Fluid-phase Endocytosis Research Articles

The role of seawater endocytosis in the biomineralization process in calcareous foraminifera

Foraminifera are unicellular organisms that inhabit the oceans in various ecosystems. The majority of the foraminifera precipitate calcitic shells and are among the major CaCO(3) producers in the oceans. They comprise an important component of the global carbon cycle and also provide valuable paleoceanographic information based on the relative abundance of stable isotopes and trace elements (proxies) in their shells. Understanding the biomineralization processes in foraminifera is important for predicting their calcification response to ocean acidification and for reliable interpretation of the paleoceanographic proxies. Most models of biomineralization invoke the involvement of membrane ion transporters (channels and pumps) in the delivery of Ca(2+) and other ions to the calcification site. Here we show, in contrast, that in the benthic foraminiferan Amphistegina lobifera, (a shallow water species), transport of seawater via fluid phase endocytosis may account for most of the ions supplied to the calcification site. During their intracellular passage the seawater vacuoles undergo alkalization that elevates the CO(3)(2-) concentration and further enhances their calcifying potential. This mechanism of biomineralization may explain why many calcareous foraminifera can be good recorders of paleoceanographic conditions. It may also explain the sensitivity to ocean acidification that was observed in several planktonic and benthic species.

On Some Aspects of the Thermodynamic of Membrane Recycling Mediated by Fluid Phase Endocytosis: Evaluation of Published Data and Perspectives

The theoretical and experimental description of fluid phase endocytosis (FPE) requires an asymmetry in phospholipid number between the two leaflets of the cell membrane, which provides the biomechanical torque needed to generate membrane budding. Although the motor force behind FPE is defined, its kinetic has yet to be determined. Based on a body of evidences suggesting that the mean surface tension is unlikely to be involved in endocytosis we decided to determine whether the cytosolic hydrostatic pressure could be involved, by considering a constant energy exchanged between the cytosol and the cell membrane. The theory is compared to existing experimental data obtained from FPE kinetic studies in living cells where altered phospholipid asymmetry or changes in the extracellular osmotic pressure have been investigated. The model demonstrates that FPE is dependent on the influx and efflux of vesicular volumes (i.e. vesicular volumes recycling) rather than the membrane tension of cells. We conclude that: (i) a relationship exists between membrane lipid number asymmetry and resting cytosolic pressure and (ii) the validity of Laplace's law is limited to cells incubated in a definite hypotonic regime. Finally, we discuss how the model could help clarifying elusive observations obtained from different fields and including: (a) the non-canonical shuttling of aquaporin in cells, (b) the relationship between high blood pressure and inflammation and (c) the mechanosensitivity of the sodium/proton exchanger.

The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Sterol and Diacylglycerol Acyltransferase Deficiency Triggers Fatty Acid-mediated Cell Death

Deletion of the acyltransferases responsible for triglyceride and steryl ester synthesis in Saccharomyces cerevisiae serves as a genetic model of diseases where lipid overload is a component. The yeast mutants lack detectable neutral lipids and cytoplasmic lipid droplets and are strikingly sensitive to unsaturated fatty acids. Expression of human diacylglycerol acyltransferase 2 in the yeast mutants was sufficient to reverse these phenotypes. Similar to mammalian cells, fatty acid-mediated death in yeast is apoptotic and presaged by transcriptional induction of stress-response pathways, elevated oxidative stress, and activation of the unfolded protein response. To identify pathways that protect cells from lipid excess, we performed genetic interaction and transcriptional profiling screens with the yeast acyltransferase mutants. We thus identified diacylglycerol kinase-mediated phosphatidic acid biosynthesis and production of phosphatidylcholine via methylation of phosphatidylethanolamine as modifiers of lipotoxicity. Accordingly, the combined ablation of phospholipid and triglyceride biosynthesis increased sensitivity to saturated fatty acids. Similarly, normal sphingolipid biosynthesis and vesicular transport were required for optimal growth upon denudation of triglyceride biosynthesis and also mediated resistance to exogenous fatty acids. In metazoans, many of these processes are implicated in insulin secretion thus linking lipotoxicity with early aspects of pancreatic beta-cell dysfunction, diabetes, and the metabolic syndrome.

Rab5 Isoforms Differentially Regulate the Trafficking and Degradation of Epidermal Growth Factor Receptors

Ligand-mediated endocytosis is an intricate regulatory mechanism for epidermal growth factor receptor (EGFR) signal transduction. Coordinated trafficking of EGFR ensures its temporal and spatial communication with downstream signaling effectors. We focused our work on Rab5, a monomeric GTPase shown to participate in early stages of the endocytic pathway. Rab5 has three isoforms (A, B, and C) sharing more than 90% of sequence identity. We individually ablated endogenous isoforms in HeLa cells with short interfering RNAs and examined the loss-of-function phenotypes. We found that suppression of Rab5A or 5B hampered the degradation of EGFR, whereas Rab5C depletion had very little effect. The differential delay of EGFR degradation also corresponds with retarded progression of EGFR from early to late endosomes. We investigated the activators/effectors of Rab5A that can potentially separate its potency in EGFR degradation from other isoforms and found that Rin1, a Rab5 exchange factor, preferably associated with Rab5A. Moreover, Rab5A activation is sensitive to EGF stimulation, and suppression of Rin1 diminished this sensitivity. Based on our results together with previous work showing that Rin1 interacts with signal transducing adapter molecule to facilitate the degradation of EGFR (Kong, C., Su, X., Chen, P. I., and Stahl, P. D. (2007) J. Biol. Chem. 282, 15294-15301), we hypothesize that the selective association of Rab5A and Rin1 contributes to the dominance of Rab5A in EGFR trafficking, whereas the other isoforms may have major functions unrelated to the EGFR degradation pathway.

Actomyosin contractility controls cell surface area of oligodendrocytes

BackgroundTo form myelin oligodendrocytes expand and wrap their plasma membrane multiple times around an axon. How is this expansion controlled?ResultsHere we show that cell surface area depends on actomyosin contractility and is regulated by physical properties of the supporting matrix. Moreover, we find that chondroitin sulfate proteoglycans (CSPG), molecules associated with non-permissive growth properties within the central nervous system (CNS), block cell surface spreading. Most importantly, the inhibitory effects of CSPG on plasma membrane extension were completely prevented by treatment with inhibitors of actomyosin contractility and by RNAi mediated knockdown of myosin II. In addition, we found that reductions of plasma membrane area were accompanied by changes in the rate of fluid-phase endocytosis.ConclusionIn summary, our results establish a novel connection between endocytosis, cell surface extension and actomyosin contractility. These findings open up new possibilities of how to promote the morphological differentiation of oligodendrocytes in a non-permissive growth environment.See related minireview by Bauer and ffrench-Constant:

Negative Guidance Factor-Induced Macropinocytosis in the Growth Cone Plays a Critical Role in Repulsive Axon Turning

Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth, including growth cone collapse and chemorepulsive axon turning but not turning per se. Conversely, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons and elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2, and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.

Fluid Phase Endocytosis Contributes to Transfection of DNA by PEI-25

The understanding of internalization pathways of lipo- or polyplexes is crucial for engineering successful reagents for nonviral gene transfection. A known inhibitor of fluid phase endocytosis (FPE), rottlerin, was used to quantify the contribution of this pathway by flow cytometric and fluorescence assays. Rottlerin was shown to be a specific inhibitor of transfection by polyethylene imine (PEI-25)/DNA complexes, leading to a decrease in the amount of transfected HeLa and CHO-K1 cells and a decrease in the expression of enhanced green fluorescent protein (EGFP) reporter gene by up to 50%. Experiments using fluorescently labeled polyplexes result in a decrease of uptake by up to 40%. Additionally, rottlerin does not cross-inhibit clathrin- and caveolin-mediated endocytotic pathways of internalization, consistent with direct uptake inhibition by rottlerin. Nonspecific effects as a result of toxicity were ruled out by control experiments at concentrations where rottlerin inhibition was specific. These findings suggest that for CHO-K1 and HeLa cells, internalization of PEI-25/DNA complexes by FPE plays a decisive role in gene transfection. The establishment of an additional pathway that is independent of clathrin- and caveolin-mediated endocytotic uptake may have an impact on the design of future reagents of nonviral gene therapy and investigations of the uptake pathways and intracellular trafficking involved.

Evidence for two endocytic transport pathways in plant cells

Summary Intracellular trafficking of endocytic vesicles in eukaryotes varies with the nature of the cargo molecules and the targeted organelle, and proceeds through an intricate network of internal endosomal compartments. However, the path for fluid-phase endocytosis (FPE), the internalization of external solutes from the apoplast via plasmalemma generated vesicles, remains unresolved despite some indication of a direct transport route to the vacuole. To test this hypothesis, we made use of the membrane-impermeable Na-dependent fluorescent marker Coro-Na in combination with the fluorescent membrane marker FM 4-64 and confocal laser scanning microscopy. When protoplasts from sweet lime juice cells were incubated in Na-free solution, FM 4-64, Coro-Na, and 200 mM sucrose, two distinct types of labeled vesicles were evident. A set of vesicles (∼1 μm in diameter) was intensely labeled with Coro-Na and to a lesser extent with FM 4-64, whereas the second type of 1–7 μm structures appeared exclusively labeled with FM 4-64. These data demonstrate the parallel functioning of two endocytic pathways in plant cells. In one system, a set of small endocytic vesicles merge with the endosome, whereas a separate set of vesicles fuse to form larger vesicles independent from the endosome. Although it is likely that both vesicle systems eventually contribute to solutes reaching the vacuole, given their size (1–7 μm), and based on previous observations of endocytic vesicle formation protruding from the plasmalemma and merging with the vacuole, we conclude that these latter vesicles constitute the primary FPE vesicle system.

551 Shiga Toxin 1 Uptake By Intestinal Epithelial Cells Is CDC-42 Mediated and Clathrin-Independent

Shiga toxins (Stx) 1 and 2 are major contributors to foodborne disease caused by enterohemorrhagic E.coli (EHEC). Human intestinal epithelial cells (IEC) do not normally express Stx receptor (the glycosphingolipid Gb3), leaving the mechanism of Stx uptake poorly understood. Inhibition of clathrin-mediated endocytosis significantly increases Stx1 uptake in Gb3-free IEC. We thus now test the hypothesis that cell division cycle 42 (Cdc-42) dependent fluid phase endocytosis might be involved in clathrin-independent Stx1 uptake by IEC In Vitro and In Vivo. Methods: In Vivo: We compared pathologies in New Zealand white rabbits fed with either a) an experimental RDEC-H19A strain engineered to express Stx1, since it produces intestinal pathology quite similar to that noted in human EHEC or b) a control RDEC-1 E. coli strain that adheres to rabbit mucosa to provide an attaching and effacing lesion or c) PBS, to provide a negative control for effects of bacterial infection. Immunofluorescence of frozen sections from cecal tissue of rabbits studied at times up to 7 days after infection assessed a) uptake of Stx1 and b) presence of activated Cdc-42 in IEC. In Vitro: The apical uptake of the recombinant B-subunit of Stx1 labeled with Alexa 680 fluorescent dye (Stx1B) was assessed in T84 IEC that were: a) treated with agents known to inhibit clathrin-mediated endocytosis, b) depleted of Cdc42 by shRNA or c) untreated controls using biochemical and immunofluorescence methods. Results: 1) Cecal tissues from rabbits treated with either RDEC-1 or RDEC-H19A bacteria, but not those from PBS-treated animals, displayed Cdc42 activation in subsets of epithelial cells at sites near and those distant from attached bacteria. 2) T84 cells incubated with the inhibitors of clathrin mediated endocytosis chlorpromazine or low potassium increased Stx1B uptake in a time-dependent fashion. This stimulation was virtually eliminated, however, in T84 cells in which Cdc-42 was downregulated using shRNA. 3) The specific blocker of Cdc-42 function and nucleotide exchange, Pirl-1, also prevented stimulation of Stx1B uptake in concentration-dependent way in cells treated with inhibitors of clathrin-mediated endocytosis. 4) This clathrin independent Cdc-42 mediated Stx1B uptake was also dynamin dependent. Dynasore, a specific inhibitor of dynamin GTPase activity, inhibited the Stx1B uptake in cells with inactivated clathrin induced endocytic pathway. Conclusions: 1) Basal Stx1uptake by Gb3 negative T84 cells is regulated by Cdc-42. 2) Cdc-42 dependent clathrin independent pathway is likely to be involved in IEC uptake of Stx1 during EHEC infections.

Stress-induced Ceramide-activated Protein Phosphatase Can Compensate for Loss of Amphiphysin-like Activity In Saccharomyces cerevisiae and Functions to Reinitiate Endocytosis

Saccharomyces cerevisiae cells lacking the amphiphysin-like orthologs, Rvs161 or Rvs167, are unable to thrive under many stress conditions. Here we show cells lacking Rvs161 require Cdc55, the B subunit of the yeast ceramide-activated protein phosphatase, for viability under heat stress. By using specific rvs mutant alleles, we linked this lethal genetic interaction to loss of Rvs161 endocytic domain function. Recessive mutations in the sphingolipid pathway, such as deletion of the very long-chain fatty acid elongase, Sur4, suppress the osmotic growth defect of rvs161 cells. We demonstrate that Cdc55 is required for sur4-dependent suppressor activity and that protein phosphatase activation, through overexpression of CDC55 alone, can also remediate this defect. Loss of SUR4 in rvs161 cells reinitiates Ste3 a-factor receptor endocytosis and requires Cdc55 function to do so. Moreover, overexpression of CDC55 reinitiates Ste3 endocytic-dependent degradation and restores fluid phase endocytosis in rvs161 cells. In contrast, loss of SUR4 or CDC55 overexpression does not remediate the actin polarization defects of osmotic stressed rvs161 cells. Importantly, remediation of rvs161 defects by protein phosphatase activation requires the ceramide-activated protein phosphatase catalytic subunit, Sit4, and the protein phosphatase 2A catalytic subunits, Pph21/Pph22. Finally, genetic analyses reveal a synthetic lethal interaction between loss of CDC55 and gene deletions lethal with rvs161, all of which function in endocytosis.

Propagation of Tau Misfolding from the Outside to the Inside of a Cell

Tauopathies are neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau in neurons and glia. Although Tau is normally considered an intracellular protein, Tau aggregates are observed in the extracellular space, and Tau peptide is readily detected in the cerebrospinal fluid of patients. Tau aggregation occurs in many diseases, including Alzheimer disease and frontotemporal dementia. Tau pathology begins in discrete, disease-specific regions but eventually involves much larger areas of the brain. It is unknown how this propagation of Tau misfolding occurs. We hypothesize that extracellular Tau aggregates can transmit a misfolded state from the outside to the inside of a cell, similar to prions. Here we show that extracellular Tau aggregates, but not monomer, are taken up by cultured cells. Internalized Tau aggregates displace tubulin, co-localize with dextran, a marker of fluid-phase endocytosis, and induce fibrillization of intracellular full-length Tau. These intracellular fibrils are competent to seed fibril formation of recombinant Tau monomer in vitro. Finally, we observed that newly aggregated intracellular Tau transfers between co-cultured cells. Our data indicate that Tau aggregates can propagate a fibrillar, misfolded state from the outside to the inside of a cell. This may have important implications for understanding how protein misfolding spreads through the brains of tauopathy patients, and it is potentially relevant to myriad neurodegenerative diseases associated with protein misfolding.

A simple method for cryopreservation of MDBK cells using trehalose and storage at −80°C

Madin Darby bovine kidney cells were stored at -80 degrees C using trehalose. Trehalose was loaded into the cells by fluid-phase endocytosis that was facilitated by heat shock at 40 degrees C for 1 h. Loaded cells were gradually frozen and stored at -80 degrees C. Revival of cells was done by quick thawing and immediately seeded in the tissue culture flasks. The membrane integrity of cells was measured at different times post-storage by trypan blue dye exclusion method. It was estimated to be 96.23, 73.84, 57.33, 54.36, 25.47, 50.53 and 46.86% at 0, 7, 60, 90, 120, 160 and 180-day post-storage, respectively. Cryostorage of cells at -80 degrees C may help to reduce the use of liquid nitrogen.

Gel Electrophoresis Method to Measure the Concentration of Single-Walled Carbon Nanotubes Extracted from Biological Tissue

A rapid and sensitive method to detect single-walled carbon nanotubes (SWNTs) in biological samples is presented. The method uses polyacrylamide gel electrophoresis (PAGE) followed by quantification of SWNT bands. SWNTs dispersed in bovine serum albumin (BSA) were used to develop the method. When BSA-SWNT dispersions were subjected to sodium dodecyl sulfate (SDS)-PAGE, BSA passed through the stacking gel, entered the resolving gel, and migrated toward the anode as expected. The SWNTs, however, accumulated in a sharp band at the interface between the loading well and the stacking gel. The intensities from digitized images of these bands were proportional to the amount of SWNTs loaded onto the gel with a detection limit of 5 ng of SWNTs. To test the method, normal rat kidney (NRK) cells in culture were allowed to take up SWNTs upon exposure to medium containing various concentrations of BSA-SWNTs for different times and temperatures. The SDS-PAGE analyses of cell lysate samples suggest that BSA-SWNTs enter NRK cells by fluid-phase endocytosis at a rate of 30 fg/day/cell upon exposure to medium containing 98 microg/mL SWNTs.

Cell polarity emerges at first cleavage in sea urchin embryos

In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers GM1, integrins, and the aPKC–PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical–basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the GM1, integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow.

Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

BackgroundEnhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2).Methodology/Principal FindingsMass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm.Conclusions/SignificanceOur data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.

Scrapie Pathogenesis: The Role of Complement C1q in Scrapie Agent Uptake by Conventional Dendritic Cells

Mice lacking complement components show delayed development of prion disease following peripheral inoculation. The delay could relate to reduced scrapie prion protein (PrP(Sc)) accumulation on follicular dendritic cells (DCs). However conventional DCs (cDCs) play a crucial role in the early pathogenesis of prion diseases and complement deficiency could result in decreased PrP(Sc) uptake by cDCs in the periphery. To explore this possibility, we cultured murine splenic or gut-associated lymph node cDCs with scrapie-infected whole brain homogenate in the presence or absence of complement. Uptake decreased significantly if the serum in the cultures was heat-inactivated. Because heat inactivation primarily denatures C1q, we used serum from C1q(-/-) mice and showed that PrP(Sc) uptake was markedly decreased. PrP(Sc) internalization was saturable and temperature-dependent, suggesting receptor-mediated uptake. Furthermore, uptake characteristics differed from fluid-phase endocytosis. Immunofluorescence showed colocalization of C1q and PrP(Sc), suggesting interaction between these molecules. We evaluated the expression of several complement receptors on cDCs and confirmed that cDCs that take up PrP(Sc) express one of the C1q receptors, calreticulin. Our results show that C1q participates in PrP(Sc) uptake by cDCs, revealing a critical role for cDCs in initial prion capture, an event that takes place before the PrP(Sc) accumulation within the follicular DC network.

The C. elegans P4‐ATPase TAT‐1 Regulates Lysosome Biogenesis and Endocytosis

P-type adenosine triphosphatases (ATPases) of the Drs2p family (P4-ATPases) are multipass transmembrane proteins required to generate and maintain phospholipid asymmetry in membrane bilayers. In Saccharomyces cerevisiae, several members of this family control distinct transport events within the endosomal and secretory pathways. Comparatively, little is known about the functions of P4-ATPases in multicellular organisms. In this study, we analyzed the role of the Caenorhabditis elegans Drs2p homologue transbilayer amphipath transporter (TAT)-1 in intracellular trafficking. tat-1 is expressed in many tissues including the intestine, the epidermis and the nervous system. In intestinal cells, tat-1 loss-of-function mutants accumulate large vacuoles of mixed endolysosomal identity positive for the lysosomal protein LMP-1. In addition, they lack the same class of storage granules as lmp-1 mutants, suggesting that part of the tat-1 phenotype might result from LMP-1 sequestration in an aberrant compartment. Epidermal cells mutant for tat-1 contain acidified giant hybrid multivesicular bodies probably corresponding to endolysosomal intermediate compartments or deficient lysosomes. Finally, TAT-1 is required for yolk uptake in oocytes and an early step of fluid-phase endocytosis in the intestine. Hence, TAT-1 is required at multiple steps of the endolysosomal pathway, at least in part by ensuring proper trafficking of cell-specific effector proteins.

Glutathione depletion reveals impairment of antigen processing and inhibition of cathepsin activity by nitric oxide in antigen-presenting cells

Nitric oxide has been shown to induce immunosuppression by inhibiting class II MHC molecule expression and T-lymphocyte proliferation. However, much less is known about the ability of NO to interfere with antigen processing and presentation. So we questioned whether B lymphoma cells exposed to NO could be impaired in their ability to process lysozyme and to stimulate proliferation of a syngeneic T-cell hybridoma. As immunosuppressive pathological conditions are often associated with a pro-oxidative milieu, we also examined the influence of intracellular GSH levels on NO responsiveness. Exposure of GSH-depleted B cells to NO-releasing compounds lowered their capacity to present a reduced and alkylated lysozyme (TAP–HEL), although presentation of a lysozyme-derived peptide was unaffected. Cells with a normal GSH content were protected from this inhibition. Fluid phase endocytosis, protein synthesis and expression of class II molecules remained normal in GSH-depleted cells. However, proteolysis of a dye conjugate of ovalbumin was strongly inhibited, suggesting that protease inhibition might be involved. Cathepsin B activity, which was necessary to TAP–HEL processing, was inhibited by the NO-donors. The inhibition was higher in GSH-depleted cells and reproduced by treatment of A20 B cells by two cathepsin inhibitors. These results show that, in addition to cytostasis and reduction in class II expression, NO-induced immunosuppression could also implicate inhibition of antigen processing under oxidative stress conditions.