Figure 6.
Figure 6. Patronin is necessary and sufficient to add minus-end-out microtubules to terminal dendrites. (A) Representative image of γtubulin-GFP coexpressed with mCD8-RFP in class I neurons. Note that γtubulin is concentrated at branch points. (B) Schematic diagram showing the hypothesis that the γtubulin ring complex (γ-TuRC) and Patronin both contribute to dendritic microtubule polarity, and that Patronin is particularly important beyond branch points. (C) Representative overview image of a ddaE neuron expressing EB1-TagRFP-T and YFP-Patronin. Bar, 10 μm. Red rectangular region was enlarged to show two secondary dendrites. White dashed lines mark examples of terminal dendrite branches used for analyzing microtubule polarity here. Dendrite tips are indicated with orange arrowheads. Bar, 5 μm. (D) Representative kymographs showing microtubule polarity of terminal dendrites of control or Patronin RNAi neurons. Turquoise lines indicate plus ends. Horizontal bar, 5 μm. Vertical bar, 60 s. (E) Quantification of microtubule polarity in terminal dendrites of ddaE neurons is shown in the left graph. Numbers on the graph are total comet numbers collected from 18 and 23 neurons. *, P < 0.05 with Fisher’s exact test. Quantification of overall density of minus ends is shown in the right graph. Data were collected from 24 and 27 terminal dendrites from 18 and 23 neurons, respectively. **, P < 0.01 with Mann–Whitney U test. (F) Example kymographs of EB1-TagRFP-T showing microtubule polarity in terminal dendrites of ddaE neurons expressing CRISPR-tagged Patronin-Venus or UAS-YFP-Patronin. Turquoise lines indicate plus ends. Pink line indicates minus ends. Horizontal bar, 5 μm. Vertical bar, 60 s. (G) FI-based estimation of total Patronin protein levels when crossing with tagged Patronin fly lines. FIs were measured from 12 neurons of each genotype. Mean FI was used for normalization and plotted in the graph. See Materials and methods for information about how FI was normalized across fluorophores. (H) Quantification of microtubule polarity in terminal dendrites. Numbers on the graph are total comet numbers collected from 15, 15, and 14 neurons. ****, P < 0.0001 with Fisher’s exact test. (I) Quantification of events occurring when growing minus ends reach the dendrite tip. Numbers on the graph are the numbers of minus ends encountering the end. Data were collected from 16 neurons. Minus ends were classified as remaining at the tip if they persisted there for ≥50 s. These data were collected from cells expressing YFP-Patronin with EB1-TagRFP-T, so the number of minus ends reaching tips is higher than in control neurons because more minus-end-out microtubules are present in terminal dendrites when extra Patronin is present. (J) Representative kymographs of EB1-TagRFP-T with mild overexpression of YFP-Patronin. Only EB1-TagRFP-T traces are shown. Solid orange lines in kymograph indicate the tip of the dendrite. Minus ends are marked with pink lines and plus ends with turquoise lines. Depolymerized and persistent events are shown. Horizontal bar, 2 μm. Vertical bar, 60 s.

Patronin is necessary and sufficient to add minus-end-out microtubules to terminal dendrites. (A) Representative image of γtubulin-GFP coexpressed with mCD8-RFP in class I neurons. Note that γtubulin is concentrated at branch points. (B) Schematic diagram showing the hypothesis that the γtubulin ring complex (γ-TuRC) and Patronin both contribute to dendritic microtubule polarity, and that Patronin is particularly important beyond branch points. (C) Representative overview image of a ddaE neuron expressing EB1-TagRFP-T and YFP-Patronin. Bar, 10 μm. Red rectangular region was enlarged to show two secondary dendrites. White dashed lines mark examples of terminal dendrite branches used for analyzing microtubule polarity here. Dendrite tips are indicated with orange arrowheads. Bar, 5 μm. (D) Representative kymographs showing microtubule polarity of terminal dendrites of control or Patronin RNAi neurons. Turquoise lines indicate plus ends. Horizontal bar, 5 μm. Vertical bar, 60 s. (E) Quantification of microtubule polarity in terminal dendrites of ddaE neurons is shown in the left graph. Numbers on the graph are total comet numbers collected from 18 and 23 neurons. *, P < 0.05 with Fisher’s exact test. Quantification of overall density of minus ends is shown in the right graph. Data were collected from 24 and 27 terminal dendrites from 18 and 23 neurons, respectively. **, P < 0.01 with Mann–Whitney U test. (F) Example kymographs of EB1-TagRFP-T showing microtubule polarity in terminal dendrites of ddaE neurons expressing CRISPR-tagged Patronin-Venus or UAS-YFP-Patronin. Turquoise lines indicate plus ends. Pink line indicates minus ends. Horizontal bar, 5 μm. Vertical bar, 60 s. (G) FI-based estimation of total Patronin protein levels when crossing with tagged Patronin fly lines. FIs were measured from 12 neurons of each genotype. Mean FI was used for normalization and plotted in the graph. See Materials and methods for information about how FI was normalized across fluorophores. (H) Quantification of microtubule polarity in terminal dendrites. Numbers on the graph are total comet numbers collected from 15, 15, and 14 neurons. ****, P < 0.0001 with Fisher’s exact test. (I) Quantification of events occurring when growing minus ends reach the dendrite tip. Numbers on the graph are the numbers of minus ends encountering the end. Data were collected from 16 neurons. Minus ends were classified as remaining at the tip if they persisted there for ≥50 s. These data were collected from cells expressing YFP-Patronin with EB1-TagRFP-T, so the number of minus ends reaching tips is higher than in control neurons because more minus-end-out microtubules are present in terminal dendrites when extra Patronin is present. (J) Representative kymographs of EB1-TagRFP-T with mild overexpression of YFP-Patronin. Only EB1-TagRFP-T traces are shown. Solid orange lines in kymograph indicate the tip of the dendrite. Minus ends are marked with pink lines and plus ends with turquoise lines. Depolymerized and persistent events are shown. Horizontal bar, 2 μm. Vertical bar, 60 s.

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