Figure 5.
RhoGEF2 promotes assembly of medioapical actomyosin and apical cell area contraction. (A) RhoGEF2::GFP and F-actin labeled with Lifeact::Ruby. Snapshots from a time-lapse movie of a lattice edge (top) and magnified views of a 2° LC demarcated with a box. RhoGEF2::GFP (yellow arrow) accumulates medioapically in pulses that correlate with F-actin node enrichment (green arrowhead). (A′) Left: Time-shifted correlation plot between RhoGEF2::GFP and apical cell area. Right: RhoGEF2::GFP accumulation correlates negatively with cell area at a peak of +15 s (R = −0.1361, N = 12 from three eyes, one-sample t test P = 0.0250). (B–B‴) Clonal depletion of RhoGEF2 by RNAi reduces medioapical MyoII assembly compared with the WT counterparts (Video 6). (B) Ommatidium with RhoGEF2RNAi-expressing cells (green, upper boxed LC) and WT counterpart (lower boxed LC). (B′ and B″) Time series showing (B′) a RhoGEF2RNAi-expressing cell in which MyoII assembly does not occur compared to (B″) a WT cell in which MyoII assembles in pulses (red arrow). (B‴) Left: Time-shifted correlation plots between medioapical MyoII levels and LC area in RhoGEF2 RNAi-expressing cells (pink) compared with WT (green). Right: Peak correlation is weaker in RhoGEF2 RNAi-expressing cells compared with WT counterparts (paired t test, P = 0.0281, N = 8 pairs in four eyes). (C) RhoGEF2 overexpression enhances medioapical actomyosin network assembly and apical cell area changes (Video 6). Top: Snapshots of a lattice edge. Bottom: Magnified views of the 2° LC show the assembly (red arrow) and disassembly (green arrow) of the medioapical actomyosin network, corresponding to cell area contraction (red line) and expansion (green line). (C′) Medioapical F-actin levels correlate with apical area contraction in 2° LCs. (C″ and C‴) Medioapical F-actin pulse frequency is ∼3 min (215 ± 56 s, N = 12 from three eyes) in RhoGEF2-overexpressing eyes, with a (C‴) 1.9-fold change at their peak than at their trough levels. (D) RhoGEF2 expression increases the peak correlation between F-actin and cell area (peak correlation at −15 s, N = 11 from three eyes, t test P = 0.0337). (E and F) Rho1 and RhoGEF2 expression increase the frequency and amplitude of F-actin pulses compared with WT. (E) Pulse frequencies of medioapical F-actin in WT eyes compared to eyes overexpressing Rho1 and RhoGEF2. One-way ANOVA with Tukey’s multiple comparisons. WT versus GMR > Rho1, difference of means = 104 s, P = 0.0010. WT versus GMR > RhoGEF2, difference of means = 133 s, P = 0.0001. GMR > Rho1 versus GMR > RhoGEF2, difference of means = 29 s, P = 0.5084, N = 11, 15, and 16, respectively, from three eyes each. (F) Pulse amplitudes of F-actin in the above genotypes. Welch one-way ANOVA with Dunnett’s comparison between WT and GMR > Rho1, mean difference of R = −0.3139, P < 0.0001. WT versus GMR > RhoGEF2, mean difference of R = −0.6155, P = 0.0218. GMR > Rho1 versus GMR > RhoGEF2, mean difference of R = 2.523, P = 0.0011, N = 14, 13 and 13, respectively, from three eyes each. Scale bar = 3 µm.

RhoGEF2 promotes assembly of medioapical actomyosin and apical cell area contraction. (A) RhoGEF2::GFP and F-actin labeled with Lifeact::Ruby. Snapshots from a time-lapse movie of a lattice edge (top) and magnified views of a 2° LC demarcated with a box. RhoGEF2::GFP (yellow arrow) accumulates medioapically in pulses that correlate with F-actin node enrichment (green arrowhead). (A′) Left: Time-shifted correlation plot between RhoGEF2::GFP and apical cell area. Right: RhoGEF2::GFP accumulation correlates negatively with cell area at a peak of +15 s (R = −0.1361, N = 12 from three eyes, one-sample t test P = 0.0250). (B–B‴) Clonal depletion of RhoGEF2 by RNAi reduces medioapical MyoII assembly compared with the WT counterparts (Video 6). (B) Ommatidium with RhoGEF2RNAi-expressing cells (green, upper boxed LC) and WT counterpart (lower boxed LC). (B′ and B″) Time series showing (B′) a RhoGEF2RNAi-expressing cell in which MyoII assembly does not occur compared to (B″) a WT cell in which MyoII assembles in pulses (red arrow). (B‴) Left: Time-shifted correlation plots between medioapical MyoII levels and LC area in RhoGEF2 RNAi-expressing cells (pink) compared with WT (green). Right: Peak correlation is weaker in RhoGEF2 RNAi-expressing cells compared with WT counterparts (paired t test, P = 0.0281, N = 8 pairs in four eyes). (C) RhoGEF2 overexpression enhances medioapical actomyosin network assembly and apical cell area changes (Video 6). Top: Snapshots of a lattice edge. Bottom: Magnified views of the 2° LC show the assembly (red arrow) and disassembly (green arrow) of the medioapical actomyosin network, corresponding to cell area contraction (red line) and expansion (green line). (C′) Medioapical F-actin levels correlate with apical area contraction in 2° LCs. (C″ and C‴) Medioapical F-actin pulse frequency is ∼3 min (215 ± 56 s, N = 12 from three eyes) in RhoGEF2-overexpressing eyes, with a (C‴) 1.9-fold change at their peak than at their trough levels. (D) RhoGEF2 expression increases the peak correlation between F-actin and cell area (peak correlation at −15 s, N = 11 from three eyes, t test P = 0.0337). (E and F) Rho1 and RhoGEF2 expression increase the frequency and amplitude of F-actin pulses compared with WT. (E) Pulse frequencies of medioapical F-actin in WT eyes compared to eyes overexpressing Rho1 and RhoGEF2. One-way ANOVA with Tukey’s multiple comparisons. WT versus GMR > Rho1, difference of means = 104 s, P = 0.0010. WT versus GMR > RhoGEF2, difference of means = 133 s, P = 0.0001. GMR > Rho1 versus GMR > RhoGEF2, difference of means = 29 s, P = 0.5084, N = 11, 15, and 16, respectively, from three eyes each. (F) Pulse amplitudes of F-actin in the above genotypes. Welch one-way ANOVA with Dunnett’s comparison between WT and GMR > Rho1, mean difference of R = −0.3139, P < 0.0001. WT versus GMR > RhoGEF2, mean difference of R = −0.6155, P = 0.0218. GMR > Rho1 versus GMR > RhoGEF2, mean difference of R = 2.523, P = 0.0011, N = 14, 13 and 13, respectively, from three eyes each. Scale bar = 3 µm.

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