Shot is required for c4da neuron developmental dendrite pruning.

A Schematic showing the progression of c4da neuron dendrite pruning via hormonal induction (0 h APF) to severing (12 h APF) and clearance (16 h APF). B – G’ C4da neurons of the indicated genotypes were imaged at the third instar larval stage (B - G) and at 16 hours after puparium formation (h APF) (B’ - G’). B, B’ Control c4da neurons expressing Orco dsRNA under the control of ppk-GAL4. C, C’ C4da neurons expressing Shot dsRNA under the control of ppk-GAL4. D, D’ Control c4da neurons labeled by MARCM. E, E’ shot3mutant c4da neurons labeled by MARCM. F, F’ Control c4da neurons expressing Cas9P2 under ppk-GAL4 in a shot3 heterozygous background. G, G’ C4da neurons coexpressing Cas9P2 and Shot sgRNA under the control of ppk-GAL4 in a shot3 heterozygous background. H Phenotypic penetrance of pruning defects in B’ - G’. Number of neurons for each sample are indicated above the graph. *** P<0.001, **** P<0.0001, two-tailed Fisher’s exact test. I Average number of primary and secondary dendrites attached to the soma at 16 h APF in samples B’ - G’. Values are mean +/- s. e. m., **** P<0.0001, Mann-Whitney U test. J Total length of remaining dendrites at 16 h APF in samples B’ - G’. Values are mean +/- s. e. m., **** P<0.0001, Mann-Whitney U test. Scale bars in B and B’ are 50 µm.

Shot is required for uniform plus end-in orientation of dendritic microtubules.

A - C Delayed microtubule disassembly in neurons lacking Shot. Microtubules in control or Shot knockdown c4da neurons (green) were visualized by futsch/22C10 staining (magenta) at 6 h APF. Smaller panels below show futsch/22C10 signal in boxed areas in A, B, arrows indicate c4da neuron dendrites. A Control c4da neuron. B C4da neuron expressing Shot dsRNA. C Graph depicting the average number of dendrites with continuous futsch/22C10 staining per neuron in A, B. Box represents s. d., N = 10 neurons each, ** P<0.01, student’s t-test. D, E EB1::GFP comets were imaged in primary dendrites of third instar larval c4da neurons, and comet movement was depicted in kymographs. Time and direction of the soma are indicated by arrows. D Upper panel, EB1::GFP comets in control neuron expressing Orco dsRNA; lower panel, EB1::GFP comets in neuron expressing Shot dsRNA. E Upper panel, EB1::GFP comets in control c4da neuron MARCM clone; lower panel, EB1::GFP comets in shot3mutant c4da neuron MARCM clone. F Percentage of anterogradely moving comets in panels D, E. Numbers of comets per sample are indicated above the graph. ** P<0.01, **** P<0.0001, two-tailed Fisher’s exact test. G EB1::GFP comet speed in panels D, E. **** P<0.0001, Wilcoxon’s test. Scale bars in A and D are 20 and 5 µm, respectively.

Shot domains important for pruning and microtubule organization.

A Schematic of Shot domain structure and used UAS-Shot constructs. Right, summary of the phenotypic characterization below. CH, Calponin homology. B - J Ability of the indicated Shot UAS constructs to rescue the pruning defects of shot3 mutant c4da neuron MARCM clones at 16 h APF. B shot3 mutant c4da neuron. C shot3mutant c4da neuron rescued by a genomic Shot::YFP BAC construct. D shot3 mutant c4da neuron expressing full-length Shot::GFP. E shot3mutant c4da neuron expressing ShotΔCH1::GFP. F shot3mutant c4da neuron expressing ShotΔABD::GFP. G shot3 mutant c4da neuron expressing ShotΔCTail::GFP. H shot3 mutant c4da neuron expressing Shot C-term::YFP. I Penetrance of pruning defects. Number of neurons per sample is indicated above the graph. * P<0.05, **** P<0.0001, two-tailed Fisher’s exact test. J Number of primary and secondary dendrites attached to the cell body at the indicated timepoints. Values are mean +/- s. e. m., **** p<0.0001, Mann-Whitney U test. K – R Kin::lacZ localization in third instar shot3 c4da neuron MARCM clones. K shot3 mutant c4da neuron. L shot3 mutant c4da neuron rescued by a genomic Shot::YFP BAC construct. M shot3 mutant c4da neuron expressing full-length Shot::GFP. N shot3mutant c4da neuron expressing ShotΔCH1::GFP. O shot3mutant c4da neuron expressing ShotΔABD::GFP. P shot3 mutant c4da neuron expressing ShotΔCtail::GFP. Q shot3 mutant c4da neuron expressing Shot C-term::YFP. Asterisks in K – Q denote soma position, and arrows mark dendritic kin::lacZ puncta. R Number of dendritic kin::lacZ puncta in third instar c4da neurons expressing the indicated Shot UAS constructs. Number of neurons per sample is indicated above the graph. Values are mean +/- s. e. m., ** p<0.01, Mann-Whitney U test. Scale bars in B and K are 50 μm.

Shot and actin cooperatively regulate the behavior of dendritic microtubules.

A 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. B Penetrance of anterograde dendritic EB1 comets in control c4da neuron expressing orco dsRNA, or in neurons expressing shot dsRNA, Mical, or both (kymographs for the latter shown in A). Number of comets per sample is given in the graph. * P<0.05, Fisher’s exact test. C Speed of dendritic EB1 comets in A, B. **** P<0.0001, Wilcoxon’s test. D - F Synergistic effects of Shot knockdown and Mical overexpression on c4da neuron dendrite pruning. D C4da neuron expressing shot dsRNA and a control UAS construct at 16 h APF. E C4da neuron expressing shot dsRNA and UAS-Mical at 16 h APF. F Number of primary and secondary dendrites attached to the soma in c4da neurons overexpressing Mical, upon Shot knockdown, or both at 16 h APF. Number of neurons per sample is given in the graph. *** P<0.001, Mann-Whitney U test. Scale bars are 5 μm in A and 50 μm in B.

Shot is recruited to tips of growing dendrites via its actin binding 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 - D Actin binding is not required for Shot localization in shafts of mature dendrites. The indicated Shot::GFP constructs were visualized in third instar primary c4da neuron dendrites by immunofluorescence followed by structured illumination microscopy. Borders of dendrites are indicated by white broken lines. A Endogenously tagged Shot::GFP. B Localization of transgenic full-length Shot (ShotFL::GFP) expressed under ppk-GAL4. C Localization of ShotΔCH1::GFP expressed under ppk-GAL4. D Localization of ShotΔABD::GFP expressed under ppk-GAL4. E - K Localization of the indicated Shot::GFP variants in first instar c4da neurons. Images on the right show close-ups of dendrite tip (1) and soma regions (2). Asterisks in the dendrite close-ups mark the position of the tip. E Localization of ShotFL::GFP. F Localization of ShotΔCH1::GFP. G Localization of ShotΔABD::GFP. H Fluorescence intensity profiles of Shot::GFP variants in the distal 10 μm of first instar dendrites in E - G. Intensity values are normalized to Shot::GFP intensity in the cell body. Solid lines indicate average, envelopes indicate s. d. (N=10 neurons). Compared to full length Shot, GFP intensities of ShotΔCH1 and ShotΔABD in the 0-2 μm and 2-4 μm brackets from the tip were significantly reduced (P<0.0001, Mann Whitney U test). I Dendrite tip occupancy of Shot::GFP variants in E - G. ** P<0.01, Fisher’s exact test. J, K Effect of Mical overexpression on ShotFL::GFP localization. J Localization of ShotFL::GFP in a c4da neuron overexpressing Mical. K Fluorescence intensity profiles of ShotFL::GFP in the distal 10 μm of first instar dendrites with or without Mical overexpression. Shot::GFP intensity upon Mical overexpression was not significantly different from Shot::GFP without Mical in the 0-2 μm bracket, but highly significant in the 2-4 μm bracket (N=10 neurons, P<0.0001, Mann Whitney U test). Scale bars are 5 μm in A and 10 μm in E (larger image and close-up).

Shot acts during early development and interacts with end binding proteins.

A Frequency of anterograde EB1 comets at the L1 stage in control (Orco dsRNA) and shot knockdown neurons (shot dsRNA, shot3/+). Number of comets per sample is shown in the graph, * P<0.05, Mann Whitney U test. B Microtubules in dendrites of first instar control neurons or neurons expressing Shot::GFP were visualized using mCherry-tagged α-tubulin in single confocal slices. C Dendritic microtubule density for samples in B. Dendritic mCherry-α-tubulin 84B fluorescence intensity was normalized to that of the soma. N= 14 - 22 dendrites from 9-14 neurons, * P<0.05, Student’s t-test. D Shot can recruit EB1 to dendrite tips. EB1::mScarlet3 was expressed in c4da neurons alone, with ShotFL::GFP, or with ShotΔCTail::GFP, and visualized at the first instar larval stage. Positions of dendrite tips are indicated by asterisks. E Fluorescence intensity profiles of EB1::mScarlet3 in the distal 10 μm of first instar dendrites. Solid lines indicate average, envelopes indicate s. e. m. (N=9-10 dendrites, 3 neurons each). Significance between genotypes was calculated for 0 - 2 and 2 - 4 μm distances from the tip. **** P<0.0001, Mann Whitney U test. F Frequency of anterograde EB1 comets at the L1 stage in control (Orco dsRNA) and patronin knockdown neurons (patronin dsRNA). Number of comets is shown in the graph. ** P<0.01, Mann Whitney U test. G Frequency of anterograde EB1 comets at the L3 stage in shot knockdown neurons (shot dsRNA) and shot knockdown neurons in a heterozygous patronin mutant background. Number of comets is shown in the graph. * P<0.05, Mann Whitney U test. H - K Synergistic genetic interactions between shot and end binding proteins during c4da neuron dendrite pruning. H - J shot dsRNA was expressed in c4da neurons of animals in the indicated backgrounds, and pruning defects were quantified at 16 h APF. H C4da neuron expressing shot dsRNA. I C4da neuron expressing shot dsRNA in a patronin/+ background. J C4da neuron co-expressing shot and EB1 dsRNAs. I Severity of pruning defects in H - J and in respective controls. N = 33 - 52 neurons, ** P<0.01, **** P<0.0001, Wilcoxon’s test. Scale bars are 5 μm in B, 2 μm in D and 50 μm in E.

Evidence that Rab11 is linked to developmental dendritic microtubule regulation.

A 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. B 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. C Microtubules can originate 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). D Percentage of EB1 comets arising from dendritic Rab11 puncta at the first and third instars. Number of comets is shown in the graph. * P<0.05, Fisher’s exact test. E 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. F Penetrance of anterograde comets in D. Number of comets in E and F is shown in the graph. ** P<0.01, two-tailed Fisher’s exact’s test. G Speed of EB1::GFP comets in D. **** P<0.0001, Wilcoxon’s test. H - J Rab11 and Shot interact genetically during dendrite pruning. Rab11 dsRNA was expressed in c4da neurons of control animals (H) or in a shot3/+ heterozygous background (I), and neurons were visualized at 16 h APF. J Severity of pruning defects in H, I. N = 41 and 42 neurons. **** P<0.0001, Wilcoxon’s test. K Kymograph analysis of Rab11::GFP movement in dendrites of first instar control c4da neurons or shot knockdown neurons (lower panel). L Fraction of mobile Rab11::GFP particles in samples in K. Number of Rab11::GFP particles is shown in the graph. n. s., not significant, Fisher’s exact test. Scale bars are 5 μm in B, C, E, and K, and 20 μm in H.