and G.D. seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking. infection (Genisset et al., 2007; Haas et al., 1995; Papini et al., 1997; Schimmoller and Riezman, 1993). Given that we observed Rab7 hyperactivation in Vps34?/? cells, we tested whether the vacuolization in Vps34?/? cells was due to reduced Armus recruitment and Rab7 hyperactivation. Indeed, silencing Rab7 (shRab7) or ectopic Capsazepine expression of Armus in Vps34?/? MEFs led to a complete reversal Capsazepine of vacuolization (Fig.?5BCD). Taken together, these data suggest that Vps34 regulates the intracellular localization of Rab7 GAP Armus through PI(3)P production to modulate Rab7 activity. Open in a separate window Fig. 5. Inhibition of Rab7 or Armus expression leads to the disappearance of intracellular vacuoles. (A) GFPC2xFYVE and RFPCArmus were expressed in shNTC- or shVps34-expressing HeLa cells. Cells were observed under a deconvolution microscope. Some large GFPC2FYVE aggregates are observed in shNTC cells, which is common with GFPC2xFYVE overexpression. Note that expression of GFPC2FYVE leads to the loss of punctate Armus localization in shNTC cells, and that both GFPC2FYVE and RFPCArmus show a diffuse pattern in shVps34 cells. (where the Armus homolog TBC-2 was found to be a PI3P-binding protein (C. Rocheleau, personal communication). Silencing Vps34 or overexpression of GFPC2xFYVE inhibits the punctate localization of Armus (Figs?4G and ?and5A).5A). Under normal conditions, Vps34 might recruit Armus to Rabbit Polyclonal to JNKK late endosomes to keep Rab7-GTP levels in check. Given that PI(3)P is present on late endosomes (Cao et al., 2008) and might not be a limiting factor, a second signal might serve to recruit Armus at the precise time to regulate Rab7. Rab7 regulates the homotypic fusion of late endosomes and the heterotypic fusion of late endosomes with lysosomes (Kmmel and Ungermann, 2014). Although previous work has established a role for Armus in regulating Rab7 Capsazepine activity during autophagy and mitophagy, we believe our findings support a role for Armus-regulated Rab7 activity in the late endocytic pathway (Carroll et al., 2013; Yamano et al., 2014). Our previous work characterizing the Vps34?/? MEFs established that the autophaghic pathway is defective in these cells, beginning from the early stages of autophagosome biogenesis (Jaber et al., 2012). Thus, any Rab7 activity in the cells is likely to be associated with other pathways. However, we cannot rule out the possibility that a proportion of Rab7 activity might be related to an early step in autophagosome biogenesis. It is curious that the deletion or knockdown of Vps34 and subsequent increase in Rab7-GTP leads to the same enlarged late endosome phenotype as Rab7 knockdown. Others have observed that expression of both constitutively active and dominant-negative Rab7 leads to an enlarged late endosome phenotype (Stein et al., 2003). In comparison to the endosomes we observed in Vps34?/? cells, which do not contain ILVs, enlarged late endosomes in Rab7-knockdown cells were observed to contain increased numbers of ILVs. It has been suggested that the late endosomes in Rab7-knockdown cells expand to accommodate the increased number of internal vesicles (Vanlandingham and Ceresa, 2009). Therefore, in the case of Vps34 deletion, although it might lead to the failure of ILV formation through a Rab7-independent mechanism, the enlarged late endosomes might be the result of increased homotypic late endosome fusion due to increased Rab7-GTP levels. We also observed intracellular vacuolization, which has also been attributed to Rab7 hyperactivation (Genisset et al., 2007; Haas et al., 1995; Papini et al., 1997; Schimmoller and Riezman, 1993). We were unable to rescue the defects in EGFR degradation and lysosomal function by Rab7 silencing or by expressing its dominant-negative mutant (data not shown), possibly due to the requirement of a delicate level of Rab7 activation for these processes. Nonetheless, Rab7 silencing or Armus overexpression suppressed vacuolization in Vps34-deficient cells (Fig.?5BCD), indicating that at least some of the late endosomal trafficking defects in Vps34-deficient cells can be rescued by Armus-mediated Rab7 inactivation. Given that we believe Armus plays a role in the phenotype of Vps34?/? cells, we expect that knockdown of Armus would generate a similar phenotype. Unfortunately, we were unable to test this hypothesis because shRNA against Armus is not yet available. Moreover, we cannot rule out the possibility that the phenotypes we observe in Vps34?/? cells are partially due to the loss of PI(3,5)P2, a downstream.
