Cyst preferentially targets Rho1. (A) Stills of time-lapse videos (Videos 3 and 4) showing the Rho activity probe Anillin-RBD::GFP in a control and in an embryo derived from a cyst RNAi Df(cysts)/+ mother. Time is minutes after the onset of germband extension. Junctional levels of Anillin-RBD::GFP are reduced in cyst-depleted embryos. In ventral midline cells, the probe also accumulates in midbodies after cell division (arrowheads) in control and cyst-depleted embryos. Scale bar, 10 µm. (B) Quantification of junctional levels of Anillin-RBD::GFP in control and cyst-depleted embryos at the time points indicated in A. Quantifications at 45 min were not included, as the probe’s junctional signal was undetectable in cyst-depleted embryos. Bars indicate mean ± SEM. n = number of cell edges analyzed in four embryos per condition. ****, From left to right: P = 1.5 × 10−60, 8.3 × 10−42, 1.2 × 10−9, and 1.3 × 10−10 (Kolmogorov–Smirnov test). (C) Schematic of GFP-tagged fragments of Cyst used in D and E. (D) Activation of Drosophila Rho1, Rac1, or Cdc42 by the indicated fragments of Cyst protein depicted in C. GTPases were immunoprecipitated from HEK293T cells coexpressing the Cyst fragments. GTP and GDP were eluted and subsequently analyzed by LC-MS/MS. The absolute amounts of GTP and GDP are shown as a percentage normalized to coexpression with GFP::Cyst-N. GFP::Cyst-DH-PH showed a twofold relative increase in Rho1-associated GTP and a 1.5-fold relative increase in GTP-associated Rac1. No detectable activation of Cdc42 was found. GFP::Cyst-N and GFP::Cyst-C act as negative controls. n = 4 for all measurements. Data are presented as mean ± SD. ****, P < 0.0001; **, P < 0.01; ns, not significant (Student’s t test). (E) Positive controls for D using mammalian GEF proteins. The DH-PH domain of LARG (LARG-DH-PH), an active version of STEF (STEF-ΔN), and the DH-PH domain of INTS2 (INTS2-DH-PH) are known to target Rho1, Rac1, and Cdc42, respectively. GFP::Cyst-DH-PH and LARG-DH-PH show comparable targeting of Rho1. GFP::Cyst-DH-PH targeting of Rac1 was less than that seen for STEF-ΔN. Values are normalized to GFP, which acts as a negative control. n = 4 for all measurements. Data are presented as mean ± SD. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, not significant (Student’s t test).
