on the graph are the total numbers of comets collected from ≥10 neurons in each genotype. One neuron per animal was imaged. ns = not significant (P = 0.069); ****, P < 0.0001 with Fisher’s exact test. (D and E) Representative kymographs of EB1-GFP with control RNAi, Patronin RNAi, or null/hypomorph mutants. Turquoise lines mark plus ends. Horizontal bar, 5 μm. Vertical bar, 60 s. (F and G) Quantification of axonal microtubule (MT) polarity in ddaE or ddaC neurons. Numbers on the graph are the total comet numbers collected from 14, 12, 20, 20, 10, and 17 neurons, respectively (left to right). (H) Representative image of a ddaC neuron expressing EB1-GFP. Dashed red lines indicate the major dendrite branches used for quantification in J and K. (I) Representative kymographs of EB1-GFP in a branch with minus-end-out microtubule polarity (left), or in a branch with plus-end-out microtubule polarity (right). Turquoise lines mark plus end traces. Horizontal bar, 5 μm. Vertical bar, 60 s. (J) Quantification of overall microtubule polarity in ddaC dendrites. Numbers are total comet numbers collected from 18 and 7 neurons, respectively. ****, P < 0.0001 with Fisher’s exact test. (K) Quantification of ddaC major branches with plus-end-out, minus-end-out, or mixed microtubule polarity. Branches with >90% EB1-GFP comets moving toward the cell body are classified as minus-end-out branches. Dendrite branches with >90% EB1-GFP comets moving toward the distal tip are classified as plus-end-out branches. The rest are mixed polarity branches. Numbers on the graphs are the total major branches collected from 18 and 20 neurons. ****, P < 0.0001 with Fisher’s exact test.

Patronin is required for normal microtubule polarity in ddaE and ddaC neurons. (A) Schematic representation of the Patronin gene region (based on RA isoform). Target sites for RNAi lines as well as transposon insertion sites are indicated. The two VDRC lines generate long hairpin RNAs, while the Bloomington (BL) line generates a small hairpin. (B and C) Quantification of dendrite microtubule polarity in ddaE neurons expressing EB1-GFP in genetic backgrounds that reduce Patronin. Numbers on the graph are the total numbers of comets collected from ≥10 neurons in each genotype. One neuron per animal was imaged. ns = not significant (P = 0.069); ****, P < 0.0001 with Fisher’s exact test. (D and E) Representative kymographs of EB1-GFP with control RNAi, Patronin RNAi, or null/hypomorph mutants. Turquoise lines mark plus ends. Horizontal bar, 5 μm. Vertical bar, 60 s. (F and G) Quantification of axonal microtubule (MT) polarity in ddaE or ddaC neurons. Numbers on the graph are the total comet numbers collected from 14, 12, 20, 20, 10, and 17 neurons, respectively (left to right). (H) Representative image of a ddaC neuron expressing EB1-GFP. Dashed red lines indicate the major dendrite branches used for quantification in J and K. (I) Representative kymographs of EB1-GFP in a branch with minus-end-out microtubule polarity (left), or in a branch with plus-end-out microtubule polarity (right). Turquoise lines mark plus end traces. Horizontal bar, 5 μm. Vertical bar, 60 s. (J) Quantification of overall microtubule polarity in ddaC dendrites. Numbers are total comet numbers collected from 18 and 7 neurons, respectively. ****, P < 0.0001 with Fisher’s exact test. (K) Quantification of ddaC major branches with plus-end-out, minus-end-out, or mixed microtubule polarity. Branches with >90% EB1-GFP comets moving toward the cell body are classified as minus-end-out branches. Dendrite branches with >90% EB1-GFP comets moving toward the distal tip are classified as plus-end-out branches. The rest are mixed polarity branches. Numbers on the graphs are the total major branches collected from 18 and 20 neurons. ****, P < 0.0001 with Fisher’s exact test.

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