Figures and data
Shot is required for c4da neuron developmental dendrite pruning.
A – F’ C4da neurons of the indicated genotypes were imaged at the third instar larval stage (A - F) and at 16 hours after puparium formation (h APF) (A’ - F’). A, A’ Control c4da neurons expressing Orco dsRNA under the control of ppk-GAL4. B, B’ C4da neurons expressing Shot dsRNA under the control of ppk-GAL4. C, C’ Control c4da neurons labeled by MARCM. D, D’ shot3 mutant c4da neurons labeled by MARCM. E, E’ Control c4da neurons expressing Cas9P2 under ppk-GAL4 in a shot3 heterozygous background. F, F’ C4da neurons coexpressing Cas9P2 and Shot sgRNA under the control of ppk-GAL4 in a shot3 heterozygous background. G Phenotypic penetrance of pruning defects in A’ - F’. Sample sizes are indicated above the graph. *** P<0.001, **** P<0.0001, two-tailed Fisher’s exact test. H Average number of primary and secondary dendrites attached to the soma at 16 h APF in samples A’ - F’. Values are mean +/- s. e. m., **** P<0.0001, Mann-Whitney U test. I Total length of remaining dendrites at 16 h APF in samples A’ - F’. Values are mean +/- s. e. m., **** P<0.0001, Mann-Whitney U test. Scale bars in A and A’ are 50 µm.
Shot is required for uniform plus end-in orientation of dendritic microtubules.
A, B EB1::GFP comets were imaged in primary dendrites of third instar larval c4da neurons, and comet movement was depicted in kymographs. Direction of the soma and time are indicated. A Upper panel: EB1::GFP comets in control neuron expressing Orco dsRNA; lower panel: EB1::GFP comets in neuron expressing Shot dsRNA. B Upper panel: EB1::GFP comets in control c4da neuron MARCM clone; lower panel: EB1::GFP comets in shot3mutant c4da neuron MARCM clone. C Percentage of anterogradely moving comets in panels A, B. N is indicated above the graph. ** P<0.01, **** P<0.0001, two-tailed Fisher’s exact test. D EB1::GFP comet speed in panels A, B. **** P<0.0001, Wilcoxon’s test. The scale bar in A is 5 µm.
Shot domains important for pruning and microtubule organization.
A UAS-Shot constructs. CH, Calponin homology. B - E Ability of the indicated Shot UAS constructs to rescue the pruning defects of shot3 mutant c4da neurons at 16 h APF. B shot3 mutant c4da neuron labeled by MARCM. C shot3 mutant c4da neuron expressing full-length Shot::GFP. D shot3mutant c4da neuron expressing Shot::GFP lacking the CH1 domain. E shot3 mutant c4da neuron expressing Shot C-term::YFP. F Penetrance of pruning defects. N is indicated above the graph. * P<0.05, *** P<0.001, **** P<0.0001, two-tailed Fisher’s exact test. G Number of primary and secondary dendrites attached to the cell body at the indicated timepoints. Values are mean +/- s. e. m., *** p<0.001, **** p<0.0001, Mann-Whitney U test. H – L Kin::lacZ localization in third instar c4da neuron MARCM clones. H Control c4da neuron. I shot3 mutant c4da neuron. J shot3mutant c4da neuron expressing full-length Shot. K shot3mutant c4da neuron expressing Shot lacking the CH1 domain. L shot3mutant c4da neuron expressing Shot C-term::YFP. Arrows in H – L mark dendritic kin::lacZ puncta, asterisks denote soma position. M Number of dendritic kin::lacZ puncta in third instar c4da neurons expressing the indicated Shot UAS constructs. N is depicted above the graph. Values are mean +/- s. e. m., * p<0.05, ** p<0.01, *** p<0.001, Wilcoxon test. Scale bars in B and H are 50 μm.
Shot links microtubule regulation to actin.
A, A’ Localization of Shot in a third instar primary c4da neuron dendrite. Endogenously tagged Shot::GFP was labeled by immunofluorescence and visualized by structured illumination microscopy. C4da neurons were labeled by expression of cytosolic tdtomato under ppk-GAL4. A Shot::GFP. A’ merge with tdtomato. B Effect of actin severing on EB1::GFP comet movement. Mical was overexpressed in c4da neurons with or without Shot knockdown, and EB1 comets were analyzed as in Fig. 2 B. Upper panel, kymograph of EB1 comet movement in c4da neuron overexpressing Mical. Lower panel, kymograph of EB1 comet movement upon Mical overexpression and shot knockdown. C Penetrance of anterograde comets in Fig. 4 B. N is given in the graph. * P<0.05, Fisher’s exact test. D Graph depicting the effects of Shot knockdown and Mical expression on EB1 comet speed. N is given in the graph. **** P<0.0001, Wilcoxon’s test. E Synergistic effects of actin severing and Shot knockdown on c4da neuron dendrite pruning. Penetrance of pruning defects at 16 h APF in c4da neurons overexpressing Mical, upon Shot knockdown, or both. N is given in the graph. * P<0.05, Fisher’s exact test. Scale bars in A and B are 5 μm.
Shot is recruited to tips of growing dendrites via its CH1 domain.
The indicated GFP-tagged Shot constructs and tdtomato were expressed in c4da neurons under ppk-GAL4 and visualized at the indicated developmental stages. A, B Dendritic shaft localization of transgenic Shot::GFP variants in third instar c4da neuron dendrites was visualized by immunofluorescence and structured illumination microscopy (SIM). A Localization of full-length Shot (ShotFL::GFP). B Localization of Shot lacking the CH1 domain (ShotΔCH1::GFP). C - H Localization of Shot variants in first instar c4da neurons. Images on the right show close-ups of dendrite tip (1) and soma regions (2). The asterisk in the dendrite close-up marks the position of the tip. C Localization of ShotFL::GFP. D ShotΔCH1::GFP. E Fluorescence intensity profiles of Shot::GFP and ShotΔCH1::GFP in the distal 10 μm of first instar dendrites. Intensity values were normalized to Shot::GFP intensity in the cell body. Solid lines indicate average, envelopes indicate S. D. (N=10). The table shows significance between genotypes for the indicated distances from the tip **** P<0.0001, *** P<0.001, n. s. not significant, Mann Whitney U test. F Dendrite tip occupancy of ShotFL::GFP and ShotΔCH1::GFP. ** P<0.01, Fisher’s exact test. G Effect of Mical overexpression on ShotFL::GFP localization. Images on the right show close-ups of dendrite tip (1) and soma regions (2). H Fluorescence intensity profiles of ShotFL::GFP in the distal 10 μm of first instar dendrites with or without Mical overexpression. Quantification was as in E. N=10, **** P<0.0001, *** P<0.001, ** P<0.01, n. s. not significant, Mann Whitney U test. Scale bars are 5 μm in A and 10 μm in C (larger image and close-up).
Evidence that Rab11 is part of a developmentally regulated dendritic microtubule organizing center.
A Rab11 colocalizes with the MTOC marker γ-tubulin in growing c4da neuron dendrites. Rab11::mCherry and γ-tubulin23C::GFP were coexpressed under ppk-GAL4 and visualized at the first instar. Arrows show Rab11/ γ-tubulin-positive puncta at dendrite tips, the asterisk denotes a double-positive dot at a branchpoint. B Microtubules can be nucleated at Rab11 puncta in dendrites. Rab11::mCherry and EB1::GFP were coexpressed in c4da neurons, and EB1 comets were visualized at the first instar. Example kymographs show Rab11::mCherry (magenta) and EB1::GFP (green). C Percentage of EB1 comets arising from dendritic Rab11 puncta at the first and third instars. * P<0.05, Fisher’s exact test. D Effect of Rab11 knockdown on dendritic microtubule orientation. Kymographs show EB1::GFP movement in third instar c4da neuron dendrites. Upper panel, control c4da neuron expressing Orco dsRNA; middle panel, c4da neuron expressing Rab11 dsRNA; lower panel, c4da neuron expressing Rab5 dsRNA. E Penetrance of anterograde comets in D. ** P<0.01, two-tailed Fisher’s exact’s test. F Speed of EB1::GFP comets in D. **** P<0.0001, Wilcoxon’s test. G GFP-tagged Rab11 (wt or S25N) and FLAG-tagged Msps were cotransfected into S2 cells and immunoprecipitated with FLAG beads. Inputs and immunoprecipitates (IP) were blotted with the indicated antibodies. Sizes of molecular weight markers in kiloDalton (kD) are shown. IgG denotes antibody heavy chains. H Patronin localizes along dendrites and in dendritic tips in first instar c4da neurons. The side panel shows an enlarged image of a dendrite (boxed area in larger image). The asterisk denotes the position of the soma. Scale bars are 5 μm in A and D, 2 μm in B and 10 μm in G.
Evidence that Shot acts as part of a dendritic MTOC.
A Frequency of anterograde EB1 comets at the L1 stage in control (Orco dsRNA) and shot knockdown neurons (shot dsRNA, shot3/+). * P<0.05, Mann Whitney U test. B Shot recruits EB1 to dendrite tips. EB1::mScarlet3 was expressed in c4da neurons without (upper panel) or with ShotFL::GFP (lower panel) and visualized at the first instar larval stage. The position of the dendrite tip is indicated by an asterisk. C Fluorescence intensity profiles of EB1::mScarlet3 in the distal 10 μm of first instar dendrites. Solid lines indicate average, envelopes indicate S. D. (N=9 each). C’ Table showing significance between genotypes for the indicated distances from the tip. **** P<0.0001, ** P<0.01, Mann Whitney U test. D Example kymograph of EB1::mScarlet3 and Shot::GFP at a dendritic tip. Tip position is to the left, and arrows in the EB1::mScarlet3 and merge panels indicate a comet originating there. E - L Synergistic genetic interactions between shot and other dendritic microtubule orientation/MTOC factors during c4da neuron dendrite pruning. E - I shot dsRNA was expressed in c4da neurons of animals in the indicated backgrounds, and pruning defects were quantified at 16 h APF. E C4da neuron expressing shot dsRNA. F C4da neuron expressing shot dsRNA in a msps/+ background. G C4da neuron expressing shot dsRNA in a patronin/+ background. H C4da neuron co-expressing shot and EB1 dsRNAs. I Severity of pruning defects in E - G. N = 33 - 52, ** P<0.01, *** P<0.001, Wilcoxon’s test. J, K rab11 dsRNA was expressed in c4da neurons of control animals (J) or in a shot3/+ heterozygous background (K), and pruning defects were quantified at 16 h APF. L Severity of pruning defects in J, K. N = 41 and 42. **** P<0.0001, Wilcoxon’s test. Scale bars are 5 μm in B, 2 μm in D and 50 μm in E.
(related to Fig. 1). Loss of Shot, patronin and EB1 causes c4da neuron dendrite regrowth defects.
A - D The indicated microtubule regulators were knocked down in c4da neurons under ppk-GAL4, and neurons were imaged at 72 h APF. A Control c4da neuron expressing Orco dsRNA (N = 22). B C4da neuron expressing patronin dsRNA (N = 12). C C4da neuron expressing Shot dsRNA (N = 10). D C4da neuron expressing EB1 dsRNA (N = 10). E Total dendrite length in A - D. Values are mean +/- s. d., *** P<0.001, **** P<0.0001, Wilcoxon’s test. The scale bar in A is 50 μm.
(related to Fig. 2). Loss of Shot delays dendritic microtubule disassembly during dendrite pruning.
A - B Microtubules in early pupal control or Shot knockdown c4da neurons (green) were visualized by futsch/22C10 staining (magenta) at 6 h APF. A Control c4da neuron. A’ futsch/22C10 signal in boxed area in A. The arrow indicates a c4da neuron dendrite. B C4da neuron expressing Shot dsRNA. B’ futsch/22C10 signal in boxed area in B. The arrow indicates a c4da neuron dendrite. C Graph depicting the average number of neurons with continuous futsch/22C10 staining. Data are mean +/- s. d., N = 10 each, ** P<0.01, student’s t- test. The scale bar in A is 20 μm.
(related to Fig. 3). Effect of Shot overexpression on dendrite pruning.
A - E Full length or truncated Shot variants were overexpressed in c4da neurons, and the effects on dendrite pruning were assessed at 16 h APF. A Control c4da neuron (N = 30). B C4da neuron overexpressing full length Shot::GFP (N = 42). C C4da neuron overexpressing ShotΔCH1::GFP domain (N = 54). D C4da neuron overexpressing Shot Cterm::YFP, containing the microtubule binding domain of Shot (N = 54). E Penetrance of pruning defects in A - D. * P<0.05, **** P<0.0001, Fisher’s exact test. F Severity of pruning defects in A - D. Values are mean +/- s. e. m., * P<0.05, **** P<0.0001, Wilcoxon’s test. The scale bar in A is 50 μm.
(related to Fig. 3). Effect of Shot knockdown on dendritic microtubule structure.
Microtubules were visualized by immunofluorescence of mCherry::α-tubulin (under ppk-GAL4) followed by 2D STED. Images show primary dendrites. A Microtubules in control c4da neuron expressing a control dsRNA against Orco. B Microtubules upon Shot knockdown. The scale bar in A is 2 μm.
(related to Fig. 5). Shot is not enriched at dendritic tips in mature neurons.
ShotFL::GFP was expressed in c4da neurons under ppk-GAL4 and a region comprising both dendrite shafts and tips of a third instar c4da neuron was visualized by immunofluorescence and structured illumination microscopy (SIM). The scale bar is 10 μm.
