Figure 3.
Small-molecule inhibitors of the actin cytoskeleton alter Naegleria morphology.(A) Small-molecule inhibitors target actin dynamics. (B) Cells were incubated in media ± inhibitors for 10 min, then fixed and stained with phalloidin to detect F-actin. Total fluorescence was measured using flow cytometry. One representative histogram (left) is shown from a single replicate comparing LatB with its vehicle control (also see Fig. S3). Average F-actin intensities (right) were normalized to the stained control for all conditions. Each point represents the average normalized fluorescence of 20,000 cells from one experimental replicate, with each experimental replicate represented by a different shape. (C) Cells were treated as in B, but were fixed and stained for microscopy using DAPI to label DNA in addition to phalloidin. Cells were analyzed using SIM and widefield fluorescence (Fig. S3). Maximum intensity projections and single Z planes from SIM are shown for representative cells, and filopodia on a SMIFH2-treated cell are magnified in the inset (scale bar, 1 µm). (D–H) 50 cells/condition (imaged using widefield fluorescence) for three independent replicates (150 cells total/treatment) were used to quantify cell area (D) and circularity (E) or the percentage of cells with ≥1 ruffle (F), ≥1 filopodium (G), and ≥10 puncta (H). Small gray symbols represent individual cells, and larger symbols represent the averages for experimental replicates (coordinated by shape). For graphs in B (right) and D–H, black lines represent the means from experimental replicates ± SD, and statistical significance was determined using an ordinary ANOVA and Tukey’s multiple comparison test. Dashed lines indicate the control value. (I) Pixel intensity line scans for F-actin staining were drawn in a single Z-plane bisecting the cell edge, and values were normalized to the average intensity inside the cell, which was set to 1. Curves represent the average relative intensity ± SD for three experimental replicates, each encompassing five cells (also see Fig. S7). AU, arbitrary units; Max, maximum; RFU, relative fluorescence units; SM+CK, SMIFH2+CK-666 combined treatment.

Small-molecule inhibitors of the actin cytoskeleton alter Naegleria morphology. (A) Small-molecule inhibitors target actin dynamics. (B) Cells were incubated in media ± inhibitors for 10 min, then fixed and stained with phalloidin to detect F-actin. Total fluorescence was measured using flow cytometry. One representative histogram (left) is shown from a single replicate comparing LatB with its vehicle control (also see Fig. S3). Average F-actin intensities (right) were normalized to the stained control for all conditions. Each point represents the average normalized fluorescence of 20,000 cells from one experimental replicate, with each experimental replicate represented by a different shape. (C) Cells were treated as in B, but were fixed and stained for microscopy using DAPI to label DNA in addition to phalloidin. Cells were analyzed using SIM and widefield fluorescence (Fig. S3). Maximum intensity projections and single Z planes from SIM are shown for representative cells, and filopodia on a SMIFH2-treated cell are magnified in the inset (scale bar, 1 µm). (D–H) 50 cells/condition (imaged using widefield fluorescence) for three independent replicates (150 cells total/treatment) were used to quantify cell area (D) and circularity (E) or the percentage of cells with ≥1 ruffle (F), ≥1 filopodium (G), and ≥10 puncta (H). Small gray symbols represent individual cells, and larger symbols represent the averages for experimental replicates (coordinated by shape). For graphs in B (right) and D–H, black lines represent the means from experimental replicates ± SD, and statistical significance was determined using an ordinary ANOVA and Tukey’s multiple comparison test. Dashed lines indicate the control value. (I) Pixel intensity line scans for F-actin staining were drawn in a single Z-plane bisecting the cell edge, and values were normalized to the average intensity inside the cell, which was set to 1. Curves represent the average relative intensity ± SD for three experimental replicates, each encompassing five cells (also see Fig. S7). AU, arbitrary units; Max, maximum; RFU, relative fluorescence units; SM+CK, SMIFH2+CK-666 combined treatment.

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