L cells were incubated simultaneously with Wg (green) and Cy3-conjugated transferrin (red) for 1 hour at 4°C (a-c) or 37°C (d-f). Arrow-heads indicate positions of Wg and transferrin co-localization.

Wnt-stimulated accumulation of β-catenin is dependent upon dynamin activity. β-catenin levels were measured by immunostaining in L cells incubated in the presence (c, d, g, h, k and l) or absence (a, b, e, f, i and j) of Wnt-3A (~100 ng/mL) for 3 hours. L cells were transfected with either GFP alone (a-d) or GFP in combination with wild-type dynamin (e-h) or K44E-mutated dynamin (i-l). Arrows indicate transfected cells and arrow-heads indicate neighboring non-transfected cells. (m) Quantitation of immunofluorescence results from (a-l), expressed as the percentage of GFP-positive cells which are positive for β-catenin staining. (n) Quantitation of immunostaining experiments measuring Wg-stimulated (data not shown) accumulation of β-catenin in L cells transfected with the same constructs as in (a-l). In (m) and (n), values represent means ± SEM from 3 independent determinations in which approximately 30–70 cells were examined per condition.

Wg internalizes by clathrin- and dynamin-mediated endocytosis, and localizes to transferrin recycling compartments. Immunostaining for Wg was performed in L cells incubated in the absence (a) or presence of Wg (b-l, n-s). Cells were incubated with Wg at 37°C (b) or 4°C (c), and in the presence of MDC (d), hypertonic sucrose (e), or CPZ (f). Internalized Wg was assessed by immunostaining. L cells transfected with GFP alone (g and j are identical fields; transfected cells indicated by arrows and non-transfected cells indicated by arrow-heads), or in combination with either wild-type dynamin (h and k are identical fields; transfected cells indicated by arrows and non-transfected cells indicated by arrow-heads) or K44E-mutated dynamin (i and l are identical fields; transfected cells indicated by arrows) were assessed for Wg endocytosis by immunostaining. (m) Quantitation of results in (g-l) where values represent means ± SEM from 3 independent determinations.

Wnt signaling is dependent upon clathrin-mediated endocytosis. (a) L cells were stimulated for 3 hours in the presence or absence of Wnt-3A (~100 ng/mL), control conditioned medium (Cont. CM), or Wg conditioned medium (Wg CM) and subsequently harvested and assayed for β-catenin and GSK3β levels by immunoblotting. β-catenin and GSK3β levels were similarly assessed in L cells stimulated with Wnt-3A over a 2-hour time-course in the presence or absence of MDC (300 μM) (b), hypertonic sucrose (0.45 M) (c), or CPZ (30 μM) (d). (e) LSL cells were stimulated for 5 hours in the presence or absence of Wnt3A with either DMSO (Vehicle), MDC, hypertonic sucrose, or CPZ and assayed for TOPFLASH reporter activity. Shown is a representative experiment from 3 independent determinations each performed in triplicate, in which values represent means ± SEM. (f) L cells were pre-incubated with or without DMSO Vehicle (Veh), MDC, hypertonic sucrose, or CPZ for 1 hour, followed by stimulation in the presence or absence of LiCl (50 mM) for 3 hours. Cells were subsequently harvested and assayed for β-catenin and GSK3β levels by immunoblotting. (g) SW480 cells were treated for 1 hour with Vehicle, MDC, hypertonic sucrose, or CPZ and were subsequently lysed under hypotonic conditions to enable measurement of cytosolic β-catenin and GSK3β levels.

SiRNA-mediated depletion of clathrin impairs Wnt3A-stimulated reporter gene expression. LSL cells were transfected in two rounds with either empty plasmid vector (Mock) or siRNA corresponding to clathrin heavy chain (siCHC). (a) Levels of clathrin heavy chain and GSK3β were assessed by immunoblotting. (b) Cells were stimulated with purified Wnt-3A (~100 ng/mL) for approximately 8 hours, followed by lysis of cells and measurement of TOPFLASH and LacZ activities. Values represent means ± SEM from a representative experiment from 8 independent determinations.