Patching of gangliosideM1 in human erythrocytes – distribution of CD47 and CD59 in patched and curved membrane

Membrane rafts may act as platforms for membrane protein signalling. Rafts have also been implicated in the sorting of membrane components during membrane budding. We have studied by fluorescence microscopy cross-linking of ganglioside GM1 in the human erythrocyte membrane, and how membrane proteins CD47 and CD59 distribute in GM1 patched discoid cells and calcium-induced echinocytic cells. Patching of ganglioside_M1 (GM1) by cholera toxin subunit B (CTB) plus anti-CTB resulted in the formation of usually 40–60 GM1 patches distributed over the membrane in discoid erythrocytes. Pre-treatment of erythrocytes with methyl-β-cyclodextrin abolished GM1 patching. GM1 patching was insensitive to pre-fixation (paraformaldehyde) of cells. Patching of GM1 did not affect the discoid shape of erythrocytes. Membrane proteins CD47 and CD59 did not accumulate into GM1 patches. No capping of patches occurred. GM1 accumulated in calcium-induced echinocytic spiculae. Also CD59, but not CD47, accumulated in spiculae. However, CD59 showed a low degree of co-localization with GM1 and frequently accumulated in different spiculae than GM1. In conclusion, our study describes a novel method for examining properties and composition of rafts. The study characterizes raft patching in the human erythrocyte membrane and emphasizes the mobility and ‘echinophilicity’ of GM1. Glycosyl phosphatidylinositol-anchored CD59 was identified as a mobile ‘echinophilic’ but ‘raftophobic_GM1’ protein. Largely immobile CD47 showed no segregation.     

Intracellular signalling mechanisms regulating glucose transport in insulin-sensitive tissues

The rate of glucose transport into cells is of fundamental importance in whole body homeostasis and adaptation to metabolic stresses, and this review examines the signalling mechanisms controlling this process. The events that mediate the action of insulin on glucose transport, which is by far the best characterized paradigm for glucose transport regulation, are discussed. There are several excellent reviews on various aspects of this subject, which are referred to while highlighting very recent developments in the field, including the recently described CAP pathway, and emerging mechanisms for feedback regulation of insulin signalling. The manner in which hormonal signalling is modulated by stimuli such as oxidative and osmotic stress is then discussed. The second major physiological event where glucose transport regulation is critical is the contraction of skeletal muscle, due to the large metabolic demands of this activity. The mechanism of this regulation is distinct from that initiated by insulin, and recent developments will be examined that have begun to clarify how contraction stimulates glucose transport in skeletal muscle, including the roles performed by AMP-activated protein kinase and nitric oxide synthase.     

Structure of Protein Related to Dan and Cerberus: Insights into the Mechanism of Bone Morphogenetic Protein Antagonism

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Metabolites from intestinal microbes shape Treg

Intestinal bacterial metabolites are an important communication tool between the host immune system and the commensal microbiota to establish mutualism. In a recent paper published in Science, Wendy Garrett and her colleagues report an exciting role of the three most abundant microbial-derived short-chain fatty acids (SCFA), acetic acid, propionic acid and butyric acid, in colonic regulatory T cell (cTreg) homeostasis.A number of studies have shown that increased cTreg numbers and their immunoregulatory function are promoted by the presence of commensal intestinal microbes (either individual species such as Bacteroides fragilis¹, defined benign consortia of bacteria such as the altered Schaedler flora² or groups of Clostridia³). In a recent paper in Science, Garrett and colleagues report how these effects are generated through molecular exchanges between the host and the enormous load of microbes carried in the lower intestine⁴.Smith et al.⁴ investigated the role of SCFA, which are bacterial fermentation products produced by a wide variety of bacteria through anaerobic acidogenic pathways. SCFA released by colonic bacteria have long been known to be important as a carbon source for colonic epithelial cells⁵. From this new work we can now see that signaling effects of SCFA also regulate cTreg homeostasis.Microbiota-derived SCFA were found to increase total (thymic-derived) cTreg numbers. The homing characteristics to the colon and the regulatory functions of these cells (such as IL-10 production) were also enhanced through SCFA treatment.These effects are mediated by the G-protein-coupled free fatty acid receptor 43 (GPR43). Using mice that are genetically deficient in this receptor, Smith et al. showed that this signaling pathway is responsible for the increased cTreg numbers in vivo and that signaling by SCFA reduces the susceptibility to chronic intestinal inflammation. As they found GPR43 expression on cTreg (compared with lower GPR43 expression on Treg from other sites) this may be a direct effect, e.g. alterations in histone deacetylation. However, other cell types in the GI tract also express GPR43, including enteroendocrine cells and other leukocytes, therefore indirect effects are not yet excluded. In fact, Atarashi and colleagues have recently published their studies of how Clostridial species induce cTreg⁶. They found that bacterial-derived SCFA stimulate epithelial cells to produce TGFβ, contributing to Treg differentiation and expansion.Whereas other species-specific bacterial molecules, such as B. fragilis-derived PSA, have previously been demonstrated to have immunomodulatory functions², the report by Smith et al. is an elegant demonstration of the ubiquitous and pervasive bacterial metabolites that impact on the mucosal immune system. There is really a rather promiscuous exchange of metabolites between the microbiota and the host, with metabolic pathways that require components of both eukaryotic and prokaryotic cells. Bile acids are a great example of such a mixed pathway, where a dysbiosis caused by obesity promotes liver cancer through alterations in the microbial bile acid metabolism⁷. Although Smith et al. do not see any SCFA-mediated effects on central Treg compartments (outside the colon), other bacterial metabolites that reach systemic sites likely modulate adaptive or innate immune cell function at systemic sites. This may eventually rationalize the observed increased incidence of intestinal inflammation and systemic immune-mediated disorders such as autoimmune or allergic diseases (Figure 1), which are often linked to changes within the microbiota due to diet or antibiotic use⁸.Open in a separate windowFigure 1Bacterial metabolites that reach systemic sites likely modulate adaptive or innate immune cell function at systemic sites. This may eventually rationalize the observed correlation of microbiota composition and susceptibility to systemic immune-mediated disorders such as autoimmune or allergic diseases.A clinical situation in which the colon faces a deficiency of SCFA happens after surgery that diverts the fecal stream into a stoma bag, leaving the distal colon without its normal contents. This operation may be carried out to protect a low surgical anastomosis after removal of a tumor. The result is that the defunctioned colon frequently becomes inflamed, a condition recognized as ''diversion colitis''. In some cases, treatment with SCFA has been able to treat the condition successfully⁹. The lack of SCFA as a carbon source for colonocytes was previously considered as a key factor in the aetiopathogenesis of the condition, although this will need to be reviewed in the light of the new data on the effects of SFCA on colonic Treg numbers and function.Our colonic health depends on our intestinal microbiota and what we feed them. Changes in Western dietary patterns, e.g., due to reduced intake of plant fibers, might drastically impact the production of SCFA within the intestine. Furthermore, Smith et al. demonstrate a direct effect of antibiotic (vancomycin) treatment on SCFA levels, which in turn affects intestinal immune regulation by reducing the number of cTreg.Taken together, this draws a picture of a superorganism composed of the host (us) and our microbiota, with the metabolic interface as an important communication tool. This allows the host and the microbiota to adapt to and communicate with each other. Originally, germ-free animals were derived to challenge the notion that the existence of higher organisms was irrevocably linked to their associated microbiotas¹⁰. Although the germ-free program succeeded¹¹, it has provided us with powerful tools to show that the original notion was justified: pervasive metabolic interactions and signaling make us the sum of our prokaryotic and eukaryotic cellular components.     

Cyclic Di-AMP Homeostasis in Bacillus subtilis: BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH*

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.     

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.     

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.