Figure 4.
Inhibition of actin nucleation pathways impairs Naegleria cell crawling.(A) Crawling cells were imaged using phase/contrast microscopy for 5 min. After 5 min (at t = 0 min), an equal volume of each of the indicated inhibitors or controls was added (diluted in buffer; “control” indicates buffer alone), and imaged for 5 min. Arrowheads indicate the position of a representative cell over time. (B) Cells were treated as in A. 20 randomly selected cells from the center of the field of view at t = 0 were tracked to calculate average speeds before and after treatment. Each point represents the speed of a cell before treatment (x axis) and after treatment (y axis). The dashed line indicates no change. Three experimental replicates are shown using different shapes. Two data points are off the x axis: LatB (79.3, 12.9) and CK-666 (60.8, 23.6). (C) The data collected in B were used to calculate the change in cell speed after treatment (n = 3, 20 cells/trial, 60 cells total/condition). The dashed line is set to zero. (D) Data from B were plotted for 20 cells from one representative experiment. Tracks before treatment (pre-treat; gray) have the −5 min time point at the origin, while tracks after treatment (in color) have the t = 0 time point at the origin (also see Fig. S8). (E) Directional persistence was calculated for each post-treatment cell tracked in B by dividing the maximum displacement from the start by the path length. The dashed line indicates the average from the control. For graphs in C and E, small gray symbols represent the values of single cells, and larger symbols represent experimental averages. Black lines indicate the mean ± SD calculated from three experimental replicates, and statistical significance was determined using an ordinary ANOVA and Tukey’s multiple comparison test. (F) Cells were imaged using differential interference contrast microscopy and treated with CK-666 during imaging. Panels show time lapse images of a representative cell generating a filopodium-like protrusion ∼30 min after treatment. (G) Additional cells treated and imaged as in F were fixed and stained on the microscope after 2 min and 18 s of treatment (top: cell 2, short incubation time) or 53 min (bottom: cell 3, long incubation time). Cells were simultaneously fixed, permeabilized, and stained with DAPI to detect DNA (magenta) and phalloidin to detect F-actin (green). Times after treatment for F and G are in minutes:seconds.

Inhibition of actin nucleation pathways impairs Naegleria cell crawling. (A) Crawling cells were imaged using phase/contrast microscopy for 5 min. After 5 min (at t = 0 min), an equal volume of each of the indicated inhibitors or controls was added (diluted in buffer; “control” indicates buffer alone), and imaged for 5 min. Arrowheads indicate the position of a representative cell over time. (B) Cells were treated as in A. 20 randomly selected cells from the center of the field of view at t = 0 were tracked to calculate average speeds before and after treatment. Each point represents the speed of a cell before treatment (x axis) and after treatment (y axis). The dashed line indicates no change. Three experimental replicates are shown using different shapes. Two data points are off the x axis: LatB (79.3, 12.9) and CK-666 (60.8, 23.6). (C) The data collected in B were used to calculate the change in cell speed after treatment (n = 3, 20 cells/trial, 60 cells total/condition). The dashed line is set to zero. (D) Data from B were plotted for 20 cells from one representative experiment. Tracks before treatment (pre-treat; gray) have the −5 min time point at the origin, while tracks after treatment (in color) have the t = 0 time point at the origin (also see Fig. S8). (E) Directional persistence was calculated for each post-treatment cell tracked in B by dividing the maximum displacement from the start by the path length. The dashed line indicates the average from the control. For graphs in C and E, small gray symbols represent the values of single cells, and larger symbols represent experimental averages. Black lines indicate the mean ± SD calculated from three experimental replicates, and statistical significance was determined using an ordinary ANOVA and Tukey’s multiple comparison test. (F) Cells were imaged using differential interference contrast microscopy and treated with CK-666 during imaging. Panels show time lapse images of a representative cell generating a filopodium-like protrusion ∼30 min after treatment. (G) Additional cells treated and imaged as in F were fixed and stained on the microscope after 2 min and 18 s of treatment (top: cell 2, short incubation time) or 53 min (bottom: cell 3, long incubation time). Cells were simultaneously fixed, permeabilized, and stained with DAPI to detect DNA (magenta) and phalloidin to detect F-actin (green). Times after treatment for F and G are in minutes:seconds.

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