Figure 6.
RhoGAP71E promotes the disassembly of medioapical actomyosin and apical area expansion. (A–A‴) RhoGAP71E::GFP accumulates medioapically in pulses. (A) Snapshots from a time-lapse movie of an ommatidium. (A′) Lattice edge and (A″) magnified views of a 2° LC demarcated with a box. Note the punctate enrichment of RhoGAP71E at 4 min (yellow arrow) compared with low enrichment at 10 min (blue arrow). (A‴) Medioapical RhoGAP71E::GFP levels negatively correlate with apical cell area of 2° LCs at a time shift of +10 s (R = −0.1399, N = 11 from three eyes, one-sample t test P = 0.0328). (B–D) RhoGAP71E promotes apical cell area expansion. (B)RhoGAP71E mutant cells positively marked by GFP in fixed tissue stained for E-cadherin. White arrow, WT LC; yellow arrow, mutant LC; white arrowhead, WT 1° cell; yellow arrowhead, mutant 1° cell. (B′) The apical area of the RhoGAP71E mutant cells is significantly smaller than that of WT counterparts (connected lines, paired t test, P = 0.0007, N = 10 pairs from four eyes). (C) RhoGAP71E RNAi expression increases MyoII phosphorylation. (C′) p-MyoII levels are higher in RNAi-expressing clones marked with RFP compared with (C″) WT regions. (C‴)T test, P < 0.0001, N = 6 WT and RNAi-expressing clones from three eyes. (D) RhoGAP71E RNAi expression decreases apical cell area and strengthens the negative correlation between MyoII accumulation and cell area contraction (Video 7). Left: Snapshot from a time-lapse movie of MyoII (Sqh::GFP) in eyes with RhoGAP71E RNAi-expressing clones (RFP+). (D′ and D″) Time series montage of zoomed-in views of (D′) a RhoGAP71E RNAi-expressing 2° LC compared with (D″) a WT counterpart. (D‴) Left: Peak correlation (at −15 s) between MyoII accumulation and apical area contraction is higher in RhoGAP71E RNAi-expressing cells (light brown) compared with WT cells (green). Middle: MyoII correlation with cell area contraction is significantly higher in RhoGEF2RNAi-expressing cells than in their WT counterparts (paired t test, P = 0.0127, N = 10 pairs from three eyes). Right: RhoGAP71E RNAi expression decreased the time between pulses of MyoII accumulation to ∼3 min (212 ± 69 s) compared with ∼6.5 min in WT (387 ± 177 s, t test, P = 0.0109, N = 7 from three eyes. (E and F) Compared to (E) WT, (F) RhoGAP71E overexpression causes expansion of the cells’ apical area (yellow arrow), and gaps in the continuity of the AJs (yellow arrowheads) in highly expanded cells (asterisk) (Video 7). (F′) RhoGAP71E expression significantly increased the apical cell area of 2° LCs compared with WT (N = 14 from three eyes, t test, P < 0.0001). (G) RhoGAP71E overexpression reduces medioapical F-actin levels. Top: Snapshots of a lattice edge. Bottom: A magnified view of the boxed 2° LC. Lower levels of actomyosin accumulation correlate with shallower fluctuations in the apical cell area. (G′) Left: RhoGAP71E overexpression abolishes the correlation between actomyosin accumulation and cell area contraction. Right: Comparison of R-values at a time shift of −15 s between RhoGAP71E-expressing LCs and WT LCs (t test, P = 0.0186, N = 12 from three eyes). Scale bar = 3 µm.

RhoGAP71E promotes the disassembly of medioapical actomyosin and apical area expansion. (A–A‴) RhoGAP71E::GFP accumulates medioapically in pulses. (A) Snapshots from a time-lapse movie of an ommatidium. (A′) Lattice edge and (A″) magnified views of a 2° LC demarcated with a box. Note the punctate enrichment of RhoGAP71E at 4 min (yellow arrow) compared with low enrichment at 10 min (blue arrow). (A‴) Medioapical RhoGAP71E::GFP levels negatively correlate with apical cell area of 2° LCs at a time shift of +10 s (R = −0.1399, N = 11 from three eyes, one-sample t test P = 0.0328). (B–D) RhoGAP71E promotes apical cell area expansion. (B)RhoGAP71E mutant cells positively marked by GFP in fixed tissue stained for E-cadherin. White arrow, WT LC; yellow arrow, mutant LC; white arrowhead, WT 1° cell; yellow arrowhead, mutant 1° cell. (B′) The apical area of the RhoGAP71E mutant cells is significantly smaller than that of WT counterparts (connected lines, paired t test, P = 0.0007, N = 10 pairs from four eyes). (C) RhoGAP71E RNAi expression increases MyoII phosphorylation. (C′) p-MyoII levels are higher in RNAi-expressing clones marked with RFP compared with (C″) WT regions. (C‴)T test, P < 0.0001, N = 6 WT and RNAi-expressing clones from three eyes. (D) RhoGAP71E RNAi expression decreases apical cell area and strengthens the negative correlation between MyoII accumulation and cell area contraction (Video 7). Left: Snapshot from a time-lapse movie of MyoII (Sqh::GFP) in eyes with RhoGAP71E RNAi-expressing clones (RFP+). (D′ and D″) Time series montage of zoomed-in views of (D′) a RhoGAP71E RNAi-expressing 2° LC compared with (D″) a WT counterpart. (D‴) Left: Peak correlation (at −15 s) between MyoII accumulation and apical area contraction is higher in RhoGAP71E RNAi-expressing cells (light brown) compared with WT cells (green). Middle: MyoII correlation with cell area contraction is significantly higher in RhoGEF2RNAi-expressing cells than in their WT counterparts (paired t test, P = 0.0127, N = 10 pairs from three eyes). Right: RhoGAP71E RNAi expression decreased the time between pulses of MyoII accumulation to ∼3 min (212 ± 69 s) compared with ∼6.5 min in WT (387 ± 177 s, t test, P = 0.0109, N = 7 from three eyes. (E and F) Compared to (E) WT, (F) RhoGAP71E overexpression causes expansion of the cells’ apical area (yellow arrow), and gaps in the continuity of the AJs (yellow arrowheads) in highly expanded cells (asterisk) (Video 7). (F′) RhoGAP71E expression significantly increased the apical cell area of 2° LCs compared with WT (N = 14 from three eyes, t test, P < 0.0001). (G) RhoGAP71E overexpression reduces medioapical F-actin levels. Top: Snapshots of a lattice edge. Bottom: A magnified view of the boxed 2° LC. Lower levels of actomyosin accumulation correlate with shallower fluctuations in the apical cell area. (G′) Left: RhoGAP71E overexpression abolishes the correlation between actomyosin accumulation and cell area contraction. Right: Comparison of R-values at a time shift of −15 s between RhoGAP71E-expressing LCs and WT LCs (t test, P = 0.0186, N = 12 from three eyes). Scale bar = 3 µm.

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