Figure 7.
Cell area expansion triggers Rho1 activation while cell area contraction triggers RhoGAP71E recruitment. (A and B) Pulses of Rho1 activation in (A) WT and (B) Rho1-expressing eyes were detected using the Rho1 sensor Anillin Rho1 binding domain tagged with GFP (AniRBD::GFP). Left: Snapshots of several ommatidia. Right: Time series montages of (A) WT and (B) Rho1-overexpressing cone cells demarcated by the boxes over a period of 300 s (Video 8). Note widespread high levels of Rho1 activation in Rho1-expressing eyes compared with WT, where there is sporadic enrichment medioapically (purple arrowheads) and in contracting LC–LC contacts (orange arrowheads). (A) In WT, cone cells do not expand or contract on this timescale. (B) Overexpression of Rho1 induces pulsed expansion and contraction of cone cells. During early cone cell expansion (green lines), levels of Rho1 activation are low at the cell periphery and medioapically (t62; yellow and blue arrowheads, respectively). During mid-expansion Rho1 activation increases first at the cell periphery (t77) and then also in puncta medioapically (t108). As the cells begin to contract (red lines) Rho1 is activated strongly both at the cell periphery and medioapically. (C) Overexpressing Rho1 increases the negative correlation of medioapical AniRBD::GFP with cone cell radial length at a time shift of −30 s prior to peak cell expansion, and the correlation continues to rise after peak cell expansion (R = −0.3982, N = 14 for WT, N = 16 for GMR > Rho1 from three eyes each. t test, P = 0.0063). (D and D′) RhoGAP71E accumulates strongly during contraction of LCs in Rho1-overexpressing eyes (Video 8). (D) Top: Snapshots of a lattice edge. The 2° LC in the boxed region is highlighted in green. RhoGAP71E::GFP accumulation in contracted cells is indicated with red arrowheads. Low RhoGAP71E::GFP levels in expanding cells are indicated with green arrowheads. Middle: Zoomed-in images of the 2° LCs (boxed area). The bracketed red zones show medioapical RhoGAP71E::GFP levels increasing with cell area contraction, while bracketed green zones show RhoGAP71E::GFP levels decreasing when cell area expands. Bottom: Longer time series montage of medioapical RhoGAP71E::GFP dynamics over a period of 450 s. RhoGAP71E::GFP levels increase (red bracket) as the cell apical area decreases (red line) and decrease (green brackets) as cell area increases (green lines). (D′) A stronger correlation between RhoGAP71E::GFP levels and cell area contraction is observed in GMR > Rho1 compared with WT at a time-shift of +10 s (N = 11 in WT and N = 16 in GMR > Rho1 from four eyes each, t test, P = 0.0081). (E) A cartoon summarizing the cell-autonomous mechanical feedback loops affecting Rho1 activity, actomyosin contractility, and apical cell area. Low tension in expanding cells triggers Rho1 activation to increase tension and promote contraction. High tension in contracting cells triggers the recruitment of RhoGAP71E to inhibit Rho1, decrease tension, and promote expansion. Scale bar = 3 µm.

Cell area expansion triggers Rho1 activation while cell area contraction triggers RhoGAP71E recruitment. (A and B) Pulses of Rho1 activation in (A) WT and (B) Rho1-expressing eyes were detected using the Rho1 sensor Anillin Rho1 binding domain tagged with GFP (AniRBD::GFP). Left: Snapshots of several ommatidia. Right: Time series montages of (A) WT and (B) Rho1-overexpressing cone cells demarcated by the boxes over a period of 300 s (Video 8). Note widespread high levels of Rho1 activation in Rho1-expressing eyes compared with WT, where there is sporadic enrichment medioapically (purple arrowheads) and in contracting LC–LC contacts (orange arrowheads). (A) In WT, cone cells do not expand or contract on this timescale. (B) Overexpression of Rho1 induces pulsed expansion and contraction of cone cells. During early cone cell expansion (green lines), levels of Rho1 activation are low at the cell periphery and medioapically (t62; yellow and blue arrowheads, respectively). During mid-expansion Rho1 activation increases first at the cell periphery (t77) and then also in puncta medioapically (t108). As the cells begin to contract (red lines) Rho1 is activated strongly both at the cell periphery and medioapically. (C) Overexpressing Rho1 increases the negative correlation of medioapical AniRBD::GFP with cone cell radial length at a time shift of −30 s prior to peak cell expansion, and the correlation continues to rise after peak cell expansion (R = −0.3982, N = 14 for WT, N = 16 for GMR > Rho1 from three eyes each. t test, P = 0.0063). (D and D′) RhoGAP71E accumulates strongly during contraction of LCs in Rho1-overexpressing eyes (Video 8). (D) Top: Snapshots of a lattice edge. The 2° LC in the boxed region is highlighted in green. RhoGAP71E::GFP accumulation in contracted cells is indicated with red arrowheads. Low RhoGAP71E::GFP levels in expanding cells are indicated with green arrowheads. Middle: Zoomed-in images of the 2° LCs (boxed area). The bracketed red zones show medioapical RhoGAP71E::GFP levels increasing with cell area contraction, while bracketed green zones show RhoGAP71E::GFP levels decreasing when cell area expands. Bottom: Longer time series montage of medioapical RhoGAP71E::GFP dynamics over a period of 450 s. RhoGAP71E::GFP levels increase (red bracket) as the cell apical area decreases (red line) and decrease (green brackets) as cell area increases (green lines). (D′) A stronger correlation between RhoGAP71E::GFP levels and cell area contraction is observed in GMR > Rho1 compared with WT at a time-shift of +10 s (N = 11 in WT and N = 16 in GMR > Rho1 from four eyes each, t test, P = 0.0081). (E) A cartoon summarizing the cell-autonomous mechanical feedback loops affecting Rho1 activity, actomyosin contractility, and apical cell area. Low tension in expanding cells triggers Rho1 activation to increase tension and promote contraction. High tension in contracting cells triggers the recruitment of RhoGAP71E to inhibit Rho1, decrease tension, and promote expansion. Scale bar = 3 µm.

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