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1.  Contrasting effects of fish oil and safflower oil on hepatic peroxisomal and tissue lipid content 
To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we studied the effects of control, fish oil, and safflower oil diets on peroxisomal content, fatty acyl-CoA, diacylglycerol, and ceramide content in rat liver and muscle. We found that, in contrast to control and safflower oil-fed rats, fish oil feeding induced a 150% increase in the abundance of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase in liver but lacked similar effects in muscle. This was paralleled by an almost twofold increase in hepatic peroxisome content (both P < 0.002 vs. control and safflower). These changes in the fish oil-fed rats were associated with a more than twofold lower hepatic triglyceride/diacylglycerol, as well as intramuscular triglyceride/fatty acyl-CoA, content. In conclusion, these data strongly support the hypothesis that n-3 fatty acids protect against fat-induced insulin resistance by serving as peroxisome proliferator-activated receptor-α ligands and thereby induce hepatic, but not intramuscular, peroxisome proliferation. In turn, an increased hepatic β-oxidative capacity results in lower hepatic triglyceride/diacylglycerol and intramyocellular triglyceride/fatty acyl-CoA content.
doi:10.1152/ajpendo.00414.2001
PMCID: PMC2995503  PMID: 11788372
peroxisome proliferator-activating receptor-α; β-oxidation; diacylglycerol; acyl-CoA oxidase; 3-ketoacyl-CoA thiolase
2.  Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes 
Cell host & microbe  2009;6(4):367-380.
Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here we demonstrate that live influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes. Matrix protein 2 is sufficient and necessary for this inhibition of autophagosome degradation. Macroautophagy inhibition compromises cell survival of influenza virus infected cells, but does not influence viral replication. We propose that influenza A virus, which also encodes pro-apoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and blocking macroautophagy.
doi:10.1016/j.chom.2009.09.005
PMCID: PMC2774833  PMID: 19837376
influenza; macroautophagy; CD4+ T cells; Atg8/LC3; matrix protein 1
3.  CD2 distinguishes two subsets of human plasmacytoid dendritic cells with distinct phenotype and functions1 
SUMMARY
Plasmacytoid dendritic cells (pDCs) are key regulators of anti-viral immunity. They rapidly secrete IFN-α and cross-present viral antigens thereby launching adaptive immunity. Here we show that activated human pDCs inhibit replication of cancer cells, and kill them in a contact dependent fashion. Expression of CD2 distinguishes two pDC subsets with distinct phenotype and function. Both subsets secrete IFN-α and express Granzyme B and TRAIL. CD2high pDCs uniquely express lysozyme and can be found in tonsils and in tumors. Both subsets launch recall T cell response. However, CD2high pDCs secrete higher levels of IL12 p40, express higher levels of co-stimulatory molecule CD80 and are more efficient in triggering proliferation of naïve allogeneic T cells. Thus, human blood pDCs are composed of subsets with specific phenotype and functions.
doi:10.4049/jimmunol.0802008
PMCID: PMC2749454  PMID: 19454677
dendritic cells; T cells; cancer; viruses
4.  Excitatory Local Circuits and Their Implications for Olfactory Processing in the Fly Antennal Lobe 
Cell  2007;128(3):601-612.
Summary
Conflicting views exist of how circuits of the antennal lobe, the insect equivalent of the olfactory bulb, translate input from olfactory receptor neurons (ORNs) into projection neuron (PN) output. Synaptic connections between ORNs and PNs are one-to-one, yet PNs are more broadly tuned to odors than ORNs. The basis for this difference in receptive range remains unknown. Analyzing a Drosophila mutant lacking ORN input to one glomerulus, we show that some of the apparent complexity in the antennal lobe’s output arises from lateral, interglomerular excitation of PNs. We describe a previously unidentified population of cholinergic local neurons (LNs) with multiglomerular processes. These excitatory LNs respond broadly to odors but exhibit little glomerular specificity in their synaptic output, suggesting that PNs are driven by a combination of glomerulus-specific ORN afferents and diffuse LN excitation. Lateral excitation may boost PN signals and enhance their transmission to third-order neurons in a mechanism akin to stochastic resonance.
doi:10.1016/j.cell.2006.12.034
PMCID: PMC2866183  PMID: 17289577
5.  Phagosomal membrane targeting of Irgm1 via PtdIns(3,4)P2 and PtdIns(3,4,5)P3 promotes mycobacterial immunity 
Nature immunology  2009;10(8):907-917.
Vertebrate immunity to infection enlists a new family of 47 kDa immunity-related GTPases (IRGs). One IRG in particular - Irgm1 - is essential for macrophage host defense against phagosomal pathogens including Mycobacterium tuberculosis (Mtb). Here we show Irgm1 targets the mycobacterial phagosome (PG) via lipid-mediated interactions with phosphoinositide-3,4-bisphosphate (PtdIns[3,4]P2) and PtdIns(3,4,5)P3. An isolated Irgm1 amphipathic helix conferred lipid binding in vitro and in vivo. Mutations in this region blocked PG recruitment and failed to complement the antimicrobial defect in Irgm1-/- macrophages. PtdIns(3,4,5)P3 removal or class I phosphoinositide-3-OH kinase (PI(3)K) inhibition mimicked this effect in wild-type cells. Irgm1-PI(3)K co-operation further facilitated Irgm1 engaging its fusogenic effectors at the site of infection, thereby ensuring pathogen-directed responses during innate immunity.
doi:10.1038/ni.1759
PMCID: PMC2715447  PMID: 19620982
Interferon-gamma; macrophage; tuberculosis; innate immunity
6.  An Internal Domain of Exo70p Is Required for Actin-independent Localization and Mediates Assembly of Specific Exocyst Components 
Molecular Biology of the Cell  2009;20(1):153-163.
The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.
doi:10.1091/mbc.E08-02-0157
PMCID: PMC2613103  PMID: 18946089
7.  Role of an Ancestral D-Bifunctional Protein Containing Two Sterol-Carrier Protein-2 Domains in Lipid Uptake and Trafficking in Toxoplasma 
Molecular Biology of the Cell  2009;20(2):658-672.
The inability to synthesize cholesterol is universal among protozoa. The intracellular pathogen Toxoplasma depends on host lipoprotein-derived cholesterol to replicate in mammalian cells. Mechanisms of cholesterol trafficking in this parasite must be important for delivery to proper organelles. We characterized a unique d-bifunctional protein variant expressed by Toxoplasma consisting of one N-terminal d-3-hydroxyacyl-CoA dehydrogenase domain fused to two tandem sterol carrier protein-2 (SCP-2) domains. This multidomain protein undergoes multiple cleavage steps to release free SCP-2. The most C-terminal SCP-2 carries a PTS1 that directs the protein to vesicles before processing. Abrogation of this signal results in SCP-2 accumulation in the cytoplasm. Cholesterol specifically binds to parasite SCP-2 but with 10-fold lower affinity than phosphatidylcholine. In mammalian cells and Toxoplasma, the two parasite SCP-2 domains promote the circulation of various lipids between organelles and to the surface. Compared with wild-type parasites, TgHAD-2SCP-2–transfected parasites replicate faster and show enhanced uptake of cholesterol and oleate, which are incorporated into neutral lipids that accumulate at the basal end of Toxoplasma. This work provides the first evidence that the lipid transfer capability of an ancestral eukaryotic SCP-2 domain can influence the lipid metabolism of an intracellular pathogen to promote its multiplication in mammalian cells.
doi:10.1091/mbc.E08-05-0482
PMCID: PMC2626556  PMID: 19005217
8.  Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission 
Nature cell biology  2007;9(3):310-315.
The spread of retroviruses between cells is estimated to be 2–3 orders of magnitude more efficient when cells can physically interact with each other1,2. The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virological or infectious synapses3–6. Here, we report the direct visualization of cell-to-cell transmission of retroviruses in living cells. Our results reveal a mechanism of virus transport from infected to non-infected cells, involving thin filopodial bridges. These filopodia originate from non-infected cells and interact, through their tips, with infected cells. A strong association of the viral envelope glycoprotein (Env) in an infected cell with the receptor molecules in a target cell generates a stable bridge. Viruses then move along the outer surface of the filopodial bridge toward the target cell. Our data suggest that retroviruses spread by exploiting an inherent ability of filopodia to transport ligands from cell to cell.
doi:10.1038/ncb1544
PMCID: PMC2628976  PMID: 17293854
9.  Hypoxia-Inducible Factor-Dependent Degeneration, Failure, and Malignant Transformation of the Heart in the Absence of the von Hippel-Lindau Protein▿  
Molecular and Cellular Biology  2008;28(11):3790-3803.
Hypoxia-inducible transcription factor 1 (HIF-1) and HIF-2α regulate the expression of an expansive array of genes associated with cellular responses to hypoxia. Although HIF-regulated genes mediate crucial beneficial short-term biological adaptations, we hypothesized that chronic activation of the HIF pathway in cardiac muscle, as occurs in advanced ischemic heart disease, is detrimental. We generated mice with cardiac myocyte-specific deletion of the von Hippel-Lindau protein (VHL), an essential component of an E3 ubiquitin ligase responsible for suppressing HIF levels during normoxia. These mice were born at expected frequency and thrived until after 3 months postbirth, when they developed severe progressive heart failure and premature death. VHL-null hearts developed lipid accumulation, myofibril rarefaction, altered nuclear morphology, myocyte loss, and fibrosis, features seen for various forms of human heart failure. Further, nearly 50% of VHL−/− hearts developed malignant cardiac tumors with features of rhabdomyosarcoma and the capacity to metastasize. As compelling evidence for the mechanistic contribution of HIF-1α, the concomitant deletion of VHL and HIF-1α in the heart prevented this phenotype and restored normal longevity. These findings strongly suggest that chronic activation of the HIF pathway in ischemic hearts is maladaptive and contributes to cardiac degeneration and progression to heart failure.
doi:10.1128/MCB.01580-07
PMCID: PMC2423296  PMID: 18285456
10.  Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis 
The Journal of Cell Biology  2008;180(5):905-914.
Ca2+ influx through plasma membrane lesions triggers a rapid repair process that was previously shown to require the exocytosis of lysosomal organelles (Reddy, A., E. Caler, and N. Andrews. 2001. Cell. 106:157–169). However, how exocytosis leads to membrane resealing has remained obscure, particularly for stable lesions caused by pore-forming proteins. In this study, we show that Ca2+-dependent resealing after permeabilization with the bacterial toxin streptolysin O (SLO) requires endocytosis via a novel pathway that removes SLO-containing pores from the plasma membrane. We also find that endocytosis is similarly required to repair lesions formed in mechanically wounded cells. Inhibition of lesion endocytosis (by sterol depletion) inhibits repair, whereas enhancement of endocytosis through disruption of the actin cytoskeleton facilitates resealing. Thus, endocytosis promotes wound resealing by removing lesions from the plasma membrane. These findings provide an important new insight into how cells protect themselves not only from mechanical injury but also from microbial toxins and pore-forming proteins produced by the immune system.
doi:10.1083/jcb.200708010
PMCID: PMC2265401  PMID: 18316410
11.  Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells 
The Journal of Cell Biology  2007;179(6):1133-1140.
Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.
doi:10.1083/jcb.200709111
PMCID: PMC2140027  PMID: 18070914
12.  The transcription factor XBP-1 is essential for the development and survival of dendritic cells 
The Journal of Experimental Medicine  2007;204(10):2267-2275.
Dendritic cells (DCs) play a critical role in the initiation, maintenance, and resolution of an immune response. DC survival is tightly controlled by extracellular stimuli such as cytokines and Toll-like receptor (TLR) signaling, but the intracellular events that translate such extracellular stimuli into life or death for the DC remain poorly understood. The endoplasmic reticulum (ER) stress, or unfolded protein response (UPR), is a signaling pathway that is activated when unfolded proteins accumulate in the ER. The most conserved arm of the UPR involves IRE1α, an ER transmembrane kinase and endoribonuclease that activates the transcription factor XBP-1 to maintain ER homeostasis and prevent activation of cell death pathways caused by sustained ER stress. We report that XBP-1 is essential for DC development and survival. Lymphoid chimeras lacking XBP-1 possessed decreased numbers of both conventional and plasmacytoid DCs with reduced survival both at baseline and in response to TLR signaling. Overexpression of XBP-1 in hematopoietic progenitors rescued and enhanced DC development. Remarkably, in contrast to other cell types we have examined, the XBP-1 pathway was constitutively activated in immature DCs.
doi:10.1084/jem.20070525
PMCID: PMC2118458  PMID: 17875675
13.  Radixin is Required to Maintain Apical Canalicular Membrane Structure and Function in Rat Hepatocytes 
Gastroenterology  2006;131(3):878-884.
Background and Aim
Ezrin-Radixin-Moesin (ERM) proteins are cross-linkers between the plasma membrane and actin filaments. Radixin, the dominant ERM protein in hepatocytes, has been reported to selectively tether Mrp2 to the apical canalicular membrane. However it remains to be determined if this is its primary function.
Methods
An adenovirus-mediated siRNA was used to down-regulate radixin expression in collagen sandwich cultured rat hepatocytes and morphological and functional changes were characterized quantitatively.
Results
In control cultures, an extensive bile canalicular network developed with properly localized apical and basolateral transporters that provided for functional excretion of fluorescent cholephiles into the bile canalicular lumina. SiRNA induced suppression of radixin was associated with a marked reduction in the canalicular membrane structure as observed by differential interference contrast microscopy and F-actin staining, in contrast to control cells exposed to adenovirus encoding scrambled siRNA. Indirect immunofluorescence demonstrated that apical transporters (Mrp2, Bsep and Mdr1) dissociated from their normal location at the apical membrane and were found largely associated with Rab11-containing endosomes. Localization of the basolateral membrane transporter, Oatp2, was not affected. Consistent with this dislocation of apical transporters, the biliary excretion of GS-MF and CGamF was significantly decreased in the radixin-deficient cells but not in the control siRNA cells.
Conclusions
Radixin is essential for maintaining the polarized targeting and/or retaining of canalicular membrane transporters and is a critical determinant of the overall structure and function of the apical membrane of hepatocytes.
doi:10.1053/j.gastro.2006.06.013
PMCID: PMC1820831  PMID: 16952556
ERM; siRNA; bile transporter; bile canaliculi; Rab11
14.  MHC class II antigen loading compartments continuously receive input from autophagosomes 
Immunity  2006;26(1):79-92.
SUMMARY
Major histocompatibility complex (MHC) class II molecules present products of lysosomal proteolysis to CD4+ T cells. While extracellular antigen uptake is considered to be the main source of MHC class II-ligands, a few intracellular antigens have been described to gain access to MHC class II loading after macroautophagy. However, the general relevance and efficacy of this pathway is unknown. Here we demonstrate constitutive autophagosome formation in MHC class II positive cells, including dendritic, B and epithelial cells. The autophagosomes continuously fuse with multivesicular MHC class II-loading compartments (MIICs). This pathway is of functional relevance, since targeting of the Influenza Matrix Protein 1 (MP1) to autophagosomes via fusion to the autophagosome-associated protein Atg8/LC3 led to strongly enhanced MHC class II presentation to CD4+ T cell clones. We suggest that macroautophagy constitutively and efficiently delivers cytosolic proteins for MHC class II presentation and can be harnessed for improved helper T cell stimulation.
doi:10.1016/j.immuni.2006.10.018
PMCID: PMC1805710  PMID: 17182262
MHC class II; macroautophagy; CD4+ T cells; Influenza matrix protein 1; Atg8/LC3
15.  Internalization, Intracellular Trafficking, and Biodistribution of Monoclonal Antibody 806: A Novel Anti-Epidermal Growth Factor Receptor Antibody12 
Neoplasia (New York, N.Y.)  2007;9(12):1099-1110.
Overexpression of the epidermal growth factor receptor (EGFR) in epithelial tumors is associated with poor prognosis and is the target for a number of cancer therapeutics. Monoclonal antibody (mAb) 806 is a novel anti-EGFR antibody with significant therapeutic efficacy in tumor models when used as a single agent, and displays synergistic antitumor activity in combination with other EGFR therapeutics. Unlike other EGFR antibodies, mAb 806 is selective for tumor cells and does not bind to normal tissue, making it an ideal candidate for generation of radioisotope or toxin conjugates. Ideally, antibodies suited to these therapeutic applications must bind to and actively internalize their cognate receptor. We investigated the intracellular trafficking of fluorescently tagged mAb 806 in live cells and analyzed its biodistribution in a tumor xenografted nude mouse model. Following binding to EGFR, mAb 806 was internalized through dynamin-dependent, clathrin-mediated endocytosis. Internalized mAb 806 localized to early endosomes and subsequently trafficked to and accumulation in lysosomal compartments. Furthermore, biodistribution analysis in nude mice showed specific uptake and retention of radiolabeled mAb 806 to human tumor xenografts. These results highlight the potential use of mAb 806 for generation of conjugates suitable for diagnostic and therapeutic use in patients with EGFR-positive malignancies.
PMCID: PMC2134906  PMID: 18084617
Internalization; epidermal growth factor receptor; intracellular trafficking; dynamin; monoclonal antibody
16.  v-SNARE cellubrevin is required for basolateral sorting of AP-1B–dependent cargo in polarized epithelial cells 
The Journal of Cell Biology  2007;177(3):477-488.
The epithelial cell–specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B–dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.
doi:10.1083/jcb.200610047
PMCID: PMC2034334  PMID: 17485489
17.  v-SNARE cellubrevin is required for basolateral sorting of AP-1B–dependent cargo in polarized epithelial cells 
The Journal of cell biology  2007;177(3):477-488.
The epithelial cell–specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B–dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.
doi:10.1083/jcb.200610047
PMCID: PMC2034334  PMID: 17485489
18.  RNAi Screen in Drosophila Cells Reveals the Involvement of the Tom Complex in Chlamydia Infection 
PLoS Pathogens  2007;3(10):e155.
Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.
Author Summary
Chlamydia spp. are intracellular bacterial pathogens that infect a wide range of hosts and cause various diseases, including preventable blindness in developing countries, sexually transmitted disease, and pneumonia. Chlamydia spp. are able to establish their replication niche inside the host cell, residing in a membrane-bound compartment that serves as a protector shield against immune surveillance and antimicrobial agents but also acts as a “filter” to exchange factors with the host cell. Despite the primary importance of Chlamydia for human health, little is known about the mechanisms underlying the infection process. The study of Chlamydia pathogenesis is challenging because Chlamydia spp. are not amenable to genetic manipulation and it is difficult to conduct extensive genetic approaches in the mammalian host. To circumvent these difficulties, we have used Drosophila cells to model Chlamydia infection. We conducted a genome-wide RNA interference screen and identified host factors that, when depleted, reduce Chlamydia infection. Validating our approach, we further showed that the identified factors were also required for infection in mammalian cells. This work will help us better understand the complex interaction between Chlamydia and its host and potentially identify novel targets for therapeutic treatment.
doi:10.1371/journal.ppat.0030155
PMCID: PMC2042019  PMID: 17967059
19.  Ca2+ and synaptotagmin VII–dependent delivery of lysosomal membrane to nascent phagosomes 
The Journal of Cell Biology  2006;174(7):997-1007.
Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because [Ca2+]i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII−/− mice. Syt VII−/− macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII−/− macrophages is significantly less dependent on [Ca2+]i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII−/− macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis.
doi:10.1083/jcb.200605004
PMCID: PMC2064391  PMID: 16982801
20.  Aging-Associated Reductions in AMP-Activated Protein Kinase Activity and Mitochondrial Biogenesis 
Cell Metabolism  2007;5(2):151-156.
Summary
Recent studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function, dysregulated intracellular lipid metabolism, and insulin resistance. Given the important role of the AMP-activated protein kinase (AMPK) in the regulation of fat oxidation and mitochondrial biogenesis, we examined AMPK activity in young and old rats and found that acute stimulation of AMPK-α2 activity by 5′-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and exercise was blunted in skeletal muscle of old rats. Furthermore, mitochondrial biogenesis in response to chronic activation of AMPK with β-guanidinopropionic acid (β-GPA) feeding was also diminished in old rats. These results suggest that aging-associated reductions in AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging.
doi:10.1016/j.cmet.2007.01.008
PMCID: PMC1885964  PMID: 17276357
HUMDISEASE
21.  mBet3p is required for homotypic COPII vesicle tethering in mammalian cells 
The Journal of Cell Biology  2006;174(3):359-368.
TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.
doi:10.1083/jcb.200603044
PMCID: PMC2064232  PMID: 16880271
22.  CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis 
The Journal of Cell Biology  2006;172(7):1045-1056.
Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5−/− cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5−/− cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.
doi:10.1083/jcb.200509041
PMCID: PMC2063762  PMID: 16567502
23.  Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum 
Molecular Biology of the Cell  2006;17(7):3009-3020.
The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.
doi:10.1091/mbc.E06-01-0080
PMCID: PMC1483037  PMID: 16624861
24.  Mutants in trs120 disrupt traffic from the early endosome to the late Golgi 
The Journal of Cell Biology  2005;171(5):823-833.
Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.
doi:10.1083/jcb.200505145
PMCID: PMC2171297  PMID: 16314430
25.  Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells 
The Journal of Cell Biology  2005;170(2):317-325.
Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, “surfing” toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.
doi:10.1083/jcb.200503059
PMCID: PMC2171413  PMID: 16027225

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