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. 2022 Nov 18;11:e82094. doi: 10.7554/eLife.82094

Figure 3. Microglia without dynamic microtubules adopt a different engulfment strategy.

(A) Schematic showing the experimental setup: Embryos were treated for 1.5 hr with PST-1, then imaged for 1 hr with 561 nm light (PRE-PST-1) and then imaged for another hour with 561 nm and 405 nm light to activate PST-1 (PST-1 ON). (B) Left: microglia (grey; Tg(mpeg1:Gal4; UAS:lyn-tagRFPt)) before (upper) and after (lower) PST-1 activation. Right: 3D segmentation of the cells in the left panel. (C) Microglia sphericity before and 5 min after PST-1 activation. Segmentation in (B) was used to extract surface area and volume to calculate sphericity (N = 4, 6–15 microglia analysed per fish, ***p=0.0005). (D) 3D tracking of microglia for 1 hr before (left) and after (right) PST-1 activation, displayed as colour-coded tracks overlayed on top of microscopy images of the microglia. Full time lapse is found in Figure 3—video 1. (E) Microglia migration speed measured in μm/s (N = 4, 6–18 microglia analysed per fish, ****p<0.0001). (F) Upper: schematic of the ON-OFF experiment. Lower: microglia (grey; Tg(mpeg1:Gal4; UAS:lyn-tagRFPt)) shown before PST-1 activation (PRE-PST-1) and when the drug is turned ON with 405 nm light (PST-1-ON) and OFF again using 488 nm light (PST-1-OFF). A paired, non-parametric Friedman test with Dunn’s correction was used to compare the three groups (***p=0.0002). (G) Microglia migration speed during the ON-OFF experiment, measured in μm/s (N = 1, n = 14). (H) Microglia engulfment rate after DMSO (N = 3, 5–8 microglia analysed per fish) or PST-1 treatment (N = 3, 6 microglia analysed per fish). (I) Percentage of branch-mediated (BM) engulfments before (left) and after (right) activation of PST-1 (N = 3, 6 microglia analysed per fish). (J) Left: microglia migration persistence measured with the mean square displacement alpha (MSDα) before and after PST-1 activation (N = 4, 6–18 microglia analysed per fish, ***p=0.0001) Right: schematic of what the MSDα values imply. (K) Representative time lapse of a microglia (grey) treated with activated PST-1 and tracked (magenta), showing how microglia migrate towards apoptotic targets (yellow asterisk). Boxplots depict mean +/- min to max values. Groups were compared using a two-tailed, nonparametric Mann-Whitney U-test was used (C, E, H, J). When comparing reapeated measurements from the same group, a nonparametric Friedmann test with a Dunn's correction was used (G). N refers to the number of zebrafish and n to the number of microglia examined.

Figure 3—source data 1. Related to Figure 3C.
Figure 3—source data 2. Related to Figure 3E.
Figure 3—source data 3. Related to Figure 3G.
Figure 3—source data 4. Related to Figure 3H and I.

Figure 3.

Figure 3—figure supplement 1. Validation of PST-1 functionality in vivo.

Figure 3—figure supplement 1.

Zebrafish embryo’s injected with EB3-mScarlett-I mRNA, treated with 1%. DMSO (top panel) or 1 mM PST-1 (bottom panel) at two different developmental stages. Growing tips of microtubules are visualized as grey comets. Left panel: 4-hr-post-fertilization (hpf) embryos before and after 405 nm light illumination. Right panel: 24-hpf embryos before and after 405 nm light activation. Images were captured using spinning-disc microscopy.
Figure 3—figure supplement 2. Nocodazole and PST-1-treated microglia behave similarly.

Figure 3—figure supplement 2.

(A) Microglia (grey; mpeg1:Gal4; UAS:lyn-tagRFPt) treated with DMSO (left) or 10 mM nocodazole for 2 hr. Engulfment rate (B) and mode (C) of microglia treated with either DMSO (N = 2, n = 7) or nocodazole (N = 1, n = 9). Boxplots depict mean +/- min to max values. Groups were compared using a two-tailed, nonparametric Mann-Whitney U-test. N refers to the number of zebrafish and n to the number of microglia examined.
Figure 3—figure supplement 2—source data 1. Related to Figure 3—figure supplement 2B and C.
Figure 3—video 1. Microglia without dynamic microtubules become more mobile.
Download video file (1MB, mp4)
Time lapse of a representative zebrafish brain showing microglia (grey; mpeg1:Gal4; UAS:lyn-tagRFPt) treated with PST-1 before (left; Pre-PST-1) or after (right; PST-1-ON) activation with 405 nm light. 3D tracking of microglia is displayed as colour-coded tracks overlayed on top of the microscopy images. Images were captured every minute for 2 hr total using spinning-disc microscopy. Timescale is hh:mm.