The Arp2/3 activator WASH regulates α5β1-integrin-mediated invasive migration

Abstract

The actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for α5β1 integrin recycling. WASH regulates the trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.

Fig. 1.

WASH localises to early and late endosomal vesicles of A2780 ovarian cancer cells. (A) Cells were transfected with the indicated GFP-fused endosomal marker (green in ‘merge’ image) for 24 hours, fixed and stained for endogenous WASH (red in ‘merge’ image). Scale bars: 10 μm. (B) Relative colocalisation of WASH and the indicated endosomal markers was determined using an ImageJ plugin (see Materials and Methods). Values are the means of three independent experiments with n=15 each. Error bars indicate s.e.m.

Fig. 2.

WASH depletion in A2780 cells results in an increased association of α5β1 integrin with MVB/LEs. (A) Nontargeting siRNA (NT) and WASH-kd A2780 cells were immunostained for α5 integrin and WASH. Images represent z-stacks of 19 slices of 0.13 μm; n=3 experiments. Scale bars: 10 μm. (B) Control and WASH-kd cells with a pool of two oligonucleotides (siW1+4) were transfected with GFP–CD63 and stained for α5 integrin. Scale bars: 10 μm. (C) Endosomal distribution of α5β1 integrin. Colocalisation was quantified using an ImageJ plugin (see Materials and Methods). *P<0.05. Error bars indicate s.e.m.

Fig. 3.

WASH-mediated actin polymerisation is required for normal α5β1 integrin recycling to the plasma membrane. (A–C) WASH-kd A2780 cells expressing photoactivatable (pa) GFP–α5-integrin and Cherry–mWASH and were treated with cRGDfV. (A) A WASH-labelled vesicle (arrow) was photactivated at 405 nm and recorded with an Olympus Sim Scanner. Scale bars: 10 μm. (B) Integrated fluorescence intensity of photoactivated GFP–α5-integrin was recorded in two regions of interest (outlined in A, 140s): the activated vesicle and plasma membrane (Ves and PM in A). (C) α5 integrin trafficking from Cherry–mWASH- or Cherry–WASHδVCA (dVCA)-labelled vesicles in WASH-kd cells. Photoactivatable GFP–α5-integrin was activated on a WASH- or WASHδVCA-labelled vesicle and the fluorescence decrease was monitored. Results are background-corrected values; n=30. (D) α5β1 integrin internalisation time course; n=8 repeats in two experiments. Internalization rates were indistinguishable (F-test; P=0.4498) using Graphpad PRISM. (E) α5β1 integrin recycling in NT and WASH-kd A2780 cells treated with cRGDfV. NT, nontargeting siRNA; siW1, siWASH oligo 1; siW4, siWASH oligo 4. (F) Western blot showing α5 integrin and WASH expression; n=3. Error bars in C,D,E indicate s.e.m.

Fig. 4.

WASH-depleted A2780 cells are severely impaired in integrin-mediated 3D invasion. (A,B) NT-, WASH-kd- and mWASH-expressing cells invaded Matrigel containing 100 μg/ml fibronectin, for 3 days. The cells that migrated beyond 45 μm were counted; n⩾3 experiments, *P<0.05. (C–E) NT and Wash-kd cells were plated on cell-derived matrices for 3 hours before imaging for 24 hours. Speed, directionality and pseudopodium length were calculated using ImageJ manual tracking and a chemotaxis plugin; n⩾3 experiments each with ⩾150 cells. NT, nontargeting siRNA; siW1, siWASH oligo 1; siW4, siWASH oligo 4. *P<0.05; ns, not significant (P>0.05). (F) A2780 cells expressing GFP–WASH or GFP were analysed by FACS for surface expression of β1 integrin; n=3. (G) β1 integrin cell surface expression in A2780 cells with cRGDfV, measured by FACS; n=3 (siW1 and NT); n=2 (siW4). Error bars indicate s.e.m.