Fig. 5.

Biochemical and functional characterization of Lgr5-induced cytonemes. (A) VASP–GFP (green) or (B) fascin–GFP (green) were cotransfected into cells with 834del- (upper panel) or Lgr5–V2Rtail (Lgr5/V2Rtail, lower panel) HA-tagged receptors (antibody stained in red), and the cells were confocal imaged. The data are presented as single-channel images and merged images. VASP is present throughout the entire cytoneme but can concentrate at the tip (arrows). (C) Overnight treatment with 10 µM cytochalasin B ablates 834del-induced cytonemes (inset). 834del-expressing cells were treated with 10 µM cytochalasin B and imaged every 30 minutes for 5 hours (see supplementary material Movie 2 for full timecourse). The asterisk denotes a cytoneme that remains after treatment; the dotted region demonstrates consolidation and ablation of a cluster of cytonemes; the arrowhead depicts rapid loss of shorter cytonemes. (D) 834del-expressing cells were treated with 10 µM cytochalasin B overnight, washed five times and then imaged every 30 minutes for 2.5 hours (see supplementary material Movie 3 for full timecourse). The arrow denotes rapid appearance of cytonemes from an existing lamellipodial-like framework. Scale bar: 10 µm.