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. 2014 Mar 31;204(7):1209–1218. doi: 10.1083/jcb.201312098

Figure 5.

Figure 5.

UNC-60A promotes invadopodial membrane recycling through the endolysosome. (A) Focused laser photobleaching of an ∼1.0-µm region at the invasive membrane (circle) in a wild-type AC (top row) resulted in loss of the endolysosome LMP-1::GFP signal throughout the AC shortly after two and a half minutes (time points, minutes). Note, these images were captured on a laser-scanning microscope optimized for FLIP analysis, which has reduced sensitivity for LMP-1::GFP signal compared with the spinning-disk confocal image captured in Fig. 4 D (see Materials and methods). In unc-60a (RNAi) animals the LMP-1::GFP signal persisted ∼10 min after initiation of photobleaching. Graphs report LMP-1::GFP signal remaining in neighboring nonbleached invasive membrane over time (n ≥ 5 animals each genotype; P < 0.01 for 25%, and P < 0.05 for 50 and 75% signaling remaining; Student’s t test). (B) Time-lapse analysis of PI(4,5)P2 (GFP::PLCδPH) at the invasive cell membrane in a wild-type AC (top row) and unc-60a (RNAi) animals (bottom row) revealed reduced membrane dynamics after loss of unc-60a. Graphs report lifetimes of internal vesicles (n ≥ 35 vesicles each genotype; ***, P < 0.0001; Wilcoxon rank-sum test). Bars, 5 µm.