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

Figure 6. Movement of the centrosome correlates with targeted vesicular trafficking towards phagosomes.

(A) Microglia (magenta; Tg(csf1ra:GAL4-VP16; UAS:lyn-tagRFPt)) and Lamp1 labelled vesicles (green; Tg(UAS:Lamp1-mGFP)) localized within the cell soma and not moving into branches. (B) Time of Rab5 (n = 3, 8–10 phagocytic events analysed per microglia) and Lamp1 (n = 3, 9–11 phagocytic events analysed per microglia) appearance at phagosome, after its formation. Violin plots have mean and quartiles depicted. (C) Microglia (magenta; Tg(csf1ra:GAL4-VP16; UAS:lyn-tagRFPt)) and Rab5-positive endosomes (cyan; UAS:mNeonGreen-Rab5a), full time lapse in Figure 6—video 1. Endosomes form a cloud (arrowhead) that moves into phagocytic branches towards forming phagosomes (asterisk). Original images were deconvolved using Huygens deconvolution. (D) Representative microglia (magenta; Tg(csf1ra:GAL4-VP16; UAS:lyn-tagRFPt)), Rab5 endosomes (cyan; UAS:mNeonGreen-Rab5a), and centrosome (yellow; UAS:miRFP670-centrin4). The centrosome (circle) moves into phagocytic branches (star) and is followed by the cloud of Rab5 endosomes (arrowhead). Original images were deconvolved using Huygens deconvolution. n refers to the number of microglia examined.

Figure 6—source data 1. Related to Figure 6B.

Figure 6.

Figure 6—figure supplement 1. Vesicular markers do not affect microglia phagocytosis.

Figure 6—figure supplement 1.

(A) A single-image plane showing microglia (red) expressing Rab5 (green) on newly formed phagosomes (fms:Gal4-VP16; UAS:nfsB-mCherry; UAS:mNeonGreen-Rab5a). Size (B) and number (C) of phagosomes from same brain (N = 11) microglia, with (n = 62) or without (n = 29) Rab5 expression (1–18 phagosomes analysed per microglia). (D) A single-image plane showing microglia (red) expressing Lamp1 (green) on vesicles (fms:Gal4-VP16; UAS:nfsB-mCherry; UAS:Lamp1-mGFP). Size (E) and number (F) of phagosomes from same brain (N = 10) microglia, with (n = 17) or without (n = 48) Lamp1 expression (1–21 phagosomes analysed per microglia). N refers to the number of zebrafish and n to the number of microglia examined. Violin plots show mean and quartiles, boxplots depict mean +/- 95% confidence intervals. Groups were compared using a two-tailed, nonparametric Mann-Whitney U-test.
Figure 6—figure supplement 1—source data 1. Related to Figure 6—figure supplement 1B-C and E-F.
Figure 6—video 1. Rab5-positive endosomal cloud is highly dynamic and moves into phagocytic branches.
Download video file (2.1MB, mp4)
Time lapse of a representative microglia (grey; Tg(csf1ra:GAL4-VP16; UAS:lyn-tagRFPt)) and Rab5-positive endosomes (magma; UAS:mNeonGreen-Rab5a) showing how endosomes form a cloud that moves into phagocytic branches towards forming phagosomes. Original images were deconvolved using Huygens deconvolution. Images were captured every 15 s for 1 hr. Timescale is hh:mm:ss.