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Fig. S1. Stronger kd bristle phenotypes seen in UAS-dicer2; Gal80-ts/V22198 (Rab11-RNAi); neur-Gal4 flies. (A) Head. (B) Thorax (inset is a magnification of a small region). (C) Costa bristles on the wing.
Fig. S2. Rescue of Rab11 hypomorph phenotype using UAS-GFP-Rab11. (A) Rescue of Rab11 hypomorph phenotype by UAS-GFP-Rab11. Asterisks on gray bars indicate that the difference in bristle length as compared with Rab11 hypomorphs (white bars) is significant (P≤1×10−12 for all bars). Mean ± s.e. (B) The position of the analyzed macrochaetae on the thorax.
Fig. S3. Western blot to detect GFP-Rab11 in wing discs from wild type (Ore R) and GFP-Rab11-expressing larvae probed using a monoclonal anti-GFP antibody.
Fig. S4. Role of cytoskeleton in GFP-Rab11 localization. GFP-Rab11-expressing pupae cultured in the presence of drugs that inhibit either actin or microtubules and immunostained for GFP-Rab11 using anti-GFP antibody. All drugs except Latrunculin A were used at 150-300 µM; Latrunculin A was used at 50 µM. Images were taken at 40× magnification. Scale bars: 10 µm. (A-E′) GFP-Rab11 localization in bristles from control pupae not treated with drugs (A) and GFP-Rab11 localization in bristles from pupae treated with Nodocazole (B), Vinblastine (C), Cytochalisin D (D), Latrunculin A (E). (A′-E′) Magnified images of single bristles from A-E, respectively. (F-H) The effect of drugs on GFP-Rab11 localization (ratio in shaft versus cell body) calculated as intensity per unit area expressed in arbitrary units (see Materials and methods for details). Asterisks on bars indicate that the difference as compared with control is statistically significant. (F) Noc, P=1.86×10−7; Vb, P=3.25×10−7. (G) Noc, P=7.3×10−8; Vb, P=1×10−11. Mean ± s.e.
Fig. S5. Microtubule polarity in bristles. Polarity reporters were expressed for 3 hours. (A) Pupal leg bristles expressing the minus-end marker Nod-βgal (red) and tubulin (green). (B) Pupal wing bristles expressing the plus-end marker Nod-GFP (green) and tubulin (red). (C) Pupal wing bristles expressing the plus-end marker Kinesin-βgal (red) and tubulin (green). (D) Wild-type adult bristle. (E) Adult bristle expressing the minus-end marker Nod-βgal. The arrow points to the characteristic tip bulge. (Nod-βgal expression was induced by transferring pupae at 45 hours APF from 21°C to 29°C and keeping them there until they eclosed). (F,G) Adult bristle expressing the plus-end marker Kin-βgal (Kin-βgal expression was induced by transferring pupae at 45 hours APF from 21°C to 29°C and keeping them there until they eclosed).
Fig. S6. Variable patterns of bristle collapse in Rab11 mutants. Images shown are of the same pupa tracked until adult stage. Asterisks mark the bristle being tracked. Red arrows indicate regions of bristle showing deformities or signs of collapse. (A-D) Bristle 1, (E-G) Bristle 2. Images represent single optical sections. Scale bars: 10 µm.
Fig. S7. Actin filaments in bristles as visualized by phalloidin staining at 42 hours APF (prior to initiation of actin breakdown) and at 48 hours APF (during the process of actin breakdown). Arrows indicate regions showing small gaps, breaks or overlapping filaments, which are signs of actin breakdown. (A,C) Actin in wild-type bristles. (B,D) Actin in Rab11 dsRNA-expressing bristles. Images were taken at 40× magnification and represent single optical sections. Scale bars: 5 µm.
Fig. S8. Localization of chitin in bristles. Chitin arrangement in bristles visualized by staining with Rhodamine-conjugated chitin-binding domain probe (CBD-Rhodamine) with and without permeabilizing the fixed tissue with Triton X-100. (A,B) Chitin staining in the absence (A) and presence (B) of Triton X100 is the same. (C) Tubulin staining in the absence of Triton X-100 (control). Note the very poor staining. (D) Tubulin staining in the presence of Triton X-100 (control). Note the improved staining. All images were taken at 60× magnification and represent single optical sections. Scale bars: 10 µm.
Fig. S9. Quantitative analysis of chitin levels in bristles. The amount of chitin in wild-type and Rab11 kd bristles at various times of development. Chitin levels were estimated by measuring the intensity of CBD-Rhodamine staining in bristles using ImageJ.
Fig. S10. Effect of disrupting chitin synthase function on bristle development. (A-C′) kkv1 clones (A-C) and higher magnifications thereof (A′-C′). Note the reduced pigmentation in the clones in A and B. Arrow points to exudate that is often associated with kkv1 clones (C). (D-O) Bristles from flies injected as pupae. D and G are controls in which the pupae were injected with water. All others were injected with nikkomycin (see Materials and methods). Arrows in J point to sockets without shafts. The box in L highlights a region with a strong bristle phenotype. Note that the bristles in M-O show pigmentation defects that are reminiscent of those seen in Rab11 kd. Scale bars: 100 µm.
Fig. S11. dyl mutant phenotypes. (A,B) UAS-dyl was overexpressed in flies using sca-Gal4. Split or deformed thoracic bristles were observed (arrows). (C) Thoracic bristle from fly expressing dyl RNAi using ptc-Gal4. (D,E) Examples of enhancement of the dyl disruption phenotype in flies expressing dyl RNAi in a dyl deficiency background. (F) The severity of bristle phenotypes with disrupted dyl function.
