Figure 5.
Abi acts through WASp to regulate CME and crystal cell development. (A) Single confocal slices of S2 cells pretreated with 100 μM dynasore for 30 min prior to immunofluorescence analysis using anti-Abi (green) and anti-Chc (cyan) antibodies together with an anti-WASp or anti-SCAR (pseudocolored magenta) antibody. Arrowheads indicate Abi+ punctae colocalizing with WASp and Chc. Arrows indicate Abi+ punctae colocalizing with SCAR but not Chc. (B) Single confocal slices of primary hemocytes from WT, abi5/Df, UAS-HA-abiΔ30–65/+; HmlΔ-GAL4,abi5/+,Df (abi5/Df, HmlΔ > abiΔ30–65), HmlΔ-GAL4,abi5/UAS-HA-abiW452K,Df (abi5/Df, HmlΔ > abiW452K), HmlΔ-GAL4/+, HmlΔ-GAL4/UAS-SCARRNAi (HmlΔ > SCARRNAi), and HmlΔ-GAL4/UAS-WASpRNAi (HmlΔ > WASpRNAi) late-third instar larvae. Hemocytes were pulsed with Alexa Fluor 555-mBSA (red) for 5 min, chased for 5 min, and stained with DAPI (blue). (C) Quantification of the ratio of mean Alexa Fluor 555-mBSA to DAPI fluorescence intensities. HmlΔ-GAL4,abi5/UAS-HA-abi,Df (abi5/Df, HmlΔ > abi) was also analyzed. Values are presented as percentages of WT or HmlΔ-GAL4/+. n = 30 hemocytes. (D) Bright-field images of heated (70°C, 10 min) late-third instar larvae of the indicated genotypes. Dorsal views of the two most posterior segments (A7 and A8). (E) Number of heat-blackened crystal cells in the A7 and A8 segments. n = 12 larvae. (F–I) Transheterozygous interactions between abi and WASp. (F) Single confocal slices of primary hemocytes from late-third instar larvae of the indicated genotypes. Hemocytes were pulsed with Alexa Fluor 555-mBSA for 5 min and chased for 5 min. (G) Quantification of the ratio of mean Alexa Fluor 555-mBSA to DAPI fluorescence intensities. Values are presented as percentages of WT. n = 30 hemocytes. (H) Bright-field images of heated (70°C, 10 min) late-third instar larvae of the indicated genotypes. (I) Number of heat-blackened crystal cells in the A7 and A8 segments. n = 12 larvae. Data represent the mean ± SEM. Comparisons are with WT or HmlΔ-GAL4/+ (***P < 0.001). Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test. The dashed lines define cell boundaries. Scale bars: 10 μm (A); 5 μm (B and F); 200 μm (D and H).

Abi acts through WASp to regulate CME and crystal cell development. (A) Single confocal slices of S2 cells pretreated with 100 μM dynasore for 30 min prior to immunofluorescence analysis using anti-Abi (green) and anti-Chc (cyan) antibodies together with an anti-WASp or anti-SCAR (pseudocolored magenta) antibody. Arrowheads indicate Abi+ punctae colocalizing with WASp and Chc. Arrows indicate Abi+ punctae colocalizing with SCAR but not Chc. (B) Single confocal slices of primary hemocytes from WT, abi5/Df, UAS-HA-abiΔ30–65/+; HmlΔ-GAL4,abi5/+,Df (abi5/Df, HmlΔ > abiΔ30–65), HmlΔ-GAL4,abi5/UAS-HA-abiW452K,Df (abi5/Df, HmlΔ > abiW452K), HmlΔ-GAL4/+, HmlΔ-GAL4/UAS-SCARRNAi (HmlΔ > SCARRNAi), and HmlΔ-GAL4/UAS-WASpRNAi (HmlΔ > WASpRNAi) late-third instar larvae. Hemocytes were pulsed with Alexa Fluor 555-mBSA (red) for 5 min, chased for 5 min, and stained with DAPI (blue). (C) Quantification of the ratio of mean Alexa Fluor 555-mBSA to DAPI fluorescence intensities. HmlΔ-GAL4,abi5/UAS-HA-abi,Df (abi5/Df, HmlΔ > abi) was also analyzed. Values are presented as percentages of WT or HmlΔ-GAL4/+. n = 30 hemocytes. (D) Bright-field images of heated (70°C, 10 min) late-third instar larvae of the indicated genotypes. Dorsal views of the two most posterior segments (A7 and A8). (E) Number of heat-blackened crystal cells in the A7 and A8 segments. n = 12 larvae. (F–I) Transheterozygous interactions between abi and WASp. (F) Single confocal slices of primary hemocytes from late-third instar larvae of the indicated genotypes. Hemocytes were pulsed with Alexa Fluor 555-mBSA for 5 min and chased for 5 min. (G) Quantification of the ratio of mean Alexa Fluor 555-mBSA to DAPI fluorescence intensities. Values are presented as percentages of WT. n = 30 hemocytes. (H) Bright-field images of heated (70°C, 10 min) late-third instar larvae of the indicated genotypes. (I) Number of heat-blackened crystal cells in the A7 and A8 segments. n = 12 larvae. Data represent the mean ± SEM. Comparisons are with WT or HmlΔ-GAL4/+ (***P < 0.001). Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test. The dashed lines define cell boundaries. Scale bars: 10 μm (A); 5 μm (B and F); 200 μm (D and H).

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