Figure 5.

Expression of lrrk^GS drives perinuclear positioning and clustering of lysosomes. (A–C) Lysotracker staining of follicle cells from stage 12 egg chambers. In wild-type follicle cells (A), most Lysotracker-positive vesicles are individual and distributed throughout the cytosol, although small clusters are often seen. Expression of lrrk^GS (B) in follicle cells causes almost all Lysotracker-positive structures to collapse into one to four compact perinuclear clusters, while equivalent overexpression of lrrk^WT (C) results in no significant changes in the distribution of Lysotracker-positive structures relative to the wild-type. (D) Quantification of lysosome clustering from the genotypes depicted in (A)–(C). (E and F) Lysotracker staining of wild-type (E) and lrrk^GS-expressing (F) follicle cells also expressing the lysosomal marker Lamp1:GFP. Lamp1:GFP-positive vesicles are clustered in lrrk^GS-expressing cells (F′), colocalizing with Lysotracker (F″). Note that Lamp1:GFP accumulates to a much higher degree in lrrk^GS-expressing cells (F′) versus wild-type (E′) in images taken with equivalent microscope settings, suggesting that expression of lrrk^GS stabilizes Lamp1:GFP. (G–J) Labeling of early endosomes with the marker Hrs (G and H) and late endosomes with the marker Rab7 (I and J) reveals no significant difference between wild-type (G and I) and lrrk^GS-expressing cells (H and J), demonstrating that the clustering effect of lrrk^GS is specific to lysosomes. Follicle cell nuclei are outlined with dashed gray lines in all images. Scale bar in (A) represents 5 μm in (A)–(F); scale bar in (G) represents 5 μm in (G)–(J).