Figure 10. Model: VPS34 and PIKfyve regulate recycling of integrins from endosomes to the plasma membrane via promoting the ordered assembly of SNX17, WASH, Retriever, and CCC complexes.

The phosphatidylinositol 3-phosphate (PI3P) on cargo-containing endosomes is generated by VPS34 (Figure 7, Figure 7—figure supplement 1). PI3P facilitates the recruitment of SNX17 (Chandra et al., 2019; Jia et al., 2014), and SNX17 binds its cargo (Böttcher et al., 2012; Steinberg et al., 2012). The generation of PI3P likely recruits PIKfyve, via its FYVE domain (Stenmark et al., 2002), which initiates the production of PI3,5P₂ and phosphatidylinositol 5-phosphate (PI5P). The generation of PI3P, PI3,5P₂, and/or PI5P plays a role in the binding of the WASH complex: WASH also requires the retromer to be recruited to endosomes (Harbour et al., 2010; Harbour et al., 2012; Helfer et al., 2013; Jia et al., 2012; Zavodszky et al., 2014). The generation of PI3,5P₂ and/or PI5P by PIKfyve then plays a role in recruitment of the CCC complex (Figure 7). The CCC complex also binds the WASH complex via direct interaction of CCDC93 with the WASH subunit, FAM21 (Phillips-Krawczak et al., 2015). The Retriever complex may also associate with endosomes by directly binding to PI3,5P₂ or PI5P, and/or may require PIKfyve activity to recruit the CCC complex. The Retriever complex interacts with and requires the CCC complex to bind to endosomes (McNally et al., 2017; Phillips-Krawczak et al., 2015; Singla et al., 2019). In addition, the Retriever subunit VPS26C interacts directly with SNX17 (Farfán et al., 2013; McNally et al., 2017). Importantly, while SNX17 and WASH are necessary, they are not sufficient for the recruitment of either Retriever or CCC (Figure 7, apilimod treatment). Recruitment of the Retriever and CCC complexes requires PIKfyve. Furthermore, the CCC subunit CCDC22 in turn recruits MTMR2, which is required for late steps in this recycling pathway (Singla et al., 2019). Importantly, recruitment of MTMR2 lowers PI3P and PI3,5P2 (Singla et al., 2019). Together, these studies suggest that PI3P and PI3,5P₂ coordinate the SNX17-Retriever-CCC-WASH pathway. PI3P is required for initiation, PI3,5P₂ and/or PI5P act in the middle steps, and MTMR2, which removes PI3P and PI3,5P₂, acts late in the pathway. Once the SNX17-Retriever-CCC-WASH complex assembles with cargo, the WASH complex mediates actin nucleation (Derivery et al., 2009; Gomez and Billadeau, 2009) and SNX17 recruits EHD1 (Dhawan et al., 2020) to enable fission of cargo containing membranes for efficient recycling.