![Rho1 regulates actomyosin and cell area changes and reciprocal cytoskeletal and cell area changes in adjacent LCs. (A) F-actin organization and dynamics in Rho1DN-expressing eyes. RhoDN expression disrupts actin organization, pulsatile actomyosin dynamics, and cell shape changes (see text for details). Top: Snapshots of a lattice edge. Blue and green overlays highlight adjacent 3° and 2° LCs, respectively. Bottom: Zoomed-in images of the 2° LC (boxed area). The bracketed green zones show low medioapical F-actin during cell area expansion, while bracketed red zones show increased F-actin during cell area contraction. Note an interruption of junctional actomyosin (white arrow). (A′) The negative correlation between medioapical F-actin levels and cell area contraction is stronger in GMR > Rho1DN compared with WT (N = 18 in WT, N = 19 in GMR > Rho1DN from three eyes each, t test, P = 0.0013). (B and C) Rho1 overexpression has a dramatic effect on pulsatile medioapical F-actin dynamics and cell area fluctuations (Video 3). (B) Time series montage shows F-actin node fusion (tracked by the yellow arrows) during medioapical F-actin ring formation (red arrowhead). (B′) F-actin nodes are on average 40% larger in GMR > Rho1 compared with WT (N = 70 nodes in WT, N = 60 in GMR > Rho1 from three eyes each, t test P < 0.0001). (C) A time series montage of a cell overexpressing Rho1 through a cycle of cell expansion (red bar) and contraction (green bar). Low F-actin intensity within expanded cells (green arrows), high F-actin intensity within contracted cells (red arrow). (C′) Medioapical F-actin pulse frequency in 2° LCs is accelerated (245 ± 50 s, N = 16). (C″) F-actin levels increase on average 4.5-fold in 2° LCs (N = 14) during contraction compared with 1.4-fold in WT (Fig. 1 G). (C‴) Time-shifted correlation shows that medioapical F-actin levels correlate strongly with apical area contraction of 2° LCs. (D) The peak negative correlation between F-actin and cell area in Rho1-expressing eyes is significantly stronger than in WT (R = −0.6508 at peak correlation of −15 s, N = 15 from three eyes, t test P = 0.0002). (E) Contractile medioapical actomyosin dynamics and cell area fluctuations inversely coordinate between neighboring 2° cells prior to cell pruning (Video 4). When one cell has low-intensity F-actin and increasing area (green arrow), the neighboring cell has high-intensity of F-actin and decreasing area (red arrow). (E′) Cell area of neighboring 2° LCs is negatively correlated (R = −0.2020 at a peak correlation of +15 s, N = 14 [7 pairs] in three eyes, one-sample t test; P = 0.0318). (F) A schematic summarizing the reciprocal coordination of cytoskeletal and mechanical behavior between neighboring LCs. Scale bar = 3 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/223/2/10.1083_jcb.202304041/2/jcb_202304041_fig3.png?Expires=1771586988&Signature=yjr7YmEB6VMCX27CADrIj1W9XqbeM9I1PqUCBjpc04Rah3Pf1yUz-v~AtjVrXvZUlEz66KabW423uipa6-jGI-E8M8pSUGyE7WAVovW0W-otALcyWpQFIz~7jVXl078BYCEM1SPAJuxIlBEfc6S3-5l~Cap18H84oKspZqB252sO2SnAx1eIDVnci8wJ-nt~fZnZVMedYWbN3p2MN2aJ2ZwyifDdOFyu9-UhxkPm9Z7dlk0nuS586mRyeutHDr7UN0Xe1U-wKHTzqQR7cZJYjJ1xcDxBdiO4hm1RuuriSl5M319cNNALw1aX2xIrRxoGXfgCDTfDvR~3VdIrgHPMQg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Rho1 regulates actomyosin and cell area changes and reciprocal cytoskeletal and cell area changes in adjacent LCs. (A) F-actin organization and dynamics in Rho1DN-expressing eyes. RhoDN expression disrupts actin organization, pulsatile actomyosin dynamics, and cell shape changes (see text for details). Top: Snapshots of a lattice edge. Blue and green overlays highlight adjacent 3° and 2° LCs, respectively. Bottom: Zoomed-in images of the 2° LC (boxed area). The bracketed green zones show low medioapical F-actin during cell area expansion, while bracketed red zones show increased F-actin during cell area contraction. Note an interruption of junctional actomyosin (white arrow). (A′) The negative correlation between medioapical F-actin levels and cell area contraction is stronger in GMR > Rho1DN compared with WT (N = 18 in WT, N = 19 in GMR > Rho1DN from three eyes each, t test, P = 0.0013). (B and C) Rho1 overexpression has a dramatic effect on pulsatile medioapical F-actin dynamics and cell area fluctuations (Video 3). (B) Time series montage shows F-actin node fusion (tracked by the yellow arrows) during medioapical F-actin ring formation (red arrowhead). (B′) F-actin nodes are on average 40% larger in GMR > Rho1 compared with WT (N = 70 nodes in WT, N = 60 in GMR > Rho1 from three eyes each, t test P < 0.0001). (C) A time series montage of a cell overexpressing Rho1 through a cycle of cell expansion (red bar) and contraction (green bar). Low F-actin intensity within expanded cells (green arrows), high F-actin intensity within contracted cells (red arrow). (C′) Medioapical F-actin pulse frequency in 2° LCs is accelerated (245 ± 50 s, N = 16). (C″) F-actin levels increase on average 4.5-fold in 2° LCs (N = 14) during contraction compared with 1.4-fold in WT (Fig. 1 G). (C‴) Time-shifted correlation shows that medioapical F-actin levels correlate strongly with apical area contraction of 2° LCs. (D) The peak negative correlation between F-actin and cell area in Rho1-expressing eyes is significantly stronger than in WT (R = −0.6508 at peak correlation of −15 s, N = 15 from three eyes, t test P = 0.0002). (E) Contractile medioapical actomyosin dynamics and cell area fluctuations inversely coordinate between neighboring 2° cells prior to cell pruning (Video 4). When one cell has low-intensity F-actin and increasing area (green arrow), the neighboring cell has high-intensity of F-actin and decreasing area (red arrow). (E′) Cell area of neighboring 2° LCs is negatively correlated (R = −0.2020 at a peak correlation of +15 s, N = 14 [7 pairs] in three eyes, one-sample t test; P = 0.0318). (F) A schematic summarizing the reciprocal coordination of cytoskeletal and mechanical behavior between neighboring LCs. Scale bar = 3 µm.
