Membrane capacitance recordings resolve dynamics and complexity of receptor-mediated endocytosis in Wnt signalling

Abstract

Receptor-mediated endocytosis is an essential process in signalling pathways for activation of intracellular signalling cascades. One example is the Wnt signalling pathway that seems to depend on endocytosis of the ligand-receptor complex for initiation of Wnt signal transduction. To date, the roles of different endocytic pathways in Wnt signalling, molecular players and the kinetics of the process remain unclear. Here, we monitored endocytosis in Wnt3a and Wnt5a-mediated signalling with membrane capacitance recordings of HEK293 cells. Our measurements revealed a swift and substantial increase in the number of endocytic vesicles. Extracellular Wnt ligands specifically triggered endocytotic activity, which started immediately upon ligand binding and ceased within a period of ten minutes. By using specific inhibitors, we were able to separate Wnt-induced endocytosis into two independent pathways. We demonstrate that canonical Wnt3a is taken up mainly by clathrin-independent endocytosis whereas noncanonical Wnt5a is exclusively regulated via clathrin-mediated endocytosis. Our findings show that membrane capacitance recordings allow the resolution of complex cellular processes in plasma membrane signalling pathways in great detail.

Figure 1

(A) Fluorescent image of FM 4-64 endocytosis in control or Wnt-treated HEK293 cells. Cells were incubated for 1 minute with 10 µM of the FM 4-64 dye before addition of 5 ng/ml of the Wnt3a or Wnt5a ligands. Scale bar = 10 µm. (B) Quantification of the effect of canonical Wnt3a and noncanonical Wnt5a on the uptake of FM4-64. The relative fluorescent intensity is given as the ratio of intracellular fluorescence to whole-cell fluorescence. In control cells, the relative fluorescent intensity increased over time and shows the steady state uptake of FM4-64. Both ligands were independently tested against the control and found to be significantly higher (Student’s t-test, P < 0.005). Number of cells: control (n = 20), Wnt3a (n = 19) Wnt5a (n = 18). (C) Representative whole-cell capacitance recordings of control and Wnt-treated HEK293 cells. Arrow marks the time point of addition of the Wnt protein. Ga and Cm: imaginary and real part of admittance, corresponding to changes in membrane conductance (Ga lower trace) and capacitance (Cm upper trace). (D) Quantification of the effect of the Wnt ligands in whole-cell capacitance measurements. Number of measured cells: control (n = 9), Wnt3a (n = 9) Wnt5a (n = 10).

Figure 2

(A–D) Single spontaneous events (i.e. exocytosis, endocytosis and transients) in control HEK293 cells (imaginary and real part of admittance, corresponding to changes in membrane conductance (ΔG lower trace) and capacitance (ΔC upper trace)). (E) Number of permanent and transient events per patch, number of measured cells (n = 82), Each patch was measured on a separate cell and the first 500 s were used for the evaluation recording time in total.

Figure 3

(A) Enhanced rate of endocytosis in Wnt-treated HEK293 cells. Representative capacitance recordings of Wnt3a and Wnt5a-treated HEK293 cells in the cell-attached mode showing successive downward steps corresponding to permanent endocytosis. Imaginary and real part of admittance, corresponding to changes in membrane conductance (ΔG upper trace) and capacitance (ΔC lower trace) for control, Wnt3a treated and Wnt5a treated HEK293 cells. (B) Number of permanent and transient events per patch in control (n = 82), Wnt3a (n = 37) and Wnt5a-treated (n = 41) HEK293 cells. Each patch was measured on a separate cell and the first 500 s were used for the evaluation.

Figure 4

(A) Wnt-dependent size of endocytic vesicles. Left axis shows the geometrical mean (fF) and right axis the corresponding diameter (nm) of control, Wnt3a- and Wnt5a-treated patches. Endocytic vesicles stimulated by Wnt3a and Wnt5a are significantly smaller in vesicle size from the vesicles recorded in control cells (Error bars correspond to geometrical standard deviation for capacitance). Both ligands were independently tested against the control and against one another and found to be significantly different (Student’s t-test, ***P < 0.0005, **P < 0.005, *P < 0.05). Wnt3a and Wnt5a are also significantly different from each other**. (B) Distribution of different vesicle size intervals for both Wnt proteins and control condition. (C) Dose dependent increase of endocytic events per patch upon addition of different concentrations of Wnt3a and Wnt5a and simultaneous application of Wnt3a and Wnt5a. Each patch was measured on a separate cell and the first 500 s were used for the evaluation. Number of measurements: control (0 ng/ml; n = 82), Wnt3a (5 ng/ml; n = 37), Wnt5a (5 ng/ml; n = 41), Wnt3a (10 ng/ml; n = 6), Wnt3a (25 ng/ml; n = 3), Wnt5a (10 ng/ml; n = 8), Wnt5a (25 ng/ml; n = 5), Wnt3a + Wnt5a (2.5 ng/ml each; n = 19), Wnt3a + Wnt5a (5 ng/ml each; n = 4) and Wnt3a + Wnt5a (12.5 ng/ml each; n = 3). Under control conditions, 46 out of 82 measurements showed endocytotic events. In recordings with Wnt3a or Wnt5a in the pipette, we could detect endocytotic events in every measurement. (D) Temporal resolution of permanent endocytic events over 20 minutes and an exponential fit with tau 5.4 for Wnt3a and tau 7.4 for Wnt5a.

Figure 5

(A) Inhibition of Wnt-induced endocytosis by inhibitors of clathrin-dependent (Monodansylcadaverin MDC and Chlorprozamine CP) and clathrin-independent endocytosis (Genistein GE and Nystatin Nys). Number of permanent endocytic events per patch in control, Wnt3a and Wnt5a-treated HEK293 cells with the different inhibitors. Wnt3a cannot be blocked by MDC and CP, inhibitors of clathrin-dependent endocytosis, but is blocked by GE and Nys, inhibitors of clathrin-independent endocytosis. Wnt5a-induced endocytosis can only be blocked by MDC and CP, inhibitors of clathrin-dependent endocytosis. Dynasore (Dyn) blocked Wnt3a and Wnt5a endocytosis. Each patch was measured on a separate cell and the first 500 s were used for the evaluation. Measurement in total were: control (n = 82, Wnt3a (5 ng/ml; n = 37), Wnt5a (5 ng/ml; n = 41), Inactive Wnt3a (5 ng/ml; n = 8), Inactive Wnt5a (5 ng/ml; n = 7), Wnt3a + MDC (n = 9), Wnt5a + MDC (n = 9), Wnt3a + CP (n = 8), Wnt5a + CP (n = 14), Wnt3a + GE (n = 11), Wnt5a + GE (n = 12), Wnt3a + Nys (n = 13), Wnt5a + Nys (n = 11), Wnt3a + Dyn (n = 6), Wnt5a + Dyn (n = 6).