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1.  Choose Your Label Wisely: Water-Soluble Fluorophores Often Interact with Lipid Bilayers 
PLoS ONE  2014;9(2):e87649.
Water-soluble organic fluorophores are widely used as labels in biological systems. However, in many cases these fluorophores can interact strongly with lipid bilayers, influencing the interaction of the target with the bilayer and/or leading to misleading fluorescent signals. Here, we quantify the interaction of 32 common water-soluble dyes with model lipid bilayers to serve as an additional criterion when selecting a dye label.
PMCID: PMC3913624  PMID: 24503716
2.  Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4 
PLoS ONE  2014;9(2):e88147.
Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non-canonical site of μ4.
PMCID: PMC3912200  PMID: 24498434
3.  Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking 
Molecular Biology of the Cell  2013;24(17):2689-2702.
In this study reggie-1/flotillin-2 is identified as a component of the tubulovesicular sorting and recycling compartment, where it interacts with and controls the activity of Rab11a and SNX4. Evidence is given that reggie-1 expression is necessary for the proper recycling of transferrin receptor and E-cadherin in HeLa and A431 cells, respectively.
The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1–decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA–mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin–transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca2+ switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca2+ repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.
PMCID: PMC3756921  PMID: 23825023
4.  Multivalent immune complexes divert FcRn to lysosomes by exclusion from recycling sorting tubules 
Molecular Biology of the Cell  2013;24(15):2398-2405.
Study of receptor sorting between recycling and degradative pathways shows that sorting into the recycling pathway depends not only on recognition of sorting motifs by cytosolic adaptors, but also on the physical properties of the endosomal luminal complexes, as shown by the neonatal receptor for IgG FcRn.
The neonatal receptor for immunoglobulin G (IgG; FcRn) prevents IgG degradation by efficiently sorting IgG into recycling endosomes and away from lysosomes. When bound to IgG-opsonized antigen complexes, however, FcRn traffics cargo into lysosomes, where antigen processing can occur. Here we address the mechanism of sorting when FcRn is bound to multivalent IgG-opsonized antigens. We find that only the unbound receptor or FcRn bound to monomeric IgG is sorted into recycling tubules emerging from early endosomes. Cross-linked FcRn is never visualized in tubules containing the unbound receptor. Similar results are found for transferrin receptor, suggesting a general mechanism of action. Deletion or replacement of the FcRn cytoplasmic tail does not prevent diversion of trafficking to lysosomes upon cross-linking. Thus physical properties of the lumenal ligand–receptor complex appear to act as key determinants for sorting between the recycling and lysosomal pathways by regulating FcRn entry into recycling tubules.
PMCID: PMC3727932  PMID: 23741050
5.  Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis 
PLoS ONE  2013;8(10):e76405.
Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.
PMCID: PMC3789715  PMID: 24098492
6.  The N-Terminal Domains of Vps3 and Vps8 Are Critical for Localization and Function of the CORVET Tethering Complex on Endosomes 
PLoS ONE  2013;8(6):e67307.
Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.
PMCID: PMC3688683  PMID: 23840658
7.  Notch signaling from the endosome requires a conserved dileucine motif 
Molecular Biology of the Cell  2013;24(3):297-307.
Notch signaling is reliant on γ-secretase–mediated processing, although the subcellular location where it cleaves Notch to initiate signaling remains unresolved. Findings here support a model in which Notch signaling in mammalian systems is initiated from either the plasma membrane or lysosome, but not the early endosome.
Notch signaling is reliant on γ-secretase–mediated processing, although the subcellular location where γ-secretase cleaves Notch to initiate signaling remains unresolved. Accumulating evidence demonstrates that Notch signaling is modulated by endocytosis and endosomal transport. In this study, we investigated the relationship between Notch transport itinerary and signaling capacity. In doing so, we discovered a highly conserved dileucine sorting signal encoded within the cytoplasmic tail that directs Notch to the limiting membrane of the lysosome for signaling. Mutating the dileucine motif led to receptor accumulation in cation-dependent mannose-phosphate receptor–positive tubular early endosomes and a reduction in Notch signaling capacity. Moreover, truncated receptor forms that mimic activated Notch were readily cleaved by γ-secretase within the endosome; however, the cleavage product was proteasome-sensitive and failed to contribute to robust signaling. Collectively these results indicate that Notch signaling from the lysosome limiting membrane is conserved and that receptor targeting to this compartment is an active process. Moreover, the data support a model in which Notch signaling in mammalian systems is initiated from either the plasma membrane or lysosome, but not the early endosome.
PMCID: PMC3564540  PMID: 23171551
8.  SNAP-23 regulates phagosome formation and maturation in macrophages 
Molecular Biology of the Cell  2012;23(24):4849-4863.
Using macrophages overexpressing or reducing SNAP-23, this study shows that SNAP-23 is implicated in phagosome formation and maturation, presumably by mediating SNARE-based membrane traffic. Indeed, a conformational change in SNAP-23 structure based on FRET signal is observed on the phagosome membrane of cells overexpressing the lysosomal SNARE VAMP7.
Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H+-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.
PMCID: PMC3521691  PMID: 23087210
9.  CIN85 phosphorylation is essential for EGFR ubiquitination and sorting into multivesicular bodies 
Molecular Biology of the Cell  2012;23(18):3602-3611.
This study provides new insights into the mechanisms by which CIN85 regulates targeting of the EGF receptor for degradation. It is the first to demonstrate that CIN85 is phosphorylated by src, phosphorylation of CIN85 is essential for ubiquitinylation of the EGFR, and CIN85 mediates EGFR sequestration into intraluminal vesicles.
Ubiquitination of the epidermal growth factor receptor (EGFR) by cbl and its cognate adaptor cbl-interacting protein of 85 kDa (CIN85) is known to play an essential role in directing this receptor to the lysosome for degradation. The mechanisms by which this ubiquitin modification is regulated are not fully defined, nor is it clear where this process occurs. In this study we show that EGFR activation leads to a pronounced src-mediated tyrosine phosphorylation of CIN85 that subsequently influences EGFR ubiquitination. Of importance, phospho-CIN85 interacts with the Rab5-positive endosome, where it mediates the sequestration of the ubiquitinated receptor into multivesicular bodies (MVBs) for subsequent degradation. These findings provide novel insights into how src- kinase–based regulation of a cbl adaptor regulates the fate of the EGFR.
PMCID: PMC3442408  PMID: 22833562
10.  AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway 
Molecular Biology of the Cell  2012;23(18):3612-3623.
A GPCR ubiquitin-independent MVB/lysosomal sorting pathway is regulated by the adaptor protein complex-3 (AP-3) and ALIX, a noncanonical ESCRT component. AP-3 binds to a PAR1 C-tail–localized, tyrosine-based motif and mediates PAR1 lysosomal degradation. AP-3 also facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
PMCID: PMC3442409  PMID: 22833563
11.  Yeast vacuoles fragment in an asymmetrical two-phase process with distinct protein requirements 
Molecular Biology of the Cell  2012;23(17):3438-3449.
Yeast vacuoles are large organelles that fragment and fuse in response to environmental conditions, such as changes in osmotic conditions or nutrient availability. The morphological changes during salt-induced vacuole fission are characterized, different stages are identified, and the functions of known fission factors are assigned to these stages.
Yeast vacuoles fragment and fuse in response to environmental conditions, such as changes in osmotic conditions or nutrient availability. Here we analyze osmotically induced vacuole fragmentation by time-lapse microscopy. Small fragmentation products originate directly from the large central vacuole. This happens by asymmetrical scission rather than by consecutive equal divisions. Fragmentation occurs in two distinct phases. Initially, vacuoles shrink and generate deep invaginations that leave behind tubular structures in their vicinity. Already this invagination requires the dynamin-like GTPase Vps1p and the vacuolar proton gradient. Invaginations are stabilized by phosphatidylinositol 3-phosphate (PI(3)P) produced by the phosphoinositide 3-kinase complex II. Subsequently, vesicles pinch off from the tips of the tubular structures in a polarized manner, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol-3,5-bisphosphate and the Fab1 complex. It is accelerated by the PI(3)P- and phosphatidylinositol 3,5-bisphosphate–binding protein Atg18p. Thus vacuoles fragment in two steps with distinct protein and lipid requirements.
PMCID: PMC3431934  PMID: 22787281
12.  GLUT4 Traffic through an ESCRT-III-Dependent Sorting Compartment in Adipocytes 
PLoS ONE  2012;7(9):e44141.
In insulin target tissues, GLUT4 is known to traffic through multiple compartments that may involve ubiquitin- and/or SUMO-dependent targeting. During these trafficking steps, GLUT4 is sorted into a storage reservoir compartment that is acutely released by insulin signalling processes that are downstream of PI 3-kinase associated changes in inositol phospholipids. As ESCRT components have recently been found to influence cellular sorting processes that are related to changes in both ubiquitination and inositol phospholipids, we have examined whether GLUT4 traffic is routed through ESCRT dependent sorting steps. Introduction of the dominant negative inhibitory constructs of the ESCRT-III components CHMP3 (CHMP3(1–179)) and Vps4 (GFP-Vps4E235Q) into rat adipocytes leads to the accumulation of GLUT4 in large, coalesced and extended vesicles structures that co-localise with the inhibitory constructs over large parts of the extended structure. A new swollen hybrid and extensively ubiquitinated compartment is produced in which GLUT4 co-localises more extensively with the endosomal markers including EEA1 and transferrin receptors but also with the TGN marker syntaxin6. These perturbations are associated with failure of insulin action on GLUT4 traffic to the cell surface and suggest impairment in an ESCRT-dependent sorting step used for GLUT4 traffic to its specialised reservoir compartment.
PMCID: PMC3458039  PMID: 23049745
14.  An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I 
Molecular Biology of the Cell  2012;23(11):2184-2197.
HIV-1 Nef pirates PACS-1 and PACS-2 to downregulate MHC-I, but little is known about the Nef–PACS interaction. The sites on Nef and the PACS proteins required for their interaction are identified, and their importance for Nef trafficking and Nef-induced MHC-I downregulation is discussed. The results provide insight into the molecular basis of Nef action.
The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef–PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE65 acidic cluster on the N-terminal domain and W113 in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef–PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.
PMCID: PMC3364181  PMID: 22496420
15.  Phosphatidylserine dynamics in cellular membranes 
Molecular Biology of the Cell  2012;23(11):2198-2212.
The distribution and dynamics of phosphatidylserine are studied in the plasma membrane and in organellar membranes of live cells using two novel fluorescent probes in combination with various biophysical techniques, including fluorescence correlation spectroscopy and single-particle tracking.
Much has been learned about the role of exofacial phosphatidylserine (PS) in apoptosis and blood clotting using annexin V. However, because annexins are impermeant and unable to bind PS at low calcium concentration, they are unsuitable for intracellular use. Thus little is known about the topology and dynamics of PS in the endomembranes of normal cells. We used two new probes—green fluorescent protein (GFP)–LactC2, a genetically encoded fluorescent PS biosensor, and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phospho-l-serine (TopFluor-PS), a synthetic fluorescent PS analogue—to examine PS distribution and dynamics inside live cells. The mobility of PS was assessed by a combination of advanced optical methods, including single-particle tracking and fluorescence correlation spectroscopy. Our results reveal the existence of a sizable fraction of PS with limited mobility, with cortical actin contributing to the confinement of PS in the plasma membrane. We were also able to measure the dynamics of PS in endomembrane organelles. By targeting GFP-LactC2 to the secretory pathway, we detected the presence of PS in the luminal leaflet of the endoplasmic reticulum. Our data provide new insights into properties of PS inside cells and suggest mechanisms to account for the subcellular distribution and function of this phospholipid.
PMCID: PMC3364182  PMID: 22496416
16.  cPLA2α and EHD1 interact and regulate the vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes 
Molecular Biology of the Cell  2012;23(10):1874-1888.
cPLA2 hydrolyzes phospholipids and regulates membrane curvature and/or tubulation. Despite disparate roles for cPLA2 at the Golgi and early endosomes, its function in the regulation of membranes containing GPI-anchored proteins is not known. A role for cPLA2α and EHD1 is identified in the vesiculation of cholesterol-rich, GPI-AP–containing membranes.
The lipid modifier phospholipase A2 catalyzes the hydrolysis of phospholipids to inverted-cone–shaped lysophospholipids that contribute to membrane curvature and/or tubulation. Conflicting findings exist regarding the function of cytosolic phospholipase A2 (cPLA2) and its role in membrane regulation at the Golgi and early endosomes. However, no studies addressed the role of cPLA2 in the regulation of cholesterol-rich membranes that contain glycosylphosphatidylinositol-anchored proteins (GPI-APs). Our studies support a role for cPLA2α in the vesiculation of GPI-AP–containing membranes, using endogenous CD59 as a model for GPI-APs. On cPLA2α depletion, CD59-containing endosomes became hypertubular. Moreover, accumulation of lysophospholipids induced by a lysophospholipid acyltransferase inhibitor extensively vesiculated CD59-containing endosomes. However, overexpression of cPLA2α did not increase the endosomal vesiculation, implying a requirement for additional factors. Indeed, depletion of the “pinchase” EHD1, a C-terminal Eps15 homology domain (EHD) ATPase, also induced hypertubulation of CD59-containing endosomes. Furthermore, EHD1 and cPLA2α demonstrated in situ proximity (<40 nm) and interacted in vivo. The results presented here provide evidence that the lipid modifier cPLA2α and EHD1 are involved in the vesiculation of CD59-containing endosomes. We speculate that cPLA2α induces membrane curvature and allows EHD1, possibly in the context of a complex, to sever the curved membranes into vesicles.
PMCID: PMC3350552  PMID: 22456504
17.  Requirement of translocated lysosomal V1 H+-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells 
Molecular Biology of the Cell  2012;23(8):1546-1557.
The activation of translocated lysosomal H+-ATPase is attributed to FasL-induced formation and maintenance of an acid microenvironment around the endothelial cell membrane, which facilitates the activation of ASM and production of ceramide, thereby leading to MR clustering and redox signaling platform formation.
Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H+-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase–mediated production of O2−. in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H+-ATPase apparently attenuated FasL-caused pH decrease. V1 H+-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H+-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H+-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs.
PMCID: PMC3327313  PMID: 22357614
18.  EGFR-dependent phosphorylation of leucine-rich repeat kinase LRRK1 is important for proper endosomal trafficking of EGFR 
Molecular Biology of the Cell  2012;23(7):1294-1306.
Endocytosis and subsequent delivery of activated EGFR to lysosomes are essential for the termination of EGFR signaling. It is shown that EGFR regulates the kinase activity of LRRK1 via tyrosine phosphorylation and that this is required for proper regulation of endosomal trafficking of EGFR.
Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. We recently reported that leucine-rich repeat kinase 1 (LRRK1) is involved in the trafficking of EGFR from early to late endosomes. In this study, we demonstrate that EGFR regulates the kinase activity of LRRK1 via tyrosine phosphorylation and that this is required for proper endosomal trafficking of EGFR. Phosphorylation of LRRK1 at Tyr-944 results in reduced LRRK1 kinase activity. Mutation of LRRK1 Tyr-944 (Y944F) abolishes EGF-stimulated tyrosine phosphorylation, resulting in hyperactivation of LRRK1 kinase activity and enhanced motility of EGF-containing endosomes toward the perinuclear region. The compartments in which EGFR accumulates are mixed endosomes and are defective in the proper formation of intraluminal vesicles of multivesicular bodies. These results suggest that feedback down-regulation of LRRK1 kinase activity by EGFR plays an important role in the appropriate endosomal trafficking of EGFR.
PMCID: PMC3315817  PMID: 22337768
19.  SNX12 Role in Endosome Membrane Transport 
PLoS ONE  2012;7(6):e38949.
In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.
PMCID: PMC3376135  PMID: 22719997
20.  Endosome-to-cytosol transport of viral nucleocapsids 
Nature Cell Biology  2005;7(7):653-664.
During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus, they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. The latter step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1 and is regulated by PI3P signaling via the PI3P-binding protein SNX16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PI3P, and by their effectors.
PMCID: PMC3360589  PMID: 15951806
Animals; Biological Transport; physiology; Cattle; Cell Line; Cricetinae; Cytosol; metabolism; ultrastructure; Endosomal Sorting Complexes Required for Transport; Endosomes; metabolism; ultrastructure; Epithelial Cells; virology; Fibroblasts; virology; Hela Cells; Humans; Lysophospholipids; physiology; Membrane Fusion; drug effects; physiology; Microscopy, Electron; Microscopy, Fluorescence; Monoglycerides; Nucleocapsid; metabolism; Phosphatidylinositol Phosphates; physiology; Phosphoproteins; genetics; physiology; RNA, Viral; biosynthesis; metabolism; Signal Transduction; physiology; Sorting Nexins; Time Factors; Transport Vesicles; metabolism; ultrastructure; Vesicular Transport Proteins; genetics; physiology; Vesicular stomatitis Indiana virus; physiology; Virus Replication; genetics
21.  Rab20 Regulates Phagosome Maturation in RAW264 Macrophages during Fc Gamma Receptor-Mediated Phagocytosis 
PLoS ONE  2012;7(4):e35663.
Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.
PMCID: PMC3335809  PMID: 22545127
22.  Single-cell analysis of population context advances RNAi screening at multiple levels 
A large set of high-content RNAi screens investigating mammalian virus infection and multiple cellular activities is analysed to reveal the impact of population context on phenotypic variability and to identify indirect RNAi effects.
Cell population context determines phenotypes in RNAi screens of multiple cellular activities (including virus infection, cell size regulation, endocytosis, and lipid homeostasis), which can be accounted for by a combination of novel image analysis and multivariate statistical methods.Accounting for cell population context-mediated effects strongly changes the reproducibility and consistency of RNAi screens across cell lines as well as of siRNAs targeting the same gene.Such analyses can identify the perturbed regulation of population context dependent cell-to-cell variability, a novel perturbation phenotype.Overall, these methods advance the use of large-scale RNAi screening for a systems-level understanding of cellular processes.
Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.
PMCID: PMC3361004  PMID: 22531119
cell-to-cell variability; image analysis; population context; RNAi; virus infection
23.  A variable cytoplasmic domain segment is necessary for γ-protocadherin trafficking and tubulation in the endosome/lysosome pathway 
Molecular Biology of the Cell  2011;22(22):4362-4372.
The variable portion of the γ-protocadherin (Pcdh-γ) cytoplasmic domain (VCD) controls Pcdh-γ trafficking and organelle tubulation in the endolysosome system. Active VCD segments are conserved in Pcdh-γA and Pcdh-γB subfamilies.
Clustered protocadherins (Pcdhs) are arranged in gene clusters (α, β, and γ) with variable and constant exons. Variable exons encode cadherin and transmembrane domains and ∼90 cytoplasmic residues. The 14 Pcdh-αs and 22 Pcdh-γs are spliced to constant exons, which, for Pcdh-γs, encode ∼120 residues of an identical cytoplasmic moiety. Pcdh-γs participate in cell–cell interactions but are prominently intracellular in vivo, and mice with disrupted Pcdh-γ genes exhibit increased neuronal cell death, suggesting nonconventional roles. Most attention in terms of Pcdh-γ intracellular interactions has focused on the constant domain. We show that the variable cytoplasmic domain (VCD) is required for trafficking and organelle tubulation in the endolysosome system. Deletion of the constant cytoplasmic domain preserved the late endosomal/lysosomal trafficking and organelle tubulation observed for the intact molecule, whereas deletion or excision of the VCD or replacement of the Pcdh-γA3 cytoplasmic domain with that from Pcdh-α1 or N-cadherin dramatically altered trafficking. Truncations or internal deletions within the VCD defined a 26–amino acid segment required for trafficking and tubulation in the endolysosomal pathway. This active VCD segment contains residues that are conserved in Pcdh-γA and Pcdh-γB subfamilies. Thus the VCDs of Pcdh-γs mediate interactions critical for Pcdh-γ trafficking.
PMCID: PMC3216661  PMID: 21917590
24.  Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function 
Molecular Biology of the Cell  2011;22(21):4108-4123.
This study shows that impaired cholesterol egress from late endosomes in cells with high annexin A6 levels is associated with altered soluble N-ethylmaleimide–sensitive fusion protein 23 (SNAP23) and syntaxin-4 cellular distribution and assembly and accumulation in Golgi membranes. This correlates with reduced secretion of cargo along the constitutive and SNAP23/syntaxin-4–dependent secretory pathway.
Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4–dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.
PMCID: PMC3204072  PMID: 22039070
25.  Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3 
Molecular Biology of the Cell  2011;22(20):3861-3873.
FGFR3 is implicated in several human diseases. Following activation and endocytosis, FGFR3 undergoes sequential ectodomain and intramembrane cleavages to generate a soluble cytoplasmic fragment that can translocate to the nucleus.
Fibroblast growth factor receptor 3 (FGFR3) is a major negative regulator of bone growth that inhibits the proliferation and differentiation of growth plate chondrocytes. Activating mutations of its c isoform cause dwarfism in humans; somatic mutations can drive oncogenic transformation in multiple myeloma and bladder cancer. How these distinct activities arise is not clear. FGFR3 was previously shown to undergo proteolytic cleavage in the bovine rib growth plate, but this was not explored further. Here, we show that FGF1 induces regulated intramembrane proteolysis (RIP) of FGFR3. The ectodomain is proteolytically cleaved (S1) in response to ligand-induced receptor activation, but unlike most RIP target proteins, it requires endocytosis and does not involve a metalloproteinase. S1 cleavage generates a C-terminal domain fragment that initially remains anchored in the membrane, is phosphorylated, and is spatially distinct from the intact receptor. Ectodomain cleavage is followed by intramembrane cleavage (S2) to generate a soluble intracellular domain that is released into the cytosol and can translocate to the nucleus. We identify the S1 cleavage site and show that γ-secretase mediates the S2 cleavage event. In this way we demonstrate a mechanism for the nuclear localization of FGFR3 in response to ligand activation, which may occur in both development and disease.
PMCID: PMC3192865  PMID: 21865593

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