Figure 2.
Figure 2. EB1 and Patronin independently label the same growing microtubule minus ends. (A) Representative image of a ddaE neuron expressing high levels of YFP-Patronin. EB1-TagRFP-T is coexpressed to show cell shape. Bar in inset, 5 µm. (B–D) Representative images and kymographs of a neuron with low expression levels of YFP-Patronin with EB1-TagRFP-T are shown. The dashed line in B indicates the region where C and D were generated. (C) Pink arrowhead points to a minus end colabeled with EB1-TagRFP-T and YFP-Patronin. Numbers on each image indicate the time in seconds. Bar, 5 μm. (D) Kymographs of EB1-TagRFP-T and YFP-Patronin. Minus ends are marked with pink lines and plus ends with turquoise lines. The pink arrowhead indicates the minus end shown in C. (E) Schematic diagram of predicted YFP-Patronin relocalization after dendrite severing. New microtubule minus ends generated by dendrite cut will recruit Patronin to only one side of the severed dendrite indicated by green arrowhead. (F) Representative images of YFP-Patronin accumulating at the distal end of a proximal dendrite segment after severing. mCD8-RFP was used as the cell shape marker. Star indicates the cutting site of comb dendrite. Green arrowhead indicates the relocalized YFP-Patronin. Bar, 5 μm. (G) Representative kymograph of EB1-TagRFP-T colocalized with EGFP-Patronin on a growing microtubule minus end. EGFP-Patronin was expressed from its own promotor. Horizontal bar, 5 μm. Vertical bar, 60 s. (H) Representative images of YFP-Patronin, EB1-TagRFP-T in the cell body of ddaE. All images were acquired with the same settings and processed in the same way. Bar, 5 μm. (I and J) Quantification of RNAi knockdown efficiency. FI of YFP-Patronin was measured in cell body of control, Patronin RNAi VDRC27654 (Patr. RNAi 1), or Patronin RNAi BL36659 (Patr. RNAi 2) neurons. FI of EB1-TagRFP-T was measured in cell body of control or EB1 RNAi VDRC24451 neurons. Raw FI was divided by the average FI of control neurons for normalization. Numbers on the graphs are the numbers of neurons imaged. ****, P < 0.0001 with Mann-Whitney U test. (K) Example kymographs of minus-ends labeling by YFP-Patronin or EB1-TagRFP-T together with EB1 or Patronin RNAi, respectively. Pink lines mark the minus end traces, and turquoise lines mark plus end traces. The black arrow indicates an immobile dot of YFP-Patronin. Horizontal bar, 5 μm. Vertical bar, 60 s. (L) Schematic diagram showing EB1 and Patronin colabel growing minus ends of microtubules and track them independently.

EB1 and Patronin independently label the same growing microtubule minus ends. (A) Representative image of a ddaE neuron expressing high levels of YFP-Patronin. EB1-TagRFP-T is coexpressed to show cell shape. Bar in inset, 5 µm. (B–D) Representative images and kymographs of a neuron with low expression levels of YFP-Patronin with EB1-TagRFP-T are shown. The dashed line in B indicates the region where C and D were generated. (C) Pink arrowhead points to a minus end colabeled with EB1-TagRFP-T and YFP-Patronin. Numbers on each image indicate the time in seconds. Bar, 5 μm. (D) Kymographs of EB1-TagRFP-T and YFP-Patronin. Minus ends are marked with pink lines and plus ends with turquoise lines. The pink arrowhead indicates the minus end shown in C. (E) Schematic diagram of predicted YFP-Patronin relocalization after dendrite severing. New microtubule minus ends generated by dendrite cut will recruit Patronin to only one side of the severed dendrite indicated by green arrowhead. (F) Representative images of YFP-Patronin accumulating at the distal end of a proximal dendrite segment after severing. mCD8-RFP was used as the cell shape marker. Star indicates the cutting site of comb dendrite. Green arrowhead indicates the relocalized YFP-Patronin. Bar, 5 μm. (G) Representative kymograph of EB1-TagRFP-T colocalized with EGFP-Patronin on a growing microtubule minus end. EGFP-Patronin was expressed from its own promotor. Horizontal bar, 5 μm. Vertical bar, 60 s. (H) Representative images of YFP-Patronin, EB1-TagRFP-T in the cell body of ddaE. All images were acquired with the same settings and processed in the same way. Bar, 5 μm. (I and J) Quantification of RNAi knockdown efficiency. FI of YFP-Patronin was measured in cell body of control, Patronin RNAi VDRC27654 (Patr. RNAi 1), or Patronin RNAi BL36659 (Patr. RNAi 2) neurons. FI of EB1-TagRFP-T was measured in cell body of control or EB1 RNAi VDRC24451 neurons. Raw FI was divided by the average FI of control neurons for normalization. Numbers on the graphs are the numbers of neurons imaged. ****, P < 0.0001 with Mann-Whitney U test. (K) Example kymographs of minus-ends labeling by YFP-Patronin or EB1-TagRFP-T together with EB1 or Patronin RNAi, respectively. Pink lines mark the minus end traces, and turquoise lines mark plus end traces. The black arrow indicates an immobile dot of YFP-Patronin. Horizontal bar, 5 μm. Vertical bar, 60 s. (L) Schematic diagram showing EB1 and Patronin colabel growing minus ends of microtubules and track them independently.

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