Distinct Cellular Pools Research Articles

Circulating T Cells Are Not Sufficient for Protective Immunity against Virulent Francisella tularensis.

Pulmonary infections elicit a combination of tissue-resident and circulating T cell responses. Understanding the contribution of these anatomically distinct cellular pools in protective immune responses is critical for vaccine development. Francisella tularensis is a highly virulent bacterium capable of causing lethal systemic disease following pulmonary infection for which there is no currently licensed vaccine. Although T cells are required for survival of F. tularensis infection, the relative contribution of tissue-resident and circulating T cells is not completely understood, hampering design of effective, long-lasting vaccines directed against this bacterium. We have previously shown that resident T cells were not sufficient to protect against F. tularensis, suggesting circulating cells may serve a critical role in host defense. To elucidate the role of circulating T cells, we used a model of vaccination and challenge of parabiotic mice. Intranasally infected naive mice conjoined to immune animals had increased numbers of circulating memory T cells and similar splenic bacterial burdens as vaccinated-vaccinated pairs. However, bacterial loads in the lungs of naive parabionts were significantly greater than those observed in vaccinated-vaccinated pairs, but despite early control of F. tularensis replication, all naive-vaccinated pairs succumbed to infection. Together, these data define the specific roles of circulating and resident T cells in defense against infection that is initiated in the pulmonary compartment but ultimately causes disseminated disease. These data also provide evidence for employing vaccination strategies that elicit both pools of T cells for immunity against F. tularensis and may be a common theme for other disseminating bacterial infections.

The ATP binding cassette transporter, ABCG1, localizes to cortical actin filaments

The ATP-binding cassette sub-family G member 1 (ABCG1) exports cellular cholesterol to high-density lipoproteins (HDL). However, a number of recent studies have suggested ABCG1 is predominantly localised to intracellular membranes. In this study, we found that ABCG1 was organized into two distinct cellular pools: one at the plasma membrane and the other associated with the endoplasmic reticulum (ER). The plasma membrane fraction was organized into filamentous structures that were associated with cortical actin filaments. Inhibition of actin polymerization resulted in complete disruption of ABCG1 filaments. Cholesterol loading of the cells increased the formation of the filamentous ABCG1, the proximity of filamentous ABCG1 to actin filaments and the diffusion rate of membrane associated ABCG1. Our findings suggest that the actin cytoskeleton plays a critical role in the plasma membrane localization of ABCG1.

The role of lipids in host microbe interactions.

Lipids are one of the major subcellular constituents and serve as signal molecules, energy sources, metabolic precursors and structural membrane components in various organisms. The function of lipids can be modified by multiple biochemical processes such as (de-)phosphorylation or (de-)glycosylation, and the organization of fatty acids into distinct cellular pools and subcellular compartments plays a pivotal role for the morphology and function of various cell populations. Thus, lipids regulate, for example, phagosome formation and maturation within host cells and thus, are critical for the elimination of microbial pathogens. Vice versa, microbial pathogens can manipulate the lipid composition of phagosomal membranes in host cells, and thus avoid their delivery to phagolysosomes. Lipids of microbial origin belong also to the strongest and most versatile inducers of mammalian immune responses upon engagement of distinct receptors on myeloid and lymphoid cells. Furthermore, microbial lipid toxins can induce membrane injuries and cell death. Thus, we will review here selected examples for mutual host-microbe interactions within the broad and divergent universe of lipids in microbial defense, tissue injury and immune evasion.

A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.

In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

Regulated endosomal trafficking of Diacylglycerol lipase alpha (DAGLα) generates distinct cellular pools; implications for endocannabinoid signaling.

Diacylglycerol lipase alpha (DAGLα) generates the endocannabinoid (eCB) 2-arachidonylglycerol (2-AG) that regulates the proliferation and differentiation of neural stem cells and serves as a retrograde signaling lipid at synapses. Nothing is known about the dynamics of DAGLα expression in cells and this is important as it will govern where 2-AG can be made and released. We have developed a new construct to label DAGLα at the surface of live cells and follow its trafficking. In hippocampal neurons a cell surface pool of DAGLα co-localizes with Homer, a postsynaptic density marker. This surface pool of DAGLα is dynamic, undergoing endocytosis and recycling back to the postsynaptic membrane. A similar cycling is seen in COS-7 cells with the internalized DAGLα initially transported to EEA1 and Rab5-positive early endosomes via a clathrin-independent pathway before being transported back to the cell surface. The internalized DAGLα is present on reticular structures that co-localize with microtubules. Importantly, DAGLα cycling is a regulated process as inhibiting PKC results in a significant reduction in endocytosis. This is the first description of DAGLα cycling between the cell surface and an intracellular endosomal compartment in a manner that can regulate the level of the enzyme at the cell surface.

Retraction: 'An endogenous, systemic RNAi pathway in plants'.

The above article from The EMBO Journal , published online on 22 April 2010, has been retracted by agreement between the authors, the journal Chief Editor and Head of Scientific Publications, EMBO, Bernd Pulverer, and John Wiley & Sons Ltd. The authors' and editors' statements are as follows. Following an investigation conducted by the The EMBO Journal , the corresponding authors were alerted of several image irregularities found in this paper. Patrice Dunoyer, who assembled the figures, acknowledges making the following errors …

Modulation of Cellular Mg2+ Content in Cardiac Cells by α1-Adrenoceptor Stimulation and Anti-Arrhythmic Agents

Magnesium (Mg2+) is used pharmacologically to sedate specific forms of arrhythmias. Administration of pharmacological doses of catecholamine or adrenergic receptor agonists often results in arrhythmias onset. Results from the present study indicate that stimulation of cardiac adrenergic receptors elicits an extrusion of cellular Mg2+ into the extracellular space. This effect occurs in both perfused hearts and isolated cells within 5-6 min following either β- or α1-adrenergic receptor stimulation, and is prevented by specific adrenergic receptors antagonists. Sequential stimulation of the two classes of adrenergic receptor results in a larger mobilization of cellular Mg2+ provided that the two agonists are administered together or within 1-2 min from each other. A longer delay in administering the second stimulus results in the abolishment of Mg2+ extrusion. Hence, these data suggest that the stimulation of β- and α1-adrenergic receptors mobilizes Mg2+ from two distinct cellular pools, and that Mg2+ loss from either pool triggers a Mg2+ redistribution within the cardiac myocyte. At the sarcolemmal level, Mg2+ extrusion occurs through a Na+/Mg2+ exchange mechanism phosphorylated by cAMP. Administration of quinidine, a patent anti-arrhythmic agent, blocks Na+ transport in a non-specific manner and prevents Mg2+ extrusion. Taken together, these data indicate that catecholamine administration induces dynamic changes in total and compartmentalized Mg2+ pools within the cardiac myocytes, and suggest that prevention of Mg2+ extrusion and redistribution may be an integral component of the effectiveness of quinidine and possibly other cardiac anti-arrhythmic agents. Confirmation of this possibility by future experimental and clinical studies might result in new patents of these compounds as Mg2+ preserving agents.

Transport and distribution of 45Ca2 + in the perfused rat liver and the influence of adjuvant-induced arthritis

The purpose of the present work was to investigate Ca2+ transport and distribution under the conditions of the intact rat liver in health and disease (adjuvant-induced arthritis). The multiple-indicator dilution technique was used with the simultaneous injection of 45Ca2+ and indicators into the portal vein under defined conditions and analysis of the outflow profiles by means of a space-distributed variable transit time model. The best description of the 45Ca2+ outflow profiles corresponds to a model that assumes rapid distribution of 45Ca2+ between the vascular space and the cell surface and a slower transfer into the hepatocytes. In kinetic terms two distinct cellular pools were distinguishable, the cytosol and the endoplasmic reticulum. The concentration of Ca2+ in the cytosol was much lower than in the vascular space and in the endoplasmic reticulum. The most prominent modification observed in the livers of arthritic rats was the increased Ca2+ concentration in the hormone-sensitive cellular pool. Furthermore, reduced rates of Ca2+ influx and efflux between the hormone-sensitive cellular pool and the cytosolic space were also detected in combination with a significantly reduced expression of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA2) protein. All these observations mean that in livers from arthritic rats more time is required to replenish the hormone sensitive Ca2+ stores.

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.

Intracellular trafficking of RNASET2, a novel component of P‐bodies

The ribonucleases (RNases) constitute a heterogeneous group of enzymes, which exert diverse and specific biological functions. Several RNases have been shown to control gene expression and cell differentiation. RNASET2, a novel member of the Rh/T2/S family of RNases, exerts micro-environmental control of malignancy in different experimental models with a general onco-suppressor activity, since it prevents cancer proliferation. Indeed, RNASET2 was found to be downregulated at the transcript level in several primary ovarian tumours or cell lines and in melanoma cell lines. Although recent works shed light on the biological role of RNASET2 in delaying tumour growth, its trafficking within the cell is still poorly understood. RNASET2 seems to play diverse biological roles including turnover of tRNA in yeast as well as rRNA degradation in zebrafish. Here, we have studied the intracellular trafficking of RNASET2 in mammalian cells. RNASET2 co-localizes with markers for the trans-Golgi network (TGN), which is the central sorting and processing station of the secretory pathway. Moreover, using the temperature-sensitive vesicular stomatitis glycoprotein, we demonstrate that RNASET2 undergoes delivery to the plasma membrane. In contrast to other RNA-interacting proteins, RNASET2 does not accumulate in stress granules upon metabolic stress in mammalian cells. Surprisingly, RNASET2 shows co-localization with processing bodies (P-bodies), which increases upon metabolic stress. Finally, cells lacking RNASET2 show a reduced numbers of P-bodies. In this study, we have identified two distinct cellular pools of RNASET2-containing granules. One pool undergoes membrane delivery using the TGN, and it is released to the extracellular environment. The second pool is recruited into P-bodies, suggesting a possible involvement of RNASET2 in P-body formation in mammalian cells.

Distinct cellular pools of perilipin 5 point to roles in lipid trafficking

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~ 1.15 g/ml) and size (~ 575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~ 15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.

High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine

Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex.

Identification of distinct cellular pools of interleukin-1β during the evolution of the neuroinflammatory response induced by transient middle cerebral artery occlusion in the brain of rat

The proinflammatory cytokine interleukin(IL)-1β plays a crucial role in ischemic pathophysiology, since pharmacologic inhibition of its biological effects provides neuroprotection after stroke. However, there is evidence suggesting that under certain circumstances the cytokine may also exert beneficial functions on brain injury. We have investigated the regional and cellular expression of IL-1β after ischemia–reperfusion injury in the brain of rat, and correlated cytokine expression with the activation/recruitment of glial cells in the damaged tissue. By using a double immunofluorescence histochemical approach, we observed an increased cytokine immunoreactivity in the ischemic core, as early as 1 h after middle cerebral artery occlusion, in few activated OX-42-positive microglial cells and in perivascular GFAP-positive astrocytes, suggesting that the cytokine may participate in the early response of the neurovascular unit to reduced blood supply. After 2 h ischemia, followed by 2 h reperfusion, cytokine staining was evident in the astrocytes of the penumbra and in activated microglial cells of the ischemic core. Microglial activation increases with the progression of damage and, after 22 h reperfusion, OX-42-immunopositive cells were strongly labelled for IL-1β in the core and, even more intensely, in the penumbra. At this later stage, GFAP-positive cells, appearing hypertrophic and distributed in a ring-like pattern around the ischemic core, do no longer express IL-1β. Thus, a specific cellular and regional pattern of IL-1β expression characterises the progression of ischemia–reperfusion injury. Depending on the stage and intensity of the insult, the different cellular origin of the cytokine may suggest a distinct role of this neuroinflammatory mediator in ischemic pathophysiology.

Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P2

PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P2. Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P2.

Control of histone methylation and genome stability by PTIP

PTIP regulates gene transcription by controlling the methylation of histone H3, and also has important roles in cellular responses to DNA damage or to perturbed DNA replication. The available data suggest that the functions of PTIP in transcription and preserving genome stability might be independent and mediated by functionally distinct cellular pools of PTIP. Although considerable progress has been made in understanding how PTIP influences transcription, a coherent picture of how it protects cells from DNA damage at the molecular level has yet to emerge. Here, we describe recent progress made in understanding the cellular roles of PTIP and the relevance of PTIP-interacting proteins, as well as the questions that have yet to be answered.

Wnt3a promotes β‐catenin signaling and myotube formation during myogenic differentiation

Satellite cells are muscle precursor cells required for postnatal skeletal muscle growth and regeneration. Glycogen Synthase Kinase 3beta (GSK‐3beta) has been implicated in negative regulation of skeletal muscle growth and muscle differentiation. Two distinct cellular pools of GSK‐3beta are selectively inhibited by IGF‐I/Akt‐or canonical Wnt signaling, respectively. Since canonical Wnt signaling and subsequent stabilization of beta‐catenin is involved in embryonic muscle formation, the effects of Wnt3a on differentiating myoblasts were investigated, and compared to those resulting from GSK‐3beta inactivation by LiCl or IGF‐I.Wnt3a promoted C2C12 myoblast fusion, and induced beta‐catenin signaling in a time and dose dependent fashion. In addition, beta‐catenin‐dependent transcriptional activation was also induced after GSK‐3beta inhibition using LiCl, but not by IGF‐I. Furthermore, stimulation of muscle gene expression was only observed in response to LiCl but not Wnt3a, which uncouples the promoting effects of Wnt3a on myoblast fusion from muscle specific gene expression.These data suggest that myoblast fusion and muscle specific gene expression are controlled by distinct cellular pools of GSK‐3beta, and provide evidence for a role of GSK‐3beta as a central regulator of muscle differentiation.This work was supported by a research grant of the Netherlands Astma Foundation (NAF 3.2.02.63).

Fatty acids and immune function: new insights into mechanisms

Fatty acids are known to play diverse roles in immune cells. They are important as a source of energy, as structural components of cell membranes, as signaling molecules and as precursors for the synthesis of eicosanoids and similar mediators. Recent research has suggested that the localization and organisation of fatty acids into distinct cellular pools has a direct influence on the behaviour of a number of proteins involved in immune cell activation, including those associated with T cell responses, antigen presentation and fatty acid-derived inflammatory mediator production. This article reviews these studies and places them in the context of existing literature in the field. These studies indicate the existence of several novel mechanisms by which altered fatty acid availability can modulate immune responses and impact upon clinical outcomes.

ApoA-I induces a preferential efflux of monounsaturated phosphatidylcholine and medium chain sphingomyelin species from a cellular pool distinct from HDL3 mediated phospholipid efflux

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for a detailed analysis of cellular phospholipid and cholesterol efflux in free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages (HMDM) loaded with enzymatically modified LDL (E-LDL). Although both cell models differed significantly in their cellular lipid composition, a higher apoA-I specific efflux was found for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species in both cell types. Moreover, medium chain sphingomyelin (SPM) species SPM 14:0 and SPM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, HMDM displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL3 mediated lipid efflux from HMDM using D9-choline and 13C3-FC stable isotope labeling revealed significantly different D9-PC and D9-SPM species pattern for apoA-I and HDL3 specific efflux media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL3 mediated efflux. Together with a partial loading of fibroblasts and HMDM with HDL3-derived CE species, these data add further evidence for retroendocytosis of HDL. In summary, analysis of apoA-I/ABCA1 and HDL3 mediated lipid efflux by ESI-MS/MS demonstrated a preferential efflux of monounsaturated PC and medium chain SPM to apoA-I. Moreover, this is the first study, which provides evidence for distinct cellular phospholipid pools used for lipid transfer to apoA-I and HDL3 from the analysis of phospholipid species pattern in HMDM.

Cholesterol Metabolism in Brain and Skin Fibroblasts from Sarda Breed Sheep With Scrapie-resistant and Scrapie-susceptible Genotypes

Scrapie is a fatal spongiform encephalopathy of sheep, a transmissible form of prion disease caused by neuronal accumulation of the aberrantly conformed prion protein (PrPsc). Currently, no ante-mortem diagnostic tests are available to detect this untreatable disease in the pre-clinical stage, thus making difficult to control its spread. Recent evidence suggests that the production of PrPsc can be modulated by the levels of membrane cholesterol in neuronal cells. Since cholesterol levels in cell membranes are dependent on cholesterol homeostasis in the whole organism, we studied cholesterol metabolism in brain tissues, plasma and skin fibroblasts of Sarda breed sheep with scrapie-resistant (ARR/ARR) and scrapie-susceptible (ARQ/ARQ) prion protein genotypes, both not infected (ARQ/ARQ-) and infected (ARQ/ARQ+) with scrapie. We found that, the levels of cytoplasmic cholesterol esters (CE) in brains and skin fibroblasts from sheep with the ARQ/ARQ genotype were consistently higher than those from sheep with the ARR/ARR genotype. Conversely, the levels of free cholesterol (FC) were lower in ARQ/ARQ, as compared to ARR/ARR sheep, thus resulting in a sharp reduction of the FC/CE ratio. Moreover, both uninfected and infected ARQ/ARQ sheep showed abnormally low levels of high density lipoprotein-cholesterol (HDL-C) in their plasma, as compared to ARR/ARR sheep. These data other than adding new strength to the notion that altered levels of intracellular cholesterol may indicate the presence of a lipid metabolic state that predisposes to infection with, and accumulation of, PrPsc in the brain, discriminate for the first time between two distinct but related cellular pools of cholesterol, namely membrane FC on one hand and cytoplasmic CE on the other.

Disulfide isomerization switches tissue factor from coagulation to cell signaling

Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF.VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys186-Cys209 disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys186 retains TF.VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF.PAR2 complex and TF.VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF.VIIa signaling can be specifically inhibited with minimal effects on coagulation.