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. 2020 Feb 27;219(4):e201907018. doi: 10.1083/jcb.201907018

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© 2020 Deng et al.

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Figure 2. — Spectrin is required for attaching cortical F-actin to the cell membrane. (A–B’) A nonpermeabilized pupal eye disc containing RFP-positive MARCM clones with α-spec RNAi was imaged for utABD-GFP at the indicated apical-basal positions. Note normal cortical F-actin in α-spec mutant PECs. Quantification of utABD-GFP membrane intensity is shown in F. (C–D’) A Triton X-100–permeabilized pupal eye disc containing GFP-positive MARCM clones with α-spec RNAi was stained for phalloidin. C and C’ and D and D’ show z-projections around cell apical and lateral plane, respectively. Note the reduced cortical F-actin level in α-spec mutant PECs (arrowheads) in both apical and lateral membrane compared with wild-type PECs (arrows). Quantification of phalloidin membrane intensity is shown in G. (E–E”’) A Triton X-100–permeabilized pupal eye disc containing RFP-positive MARCM clones with α-spec RNAi and mCD8-GFP-utABD expression was imaged for mCD8-GFP-utABD and stained for phalloidin. Note the strong cortical F-actin staining in cell cortex where mCD8-GFP-utABD accumulated (arrowheads in E’ and E”). (F) Quantification of mean utABD-GFP intensity in the specified membrane domains analyzed in A–B’. Note no significant difference between α-spec mutant PECs and wild-type PECs. Data are means ± SEM (n ≥ 20 cells, representative of 10 pupal eyes); N.S., no significance (Student’s t test). (G) Quantification of mean phalloidin intensity in the specified membrane domains analyzed in C–D’. Note significant decrease of phalloidin membrane intensity in α-spec mutant PECs. Data are means ± SEM (n ≥ 20 cells, representative of 10 pupal eyes). ****, P < 0.0001. (H) Reduced cortical tension in spectrin mutant cells. Left: A nub-Gal4 UAS-EGFP driver showing EGFP expression in the wing pouch region in third instar wing discs. Right: Measurement of cortical tension by MPA. The Teff was measured for individual cells of the indicated genotype and plotted in the graph (means ± SEM, n = 15 for spectrin mutant cells, n = 12 for control cells). **, P < 0.01 (Student’s t test). Scale bars, 5 µm (A, C, and E) and 100 µm (H).