Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks

Abstract

The Arp2/3-activator Wiskott-Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1(+) endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex-associated proteins or EEA1. In addition, we observed global collapse of LAMP1(+) lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells.

FIGURE 1:

WASH-knockout MEFs display reduced SHRC expression and collapsed endosomes devoid of F-actin. (A) WASH^flox/flox/ER-Cre⁺ MEFs were left untreated or treated with 4-OHT to induce WASH knockout. Control WASH^+/+/ER-Cre⁺ MEFs were also treated with 4-OHT. Lysates were immunoblotted as indicated. (B) Immunoprecipitations were performed using the indicated antibodies from lysates of either 4-OHT–induced WASH^−/- (WASHout) MEFs or WASH^+/+ control MEFs. (C, D) WASH^flox/flox and WASHout MEFs were analyzed by immunofluorescence for mWASH (red) and EEA1 (green). (E-J) WASH^flox/flox and WASHout MEFs were stained with rhodamine–phalloidin for F-actin (red) and with anti-EEA1 (green). Arrows indicate distinct endosomal morphologies. (F, H) Insets from E and G as indicated. (K, L) WASH^flox/flox and WASHout MEFs were stained with anti-EEA1 (green) and anti-ARPC2 (red). (M, N) WASHout MEFs reconstituted with wild-type GFP-WASH were costained with rhodamine–phalloidin (red) or with anti-EEA1 (red), respectively. (O, P) WASHout MEFs reconstituted with GFP-WASH ΔVCA were costained with rhodamine–phalloidin (red) or with anti-EEA1 (red), respectively. (C–P) The nucleus is shown via Hoechst staining (blue).

FIGURE 2:

Collapsed endosomes in WASHout fibroblasts harbor distinct subdomains enriched in retromer CSC and SHRC components. WASH^flox/flox and WASHout MEFs were analyzed by immunofluorescence as indicated. The nucleus is shown via Hoechst staining (blue).

FIGURE 3:

Lysosomes collapse in WASHout fibroblasts. WASH^flox/flox and WASHout MEFs were analyzed by immunofluorescence as indicated. In C the arrows within the insets indicate distinct lysosomal morphologies. The nucleus is shown via Hoechst staining (blue).

FIGURE 4:

Collapsed EEA1⁺, retromer-rich, and LAMP1⁺ domains in WASHout MEFs remain distinct. WASH^flox/flox and WASHout MEFs were analyzed by immunofluorescence as indicated. Sequential insets are displayed as indicated. The nucleus is shown via Hoechst staining (blue).

FIGURE 5:

Transferrin receptor shows altered localization but normal levels in WASHout MEFs. (A–E) WASH^flox/flox and WASHout MEFs were analyzed by immunofluorescence as indicated. The nucleus is shown via Hoechst staining (blue). (F) Transferrin receptor surface levels were analyzed by flow cytometry. (G) WASH^flox/flox/ER-Cre⁺ MEFs were left untreated or treated with 4-OHT. WASH^+/+/ER-Cre⁺ MEFs were also 4-OHT treated as a control. Lysates were immunoblotted as indicated. (H) WASH^flox/flox and WASHout MEFs were analyzed by flow cytometry for uptake of AF488-Tfn (AF488 up to 30 min). AF488-Tfn was then exchanged with AF647-Tfn in order to analyze TfnR-mediated recycling of AF488-Tfn (AF488; after 30 min) and new accumulation of AF647-Tfn (AF647 after 30 min) via two-color flow-cytometric analysis. Blue arrow indicates point of AF488-Tfn and AF647-Tfn exchange.

FIGURE 6:

EGFR is reduced and localizes with lysosomes at steady state in WASHout MEFs. (A) Control WASH^+/+/ER-Cre⁺ and WASH^flox/flox/ER-Cre⁺ MEFs were treated with 4-OHT over time. Lysates were immunoblotted as indicated. (B) Basal mEGFR surface levels were analyzed by flow cytometry. (C) Basal mEGFR mRNA levels were measured with quantitative reverse transcriptase PCR. (D) Control and WASHout MEFs were treated with mEGF over time, and lysates were immunoblotted as indicated. (E) EGFR degradation was quantified from immunoblots via densitometry from three independent experiments, and the slope (m) is indicated between time points. (F) WASH^flox/flox/ER-Cre⁺/hEGFR-GFP–expressing MEFs were treated or not treated with 4-OHT, and lysates were immunoblotted as indicated. (G, H) Total hEGFR-GFP expression and surface hEGFR levels were measured by flow cytometry using hEGFR-GFP–expressing WASH^flox/flox and WASHout MEFs. (I–K) hEGFR-GFP–expressing WASH^flox/flox and WASHout MEFs were analyzed by immunofluorescence as indicated. The nucleus is shown via Hoechst staining (blue). (L) hEGFR-GFP–expressing WASHout MEFs were left untreated or treated with 400 nM bafilomycin A for 20 h and analyzed by immunoblot as indicated.

FIGURE 7:

EGFR is degraded within enlarged lysosomal structures upon activation in WASHout MEFs. hEGFR-GFP–expressing WASH^flox/flox and WASHout MEFs were analyzed by immunofluorescence after either serum starvation or stimulation with hEGF over the indicated time course. The nucleus is shown via Hoechst staining (blue).