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1.  Autophagosomes contribute to intracellular lipid distribution in enterocytes 
Molecular Biology of the Cell  2014;25(1):118-132.
Delivery of alimentary lipids induces immediate autophagic response in enterocytes. Forming autophagosomes are recruited to the ER membrane, where they capture nascent lipid droplets and later fuse with lysosomes, illustrating for the first time the role of autophagy in neutral-lipid distribution in enterocytes.
Enterocytes, the intestinal absorptive cells, have to deal with massive alimentary lipids upon food consumption. They orchestrate complex lipid-trafficking events that lead to the secretion of triglyceride-rich lipoproteins and/or the intracellular transient storage of lipids as lipid droplets (LDs). LDs originate from the endoplasmic reticulum (ER) membrane and are mainly composed of a triglyceride (TG) and cholesterol-ester core surrounded by a phospholipid and cholesterol monolayer and specific coat proteins. The pivotal role of LDs in cellular lipid homeostasis is clearly established, but processes regulating LD dynamics in enterocytes are poorly understood. Here we show that delivery of alimentary lipid micelles to polarized human enterocytes induces an immediate autophagic response, accompanied by phosphatidylinositol-3-phosphate appearance at the ER membrane. We observe a specific and rapid capture of newly synthesized LD at the ER membrane by nascent autophagosomal structures. By combining pharmacological and genetic approaches, we demonstrate that autophagy is a key player in TG targeting to lysosomes. Our results highlight the yet-unraveled role of autophagy in the regulation of TG distribution, trafficking, and turnover in human enterocytes.
doi:10.1091/mbc.E13-06-0324
PMCID: PMC3873883  PMID: 24173715
2.  PI3P regulates sorting and processing of amyloid precursor protein through the endosomal system 
Nature communications  2013;4:2250.
Defects in endosomal sorting have been implicated in Alzheimer’s disease (AD). Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that PI3P is selectively deficient in brain tissue from humans with AD and AD mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein (APP) in these organelles and reduces APP sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT pathway, including the PI3P effector Hrs and Tsg101. APP is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of APP to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing PI3P deficiency as a contributing factor in AD, these results clarify the mechanisms of APP trafficking through the endosomal system in normal and pathological states.
doi:10.1038/ncomms3250
PMCID: PMC3905799  PMID: 23907271
3.  Scavenger Receptor Class B Type I (SR-BI) is a Plasma Membrane Cholesterol Sensor 
Circulation research  2012;112(1):140-151.
Rationale
Signal initiation by the HDL receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain (CTTM). The CTTM uniquely interacts with plasma membrane (PM) cholesterol.
Objective
The molecular basis and functional significance of SR-BI interaction with plasma membrane cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a plasma membrane cholesterol sensor.
Methods and Results
In studies performed in COS-M6 cells, mutation of a highly-conserved CTTM glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin (CD) was also prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or CD in cultured enterocytes, and it is required for HDL activation of eNOS and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo.
Conclusion
Through interaction with plasma membrane cholesterol, SR-BI serves a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.
doi:10.1161/CIRCRESAHA.112.280081
PMCID: PMC3564583  PMID: 23023567
Endothelium; enterocyte; nitric oxide synthase; reverse cholesterol transport; scavenger receptor BI
4.  Characterizing the location and trafficking routes of the neuronal retromer and its role in amyloid precursor protein transport 
Neurobiology of disease  2012;47(1):126-134.
The retromer complex plays an important role in intracellular transport, is highly expressed in the hippocampus, and has been implicated in the trafficking of the amyloid precursor protein (APP). Nevertheless, the trafficking routes of the neuronal retromer and the role it plays in APP transport in neuronal processes remains unknown. Here we use hippocampal neuronal cultures to address these issues. Using fluorescence microscopy, we find that Vps35, the core element of the retromer complex, is in dendrites and axons, is enriched in endosomes and trans-Golgi network, and is found in APP-positive vesicles. Next, to identify the role the neuronal retromer plays in cargo transport, we infected hippocampal neurons with a lentivirus expressing shRNA to silence Vps35. By live fluorescence imaging, Vps35 deficiency was found to reduce the frequency, but not the kinetics, of long-range APP transport within neuronal processes. Supporting the interpretation that retromer promotes long-range transport, Vps35 deficiency led to increased APP in the early endosomes, in processes but not the soma. Finally, Vps35 deficiency was associated with increased levels of Aβ, a cleaved product of APP, increased co-localization of APP with its cleaving enzyme BACE1 in processes, and caused an enlargement of early endosomes. Taken together, our studies clarify the function of the neuronal retromer, and suggest specific mechanisms for how retromer dysfunction observed in Alzheimer’s disease affects APP transport and processing.
doi:10.1016/j.nbd.2012.03.030
PMCID: PMC3589992  PMID: 22516235
5.  Roles of the cellular prion protein in the regulation of cell-cell junctions and barrier function 
Tissue Barriers  2013;1(2):e24377.
The cellular prion protein was historically characterized owing to its misfolding in prion disease. Although its physiological role remains incompletely understood, PrPC has emerged as an evolutionary conserved, multifaceted protein involved in a wide-range of biological processes. PrPC is a GPI-anchored protein targeted to the plasma membrane, in raft microdomains, where its interaction with a repertoire of binding partners, which differ depending on cell models, mediates its functions. Among identified PrPC partners are cell adhesion molecules. This review will focus on the multiple implications of PrPC in cell adhesion processes, mainly the regulation of cell-cell junctions in epithelial and endothelial cells and the consequences on barrier properties. We will show how recent findings argue for a role of PrPC in the recruitment of signaling molecules, which in turn control the targeting or the stability of adhesion complexes at the plasma membrane.
doi:10.4161/tisb.24377
PMCID: PMC3887058  PMID: 24665391
Src family kinases; adherens junctions; cell adhesion; cell signaling; cellular prion protein PrPC; desmosomes; intestinal barrier; raft; tight junctions
6.  The Phospholipase D1 Pathway Modulates Macroautophagy 
Nature Communications  2010;1:142.
While macroautophagy is known to be an essential degradative process whereby autophagosomes mediate the engulfment and delivery of cytoplasmic components into lysosomes, the lipid changes underlying autophagosomal membrane dynamics are undetermined. Here we show that phospholipase D1 (PLD1), which is primarily associated with the endosomal system, partially relocalizes to the outer membrane of autophagosome-like structures upon nutrient starvation. The localization of PLD1, as well as the starvation-induced increase in PLD activity, are altered by wortmannin, a phosphatidylinositol 3-kinase inhibitor, suggesting PLD1 may act downstream of Vps34. Pharmacological inhibition of PLD and genetic ablation of PLD1 in the mouse decrease the starvation-induced expansion of LC3-positive compartments, consistent with a role of PLD1 in the regulation of autophagy. Furthermore, inhibition of PLD results in higher levels of tau and p62 aggregates in organotypic brain slices. Our in vitro and in vivo findings establish a novel role for PLD1 in autophagy.
doi:10.1038/ncomms1144
PMCID: PMC3328354  PMID: 21266992
7.  The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics 
Biology of the Cell  2011;103(Pt 11):499-517.
Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization.
Results. Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet.
Conclusions. We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.
doi:10.1042/BC20110024
PMCID: PMC3181828  PMID: 21787361
3-β-hydroxysteroid dehydrogenase; apolipoprotein A-IV; Caco-2/TC7 cell; cytosolic lipid droplet; enterocyte; lysophosphatidylcholine acyltransferase 2 (LPCAT2); proteome; 3BHS1, 3-β-hydroxysteroid dehydrogenase 1; ABHD5, α/β-hydrolase-domain-containing protein 5; ACSL3, acyl-CoA synthetase long-chain 3; ApoA-IV, apolipoprotein A-IV; CCT-α, choline-phosphate cytidylyltransferase A; CE, cholesterol ester; CLD, cytosolic lipid droplet; DAPI, 4′,6-diamidino-2-phenylindole; DGAT, diacylglycerol acyltransferase; DGE, diacylglyceryl ether; emPAI, exponentially modified protein abundance index; ER, endoplasmic reticulum; GM130, Golgi matrix 130; HSD17B11, 17-β-hydroxysteroid dehydrogenase type 11; HSP60, heat-shock protein 60 kDa; LC-MS/MS, liquid chromatography coupled with tandem MS; LDH, lactate dehydrogenase; LPCAT2, lysophosphatidylcholine acyltransferase 2; MALDI, matrix-assisted laser-desorption ionization; MGAT, monoacylglycerol acyltransferase; MGLL, monoacylglycerol lipase; TAG, triacylglycerol; MTTP, microsomal TAG transfer protein; NSDHL, NAD(P)-dependent steroid dehydrogenase-like; OA, oleic acid; PC, phosphatidylcholine; PDI, protein disulfide-isomerase; PFA, paraformaldehyde; PL, phospholipid; PLIN, perilipin; TOF, time-of-flight; TRL, TAG-rich lipoprotein; VLDL, very-low-density lipoprotein
10.  Hrs and SNX3 Functions in Sorting and Membrane Invagination within Multivesicular Bodies  
PLoS Biology  2008;6(9):e214.
After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.
Author Summary
The cell's genetic program is modulated by extracellular signals that activate cell surface receptors and, in turn, intracellular effectors, to regulate transcription. For cells to function normally, these signals must be turned off to avoid permanent activation—a situation often associated with cancer. For many receptors, signaling is repressed, or down-regulated, in a process that first internalizes and then degrades the receptors. After receptors are removed from the cell surface into structures called early endosomes, they are selectively incorporated within vesicles that form inside the endosome. During this process, endosomal membranes are pulled away from the cytoplasm towards the endosome lumen, against the flow of intracellular membrane traffic, eventually resulting in the formation of a “multivesicular body” (vesicles within vesicles). The common view is that these intralumenal vesicles are then delivered to lysosomes, where they are degraded along with their receptor cargo. We have investigated the mechanisms responsible for the biogenesis of intralumenal vesicles in multivesicular bodies. We find that the small protein SNX3, which binds the signaling lipid phosphatidyl inositol-3-phosphate, is necessary for the formation of intralumenal vesicles, but is not involved in the degradation of the cell surface receptor for EGF. Conversely, we find that Hrs, which also binds phosphatidyl inositol-3-phosphate and mediates receptor sorting into intralumenal vesicles, is essential for lysosomal targeting but dispensable for multivesicular body biogenesis. Phosphatidyl inositol-3-phosphate thus controls the complementary functions of Hrs and SNX3 in the sorting of signaling receptors and multivesicular body biogenesis.
SNX3 plays a direct role in the formation of intralumenal vesicles of multivesicular bodies (MVBs) but is not involved in EGF receptor degradation, whereas Hrs is essential for lysosomal targeting but dispensable for MVB biogenesis. Hence, intralumenal vesicle formation in MVB biogenesis can be uncoupled from lysosomal targeting.
doi:10.1371/journal.pbio.0060214
PMCID: PMC2528051  PMID: 18767904
11.  Video-assisted thoracoscopic surgery (VATS) for the treatment of scolioticrib hump deformity 
European Spine Journal  2007;16(9):1373-1377.
A retrospective study of 21 patients with idiopathic scoliosis who underwent endoscopic thoracoplasty was done. The objective of the study was to report and assess the morbidity and mid term outcomes of video-assisted thoracoplasty in idiopathic scoliosis. Patients with idiopathic scoliosis often present cosmetic complaints due to their rib deformity. This deformity may still exist after surgical correction of the main scoliotic curve. Endoscopic thoracoplasty has been reported as a safe method in limited cases of idiopathic scoliosis. Between 2002 and 2004, 21 patients underwent endoscopic anterior release and thoracoplasty for significant rib hump deformity associated with idiopathic scoliosis. Patients were operated on lateral position, with two endoscopic ports. Anterior release and rib resection were performed during the first stage, and instrumented posterior fusion was performed in a second stage. Patients were evaluated preoperatively, 1 week after surgery, 6 months after surgery and at their most recent follow-up with clinical and radiological measurement of the rib deformity. The mean age at surgery was 14.9 years old (range 13–17 years). The average Cobb’s angle of the main scoliotic curve was 70° (range 60°–85°). Average follow-up was 25 months (range 23–32 months). The mean number of resected ribs was five ribs (range 4–7) and the mean length of the resected rib was 4.2 cm (range 2.2–7 cm). Average operating time of endoscopic thoracoplasty (including anterior release) was 65 min (range 45–108 min). The mean preoperative height of rib hump deformity was 3.6 cm (range 2.5–5.5 cm). It was reduced to 1.5 cm at most recent follow-up. There was no significant thoracic pain necessitating medication postoperatively. No complications related to endoscopic anterior release and rib hump resection occurred in the series. Endoscopic thoracoplasty is a safe and reliable technique in idiopathic scoliosis. If indicated, the anterior release can be performed with video-assistance and the thoracoplasty can be performed on the same stage.
doi:10.1007/s00586-007-0340-z
PMCID: PMC2200748  PMID: 17356821
Scoliosis; Thoracoscopy; Thoracic deformity
12.  The Cellular Prion Protein PrPc Is Involved in the Proliferation of Epithelial Cells and in the Distribution of Junction-Associated Proteins 
PLoS ONE  2008;3(8):e3000.
Background
The physiological function of the ubiquitous cellular prion protein, PrPc, is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrPc is targeted to cell–cell junctions of polarized epithelial cells, where it interacts with c-Src.
Methodology/Findings
We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrPc is differentially targeted either to the nucleus in dividing cells or to cell–cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrPc interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrPc, desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrPc is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrPc knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi.
Conclusions/Significance
From these results, PrPc could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells.
doi:10.1371/journal.pone.0003000
PMCID: PMC2500194  PMID: 18714380
13.  The Redox Sensor TXNL1 Plays a Regulatory Role in Fluid Phase Endocytosis 
PLoS ONE  2007;2(11):e1144.
Background
Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis.
Methodology/Principal Findings
When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5.
Conclusions/Significance
Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis.
doi:10.1371/journal.pone.0001144
PMCID: PMC2043495  PMID: 17987124
14.  The p11/S100A10 Light Chain of Annexin A2 Is Dispensable for Annexin A2 Association to Endosomes and Functions in Endosomal Transport 
PLoS ONE  2007;2(10):e1118.
Background
Annexin A2 is a peripheral membrane protein that belongs to the annexin family of Ca2+ and phospholipid-binding proteins. This protein, which plays a role in membrane organization and dynamics in particular along the endocytic pathway, exists as a heterotetrameric complex, consisting of two annexin A2 molecules bound via their N-termini to a dimer of p11/S100A10 light chains. The light chain, and thus presumably formation of the heterotetramer, was reported to control annexin A2 association to the plasma membrane and to cortical actin, as well as the distribution of recycling endosomes. However, the specific role of the light chain and the functions of monomeric versus heterotetrameric annexin A2 have remained elusive in the endocytic pathway.
Methodology/Principal Findings
Here, we have investigated whether p11 plays a role in the endosomal functions of annexin A2. Using morphological and biochemical approaches, we found that p11, unlike annexin A2, was not present on early endosomes. Neither was the heterotetramer detected on purified early endosomes, while it was clearly present in total cell lysates. Moreover, knockdown of p11 with siRNAs did not affect annexin A2 targeting to early endosomes, and, conversely, binding of annexin A2 to purified endosomes or liposomes occurred without p11 in vitro. Finally, while we could confirm that annexin A2 knockdown inhibits transport beyond early endosomes, p11 knockdown had no such effects on early-to-late endosome transport.
Conclusions/Significance
Our data show that the binding of annexin A2 to endosomal membranes and its role in endosomal trafficking are independent of the p11/S100A10 light chain. We thus conclude that annexin A2 functions are fully supported by the monomeric form of the protein, at least the endocytic pathway leading to lysosomes.
doi:10.1371/journal.pone.0001118
PMCID: PMC2040519  PMID: 17971878
15.  Lipid-dependent Bidirectional Traffic of Apolipoprotein B in Polarized Enterocytes 
Molecular Biology of the Cell  2004;15(1):132-141.
Enterocytes are highly polarized cells that transfer nutrients across the intestinal epithelium from the apical to the basolateral pole. Apolipoprotein B (apoB) is a secretory protein that plays a key role in the transepithelial transport of dietary fatty acids as triacylglycerol. The evaluation of the control of apoB traffic by lipids is therefore of particular interest. To get a dynamic insight into this process, we used the enterocytic Caco-2 cells cultured on microporous filters, a system in which the apical and basal compartments can be delimited. Combining biochemical and morphological approaches, our results showed that, besides their role in protection from degradation, lipids control the intracellular traffic of apoB in enterocytes. A supply of fatty acids and cholesterol is sufficient for the export of apoB from the endoplasmic reticulum and its post-Golgi traffic up to the apical brush-border domain, where it remains until an apical supply of complex lipid micelles signals its chase down to the basolateral secretory domain. This downward traffic of apoB involves a microtubule-dependent process. Our results demonstrate an enterocyte-specific bidirectional process for the lipid-dependent traffic of a secretory protein.
doi:10.1091/mbc.E03-04-0215
PMCID: PMC307534  PMID: 14565984

Results 1-15 (15)