the endo-lysosomal pathway through various strategies including reduction of lysosomal acidification by NH4Cl or inhibition of the vacuolar H+-ATPase with bafilomycin A1 (Fig. 1b). RhoB showed a characteristic punctuate pattern in control cells (Fig. 1c). However, cells treated with inhibitors of lysosomal degradation showed large accumulations of RhoB that were also stained with the fluorescent probe for acidic vesicles Lysotracker Red (LTR). RhoB co-localized with the lysosomal protease cathepsin B, both in control and in chloroquine-treated cells (Fig. S1). NH4Cl and bafilomycin A1 also induced marked RhoB accumulation (Fig. 1d). Interestingly, osmotic shock followed by potassium depletion, which disrupts both plasma and intracellular membrane-associated clathrin lattices [22] completely stabilized RhoB (Fig. 1e). The major pathway leading to lysosomal degradation of intracellular components, including long-lived proteins and organelles, is macroautophagy [23]. Blocking autophagy by deletion of the Atg5 gene, as in Atg5(2/2) MEFs or by means of the classical inhibitor 3-methyladenine failed to increase RhoB levels or stability (Fig. S2). Thus, RhoB stabilization by inhibitors of lysosomal proteolysis is not the result of a general disruption of vesicular traffic or of the autophagic pathway, but of selective events in the endo-lysosomal pathway. A higher resolution of RhoB-positive endosomal structures could be achieved by confocal microscopy in live cells. Similarly to endogenous RhoB, GFP-RhoB and RFP-RhoB localized to intracellular vesicles and to the plasma membrane (Fig. 2a,b). The localization of RhoB along the endocytic pathway was confirmed by its presence on vesicles containing endocytosed dextran (Fig. 2a). Interestingly, RhoB was not associated with GFP-Rab5-positive structures but was clearly observed inside vesicles delimited by Rab7, a marker of late endosomes and lysosomes (Fig. 2b). RFP-RhoB was clearly localized inside Lamp1-GFP positive vesicles and GFP-RhoB-positive vesicles showed LTR staining (Fig. 2c). Thus, RhoB is associated with late- endosomes/lysosomes. Interestingly, blocking lysosomal degrada- tion with leupeptin plus E64d or with chloroquine induced the accumulation of RhoB constructs in enlarged lysosomal vesicles (Fig. 2c and S3a). Remarkably, RhoB distribution inside the vesicles showed a patchy appearance, indicative of its presence in intravesicular structures. Indeed, a greater magnification of these dilated compartments, either in GFP-RhoB or Lamp1-GFP transfected cells, revealed multiple intraluminal vesicles, typical of multivesicular bodies (MVB) (Fig. S3b). Electron microscopy (EM) clearly showed the formation of dilated multivesicular structures in chloroquine-treated cells, that could correspond to MVB or to late endosome/lysosome hybrids [24] (Fig. 2d). Immuno-EM (IEM) confirmed the presence of GFP-RhoB in the intraluminal vesicles of MVB both in control and in chloroquine- treated cells (Fig. 2e). Taken together, these results suggest that RhoB, localized in late endosomes/MVB may be delivered to the luminal space and be degraded in this compartment and/or in lysosomes. Specific Lipids Regulate RhoB Accumulation into MVB The biogenesis of MVB from late endosomes occurs through a complex mechanism involving protein and lipid-mediated pro- cesses. Lipid composition and dynamics are crucial for MVB biogenesis [25]. Thus, we explored whether altering late endosome lipids affected RhoB distribution. For this we used the compound U18666A, which causes the accumulation of the late endosome- specific lipid, 2,29-dioleoyl lysobisphosphatidic acid (LBPA) and cholesterol in late endosomal membranes [26,27] and promotes their invagination [28]. U18666A induced a massive accumulation Lysosomal Degradation Sequence of membranes and/or vesicles inside late endosomes giving rise to electron-dense structures (Fig. 3a), that were also visible by differential interference contrast imaging (DIC) (Fig. 3b). Remark- ably, U18666A-induced dense MVB showed an intense accumu- lation of GFP-RhoB (Fig. 3b). RhoB co-localized with LTR and LBPA inside U18666A-induced structures (Fig. 3c a–d). In contrast, Rab7, which is bi-geranylgeranylated, was retained at the limiting membranes (Fig. 3c e,f ). Thus, disruption of late endosome lipid dynamics promotes a selective trapping of RhoB in these structures. The presence of GFP-RhoB in the intraluminal membranes of U18666A-induced dense MVB was confirmed by IEM (Fig. 3e). U18666A did not increase RhoB levels or protein stability (Fig. S4), suggesting that RhoB degradation is not impaired in U18666A-treated cells. LBPA plays a key role in membrane invagination leading to MVB formation by promoting luminal vesicle formation due to its cone-shaped structure [29] and controls late endosome cholesterol dynamics [26]. The role of LBPA in MVB invagination can be specifically tackled in vivo by treatment with an anti-LBPA antibody, which is endocytosed and accumulates in late endo- somes blocking LBPA-dependent dynamics [26]. Anti-LBPA induced a marked distortion of GFP-RhoB-positive endosomes that appeared as dilated structures containing anti-LBPA (Fig. 3f), confirming that late endosome-specific lipids regulate RhoB sorting into MVB. Genetic alterations of the endo-lysosomal pathway cause various severe human diseases. The dynamics of late endo- some-specific lipids is profoundly altered in Niemann-Pick type C disease [26], a neurodegenerative disorder in which a mutation in the NPC1 protein, a regulator of vesicular cholesterol transport [30], causes massive accumulation of cholesterol and LBPA in late endosomes. Using fibroblasts from NPC1 patients, we observed an increase in both, the size of RhoB and LTR positive vesicles and of RhoB-LTR co-localization with respect to fibroblasts from control subjects (Fig. 3g). NPC fibroblasts also showed a marked increase in endosomal cholesterol content, as revealed by filipin staining, which co-localized with RhoB (Fig. S5). These findings show that RhoB distribution is altered in a relevant pathological condition affecting late-endosome lipid dynamics. RhoB Sorting into MVB Requires Integrity of the ESCRT Machinery MVB formation involves assembly of the multiprotein complexes known as ESCRT on the late endosomal membrane. Subsequently, recruitment of the ATPase Vps4 and ATP hydrolysis drives the dissociation of the ESCRT machinery, and allows the intraluminal release of vesicles formed by endosomal membrane invagination [31]. Vps4 transiently associates with late endosomes and is mainly cytosolic. In contrast, ATPase-defective Vps4 mutants fail to dissociate from these membranes and severely impair MVB sorting [32]. We observed that whereas GFP-Vps4 wt did not alter RhoB distribution in control or in chloroquine-treated cells (Fig. 4a), the ATPase-defective mutant GFP-Vps4 E223Q, which acts as a dominant negative (DN) [32], drastically reduced the formation of RhoB-containing dilated MVB elicited by chloroquine. Instead, RhoB appeared in small vesicles or retained at the edge of large vesicles (Fig. 4a). Vps4 DN also impaired the formation of chloroquine-elicited enlarged LTR-positive vesicles (Fig. 4b). We next explored the role of Vps4 in RhoB degradation (Fig. 4c,d). First, BAEC were transfected with the constructs of interest and incubated with CHX under conditions resulting in the degrada- tion of most of endogenous RhoB protein. Due to low PLoS ONE | www.plosone.org 3 December 2009 | Volume 4 | Issue 12 | e8117