Extended Data Figure 7: Analysis of the role of Vinc, Rho and several F-actin nucleators in MyoII accumulation in the neighbours.

a, Structure of vinc^Δ3 and GFP–Vinc alleles generated by CRISPR/Cas9-mediated homologous recombination, showing position of GFP tag insert relative to vinc coding sequence. M1, methionine 1; P2, proline 2. b, E-cad–GFP and MyoII–mChFP localization in wild-type (n = 38 cells, 2 pupae) and vinc (n = 44 cells, 2 pupae) neighbours. Filled arrowheads denote MyoII–mChFP accumulation in the neighbours. c, Rate of contractile ring constriction in wild-type and vinc dividing cells (2 and 2 pupae, respectively). d, Normalized MyoII accumulation at 80% of the initial cell diameter for wild-type and vinc neighbours (2 and 2 pupae, respectively). e, GFP–Vinc and MyoII–3 × mKate2 localization during cytokinesis. During constriction, GFP–Vinc is reduced along the ingressing AJ and does not accumulate with MyoII–3 × mKate2 at the base of the ingressing membrane. Greyscale inset shows GFP–Vinc localization upon full contractile ring constriction. n = 46 cells (3 pupae). f, Cytokinesis failure in rok^RNAi, rho and dia dividing cells. n/n indicates the number of cells that failed cytokinesis/total number of cells (10, 10 and 7 pupae, respectively). The percentage of cytokinesis failure in rho and dia dividing cells is higher than that observed in rok^RNAi dividing cells. However, loss of Rho or Dia function in the neighbours only delays MyoII accumulation in the neighbours, whereas rok^RNAi in the neighbours strongly impairs it (g–j). g, i, E-cad–GFP and MyoII–mChFP localization in a rho (g; n = 30 cells, 10 pupae) or a dia (i; n = 26 cells, 7 pupae) mutant cell neighbouring a wild-type dividing cell (dots, marked by the absence of nls–GFP). Open arrowheads denote decreased MyoII–mChFP accumulation from mid-constriction until 80% of the initial cell diameter and MyoII–mChFP accumulation following midbody formation, respectively. h, j, Normalized MyoII accumulation at 80% of the initial cell diameter and 1 min after midbody formation in wild-type (n = 53 cells, 9 pupae), rok^RNAi (n = 29 cells, 11 pupae) and rho (h; n = 30 cells, 10 pupae) or dia (j; n = 26 cells, 7 pupae) neighbouring cells facing wild-type dividing cells. k, n, Dia–eGFP (k; n = 33 cells, 9 pupae) or Arp3–GFP neighbours (n; n = 30 cells, 2 pupae) facing a dividing cell in a MyoII–3 × mKate2 tissue. Greyscale insets denote Dia–eGFP (k) or Arp3–GFP (n) localization upon full contractile ring constriction. Both Dia–eGFP and Arp3–GFP are uniformly localized along the ingressing region throughout constriction in the dividing cell. l, E-cad–GFP and MyoII–mChFP localization in wild-type and arpc1 mutant neighbours (dots, marked by the absence of nls–GFP) facing a wild-type dividing cell (n = 27 cells, 7 pupae) at 80% of the initial cell diameter. Arrowheads as in b. m, Normalized MyoII accumulation at 80% of the initial cell diameter and 1 min after midbody formation for wild-type (n = 25 cells, 5 pupae) and arpc1 (n = 27 cells, 7 pupae) neighbours facing wild-type dividing cells. o, E-cad–GFP and MyoII–mChFP localization in wild-type (n = 96 cells, 12 pupae), fhos (n = 36 cells, 3 pupae), capu (n = 29 cells, 4 pupae), daam (n = 26 cells, 5 pupae), form3^RNAi (n = 29 cells, 7 pupae), frl^RNAi (n = 35 cells, 13 pupae), ena (n = 25 cells, 8 pupae) and chic (n = 27 cells, 6 pupae) neighbours. Since Capu and fhos loss-of-function mutants are viable, MyoII accumulation was analysed in capu or fhos tissues. Notably, the rate of contractile constriction in both fhos and capu dividing cells is similar to wild-type cells and no detectable defects were observed during cytokinesis (p). For all other conditions, the dividing cell is wild-type. Dots denote daam, form3^RNAi, frl^RNAi, ena and chic mutant cells, marked by the absence of His2a–GFP, cytosolic GFP or nls–GFP. Filled and open arrowheads denote MyoII–mChFP accumulation and reduced accumulation in the neighbours, respectively. p, Rate of contractile ring constriction in wild-type, fhos or capu dividing cells (2, 3 or 4 pupae, respectively). q, Normalized MyoII accumulation at 80% of the initial cell diameter for wild-type, fhos, capu, daam, form3^RNAi, frl^RNAi, ena and chic neighbours (12, 3, 4, 5, 7, 13, 8 or 6 pupae respectively). r, Normalized MyoII accumulation at 80% of the initial cell diameter and 1 min after midbody formation for wild-type (n = 57 cells, 10 pupae), chic (n = 27 cells, 6 pupae) or rok^RNAi (n = 29 cells, 11 pupae) neighbours facing wild-type dividing cells. n denotes number of cells. *P < 0.05, ****P < 0.0001, Mann–Whitney U-test (c), Student’s t-test (d), ANOVA (m, q), Kruskal–Wallis test (h, j, p, r). Data are mean ± s.e.m. Scale bars, 5 μm.

Source data