The retromer CSC functions as a hub to recruit proteins such as the WASH complex to endosomes (Harbour et al., 2010). The increased association of the retromer CSC with the WASH complex component Fam21 could enhance the recruitment of the WASH complex to endosomes. We have therefore investigated whether silencing TBC1D5 expression can increase recruitment of Fam21 to endosomes. Cells expressing either wild-type GFP-VPS35 (Fig. 4A) or the mutant D620N GFP-VPS35 (Fig. 4B) were treated with siRNA to silence TBC1D5 expression and then fixed and labelled with antibodies against Fam21 or the CIMPR. Fam21 staining was slightly reduced in the VPS35 D620N-expressing cells compared with the control cells expressing wild-type VPS35 (as expected) but was markedly brighter following knockdown of TBC1D5. Fig. 4C shows quantification of Fam21 staining following imaging using an automated microscope. As we have shown previously (see Fig. 1), knockdown of TBC1D5 enhances the fluorescence intensity of VPS26 and the CIMPR. Fam21 staining in the VPS35 D620N-expressing cells was markedly reduced compared with levels in cells expressing wild-type VPS35. Loss of TBC1D5 expression enhances the Fam21 fluorescence
