Propagation of F-actin disassembly via Myosin15-Mical interactions

Figures

• 

  Fig. 1. The unconventional class XV myosin Myo15 controls Sema/Plexin/Mical-mediated cellular remodeling. (A) Bristle cells project a long, unbranched, F-actin–driven cellular extension. (B) Elevating bristle Mical levels (Bristle Mical+++ = UAS:Mical/+, B11-GAL4/+) generates a shorter bristle and branching at the tip (arrowhead and drawings). (C and D) Mical-driven bristle effects are strongly enhanced by the P{XP}d05943 mutant, such that the bristles are shorter and more branched (arrowhead, drawings, and graph). Means ± SEM. n ≥ 40 animals (one bristle per animal) per genotype. ****P < 0.0001, unpaired t test (two-tailed). Bristle Mical+++ + Myo15d05943/+ = UAS:Mical/+, B11-GAL4/+, P{XP}d05943/+. Bristle Myo15d05943/+ only = B11-GAL4/+, P{XP}d05943/+. (E) Myo15 genomic map and mutant generation. Exons, black boxes; 5′/3′ untranslated region, gray boxes; transposons, triangles. Only a short noncoding region remains in the Myo1521J mutant we generated by deleting the region between the transposons containing FRT sites (blue and purple). (F and G) Mical-driven bristle branching is suppressed by decreasing Myo15 (arrowhead, drawings, and graph). Means ± SEM. n ≥ 47 animals (one bristle…

• 

  Fig. 2. Myo15 associates with and distributes Mical to spatially and directionally remodel cells. (A) Drosophila Myo15’s domain organization matches mammalian Myo15. (B) Mical (red) and Myo15 (green) colocalize in vivo (UAS:mCherryMical/+, UAS:Myo15GFP/+, B11-GAL4/+). (1) to (7) show different bristles at increasing developmental stages. Mical-Myo15 overlap/colocalize within early extending bristle tips (open arrows), tracks around these tips (closed arrows), narrow tracks emanating from Mical-enriched regions (closed arrowheads), and within Mical-triggered branches (open arrowheads). Asterisks indicate adjacent bristle. (C) Myo15 immunoprecipitates (IPs) Mical in vivo. α, antibody. (D) Myo15 spatially redistributes Mical (red) to remodel cells. Bristles begin extending at 0 hour (~31 hours after puparium formation). One to13 hours show different bristles at increasing developmental stages and typical of those stages/genotypes. (1) Bristle Mical+++ (UAS:mCherryMical/+, B11-GAL4/+): Elevating Mical levels induces a branch (3 hours, arrowhead). Bristles then (5 to 10.5 hours) extend beyond (thin arrows) this branch point. A second Mical-triggered branch (open arrow) then forms (13 hours).…

• 

  Fig. 3. Myo15 and its motor and cargo-transporting regions locally expand Mical-mediated remodeling. (A) Myo15 does not alter Mical’s long-range transport. (1) Mical (red) still localizes to bristle tips (arrowheads) in Myo15 knockout/knockdown. (2) Mical’s distribution measured as in Fig. 2D (3). Means ± SEM. n ≥ 12 bristles (6 to 12 animals) per genotype. ns (not significant), P = 0.8083, unpaired t test (two-tailed). (B) Mical locally redistributes with Myo15 in vivo. Same bristle imaged over time (times from start of movie S1). Myo15 (green) and Mical (red) exhibit regions of colocalization (closed arrows) and no colocalization (open arrows). During remodeling/extension (open arrowheads and similar regions at subsequent times), Mical’s (red) movement/redistribution tracks with Myo15 (different color arrowheads), resulting in coenriched areas/nodes that enlarge (get brighter) over time. Note that a slight postimaging brightness adjustment was made to the last red channel image (5 hours, 53 min, 15 s) to better highlight the most distal labeling spot. (C) Myo15’s motor and cargo-transporting regions drive Mical’s redistribution and cellular remodeling. (1) Myo15ΔMotor, lacks the motor…

• 

  Fig. 4. Myo15 positions Mical to spatially target and expand F-actin disassembly. Genotypes are as previously described plus the addition of GFPactin transgene to visualize actin. (A) Myo15 is necessary and sufficient for Mical-mediated F-actin disassembly. (1) Normal bristle F-actin organization (B11-GAL4/+, UAS:GFPActin/+). (2) Elevating Myo15 induces regions of more (stars) and less (arrow) F-actin. Abnormal bristle bends, and other alterations accompanied these F-actin changes (e.g., stars; see also figs. S2, E and F, and S6A). (3) Elevating Mical induces regions of less F-actin (arrow) and branching (arrowhead) (4, 7). (4) Reducing Myo15 decreases Mical-triggered F-actin disassembly and branching (arrows). (5) Elevating Myo15 increases Mical-triggered F-actin disassembly (arrows) and branching (arrowheads). (B) Myo15 expands Mical-mediated F-actin disassembly. Bristle development tracked as in Figs. 2D [(1) and (2)] and 3C (2) and fig. S9B. (1) Elevating Mical induces F-actin disruptions and branching (4 hours, closed arrowhead). Bristles then extend past this branch point (9 hours, thin arrows), where F-actin disruptions (open arrow) and branching [13 hours; Fig. 2D, (1)] occur…

• 

  Fig. 5. Mical and Myo15 regulate synaptic structure and F-actin muscle organization. (A) Illustrated hemisegment showing the stereotypical synaptic innervation of intersegmental nerve (ISN) (blue) and ISNb (yellow) motor axons. Muscles 1, 2, 6, and 7, green; other muscles, light blue; dashed lines, nerve lying underneath/lateral to muscles. (B) Myo15−/− mutants, similar to Mical−/− mutants, have reduced synaptic innervation. Synapses visualized with CD8-GFP-Shaker (shGFP7A) (7, 45). (1) Compared to wild-type (shGFP7A/shGFP7A) synapses, which spread out along muscles 1 and 9 (top), Myo15−/− mutant synapses (Myo1521J/Y; shGFP7A/shGFP7A) are shorter (middle and bottom). Muscle 2 indicated for reference. (2) Both Mical−/− and Myo15−/− mutants have reduced synaptic innervation. Mical−/− (MicalI666, shGFP7A/MicalG56, shGFP7A). Means ± SEM. n ≥ 20 synapses/muscles (10 animals) per genotype. *P = 0.0243 (Myo15−/−) and *P = 0.0219 (Mical−/−), unpaired t test (two-tailed). (C) Synaptic innervation of muscles 6 and 7 is also shorter in Myo15−/− (Myo1521J/Y) mutants. Synapses/presynaptic active zones visualized with nc82 [Bruchpilot (Brp)] antibody. Muscles visualized with fluorophore-conjugated…

• 

  Fig. 6. Summary/model: Myo15 propagates Mical Redox-triggered F-actin disassembly and cellular remodeling. (A) Summary: (1) Left: Mical (red) localizes to the growing bristle tip and is spatiotemporally activated to disassemble F-actin and trigger cellular remodeling/branching. Right: As development continues, the bristle again assembles F-actin/bundled filaments, which elongate the bristle past this region of F-actin disassembly/branching. (2) Left and right: Elevating Myo15 levels distributes Mical more broadly around the branch point, spatially increasing F-actin disassembly. Right: Myo15’s expansion of Mical’s distribution and F-actin disassembly hinders new F-actin/bundled filament assembly, destabilizing and misdirecting the elongating bristle. Myo15 thereby transforms regions where Sema/Plexin/Mical activation typically induces limited disassembly/reorganization (1) into more expansive effects (2). (B) Model: (1) During outgrowth/extension (large green arrow), Mical (black) specifically localizes and is activated by Sema repellents (light brown) and their Plexin receptors (dark brown) (4, 5). Activated Mical oxidizes F-actin (gray) subunits on their pointed ends, generating…