Supplementary Figure 9: Stac vesicles show features of Secretory Lysosomes.
(a) Stills from time-lapse movie of DB FCs expressing GFP-Lamp1 (green) and mCherry-StacA (magenta). mCherry-StacA signals colocalise partially with Lamp1-GFP. Images are representative of six embryos and two experiments. (b) Live imaging of stage 16 embryos expressing either GFP-Lamp1 (green, upper panels) or EGFP-StacA (green, lower panels) in all tracheal cells. Embryos were injected with Lysotracker-TexasRed (magenta) to label acidic organelles. Most of the GFP-Lamp1-marked structures in FCs contain Lysotracker, with some structures showing low-intensity signals (red asterisks). EGFP-StacA compartments show either no (red asterisk on right FC) or weak Lysotracker staining (red asterisks on left FC). Images are representative of at least 10 embryos for each condition. (c) Stills from time-lapse movie of DB FCs expressing EGFP-StacA (green) and LifeAct-Ruby (magenta) to visualize F-actin. EGFP-StacA vesicles are visible close to the LifeAct-Ruby-marked apical cortex and F-actin track, and are associated with the F-actin track at later stages (highlighted in close-up views). Images are representative of six embryos. (d) Tracheal Expression of Dynamitin (Dmn) or of dominant negative p150-Glued (Gl[Δ84]) cause similarly penetrant fusion defects (Dmn, N = 15 embryos, P = 0.007;Gl[Δ84], N = 15 embryos, P = 0,001; control, N = 12 embryos; Student’s t-tests and KS-tests). Cumulative bar graph shows mean values; numbers of fusion positions scored for each genotype are indicated inside bars. Fusion defects fall into three categories (panels below graph): lumen defects (partner FCs established contact, but lumen remains discontinuous), breaks (partner FCs are separated) and misguided branches (FCs of non-cognate branches engage in ectopic fusion; open arrowhead). White arrowhead indicates FC-FC interface. Frequencies of each class of defect (percent of total number of defects) are indicated to the right of each bar. Note that expression of Dmn and Gl[Δ84] leads to a high incidence of specific lumen defects (46.7 and 60% of the total number of defects, respectively), consistent with effects on membrane trafficking. However, the percentage of specific lumen defects in stac3B20 embryos is higher (75.3%), indicating that interfering with Dynein motor activity may also have more general effects, possibly impacting on the motility of tracheal cells. Statistics source data are provided in Supplementary Table 3. (e) Live imaging of DB FCs expressing EGFP-StacA alone as a control (top) or in combination with Dmn (bottom). Maximum intensity projections of 46 time points are shown. In the control, EGFP-StacA vesicles cluster along the cytoskeletal track region (dashed red lines) and near the FC-FC interface (arrowhead) in each FC. In contrast, upon Dmn expression, EGFP-StacA vesicles are not tightly clustered and EGFP-StacA puncta (encircled by white dashed lines) are found distant from the central track region. Images are representative of >10 time-lapse movies from two experiments. Scale bars, 5 μm.
