Double-membrane gap junction internalization requires the clathrin-mediated endocytic machinery

Abstract

Direct cell-cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.

Fig. 1. RNAi-mediated KD of clathrin heavy chain (CHC), and the clathrin adaptors α-Adaptin (AP-2) and Dab2 in HeLa cells

(A) Over 90% of respective protein levels were depleted when assayed 72 h post oligo transfection by Western blot analyses. Equal gel loading was verified by β-tubulin (β-Tub) staining. (B) Efficient protein depletion verified by immunofluorescence staining (images were captured at identical parameters). Boxed areas are shown enlarged on the right. (C) Blots of fluorescence intensity measured along respective lines (shown in the images in B). (D) Inhibition of CME monitored by fluorescence labeled transferrin (Trfn) uptake. (E) Blots of fluorescence Trfn intensity measured along respective lines (shown in the images in D).

Fig. 2. GJ internalization is significantly reduced in CHC, α-Adaptin and Dab2 RNAi-treated cells

(A) Representative images of mock (wt), and RNAi- (KD) treated cells with Cx43-GFP GJs (arrows), and internalized GJ vesicles (arrowheads) marked. (B, C) Quantitative analyses of total and average GJ length (blue) and numbers (yellow), and numbers of internalized GJs (red) per cell pair in KD and control cells.

Fig. 3. RNAi-mediated depletion and functional inhibition of dynamin significantly reduces GJ internalization

(A) Efficient Dyn2 protein depletion verified by Western blot analysis, and (B) immunofluorescence staining (images were captured at identical parameters). Boxed areas are shown enlarged on the right. (C) Plots of fluorescence intensity measured along respective lines (shown in the images in B). (D) Inhibition of CME in HeLa cells overexpressing dominant negative Dyn2-K44A-GFP mutant, or were treated with Dynasore, or GTPγS monitored by fluorescence labeled Trfn uptake. (E) Plots of fluorescence Trfn intensity measured along respective lines (shown in the images in D). (F) Representative images of mock (wt), and Dyn2 RNAi- (KD) treated cells with Cx43-GFP GJs (arrows), and internalized GJ vesicles (arrowheads) marked. (G, H) Quantitative analyses of total and average GJ length and numbers, and numbers of internalized GJs per cell pair in treated and control cells.