Fig. 3: The role of the MVB pathway, integrins, and BM components in establishing asymmetric membrane identity and filopodia organization in PG cells.
a Silencing Rab11 results in a 190-h delay in metamorphosis onset compared to the control. Number of sample tubes: nphm>xw1118 = 8, nRNAi-Rab11xw1118 = 18, nphm>RNAi-Rab11 = 10. b Silencing Vps25, Vps36, or shrb in PG cells resulted in a several-hour delay compared to the control. Number of sample tubes: nphm> xw1118 = 8, nRNAi-Vps25xw1118 = 10, nRNAi-Vps36xw1118 = 6, nRNAi-shrbxw1118 = 4, nphm>RNAi-Vps25 = 8, nphm>RNAi-Vps36 = 2, nphm>RNAi-shrb = 2. phm > RNAi-Rab11 PG lacks microtubule-based (c) and βPS-based (d) filopodia in the sub-BM region. Filopodia are visible with the PLC-GFP membrane marker in ex vivo control PG cells (e, arrowheads) but disappear upon Rab11 downregulation (f). Comparison of control w1118 PG (g) and phm > RNAi-Rab11 PG (h) showing F-actin (single section) and PTTH staining (maximum projection of a Z-stack) processed with a depth color code from a Fiji plugin. XZ cuts reveal PTTH axons positioned between the PG bilayer in the control (g, arrowheads), whereas upon recycling pathway disruption, PTTH axons are also mislocalized just below the BM (h, arrowheads). i Axon from a phm > RNAi-Rab11 PG at the sub-BM region. The PTTH staining overlaps with its receptor Torso, suggesting that the mislocalized synaptic buttons are functional. j In phm > RNAi-Rab11 PG at the sub-BM region, mislocalized PTTH-containing synaptic buttons exclude βPS expression. Insets 1 and 2 highlight the signal exclusion between PTTH and βPS expression. k Z sections of a trolJO271#49 mutant PG reveal the loss of a bilayer organization. l αTub85E staining in trolJO271#49 mutant PG shows short microtubules in the sub-BM region, while in a lateral plane, αTub85E staining appears normal. m Down-regulation of mys impedes microtubule extension. n Developmental timing of trolJO271#49 mutant vs w1118 individuals. The trolJO271#49 mutant enters pupariation 24 h after the control line. nw1118 = 10, ntrolJO271#49 = 6. White scale bars: 20 μm; yellow scale bars: 5 μm. Data are presented as means ± SEM in (a, b, n). Source data are provided as a Source Data file.
