Specialized Membrane Regions Research Articles

An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry

The STIM1-ORAI1 pathway of store-operated Ca(2+) entry is an essential component of cellular Ca(2+) signalling. STIM1 senses depletion of intracellular Ca(2+) stores in response to physiological stimuli, and relocalizes within the endoplasmic reticulum to plasma-membrane-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca(2+) channels. Here we use a genome-wide RNA interference screen in HeLa cells to identify filamentous septin proteins as crucial regulators of store-operated Ca(2+) entry. Septin filaments and phosphatidylinositol-4,5-bisphosphate (also known as PtdIns(4,5)P2) rearrange locally at endoplasmic reticulum-plasma membrane junctions before and during formation of STIM1-ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PtdIns(4,5)P2 reorganization and efficient STIM1-ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud and the primary cilium, and to serve as membrane diffusion barriers and/or signalling hubs in cellular processes such as vesicle trafficking, cell polarity and cytokinesis. Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1-ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes.

Organization of cytochrome P450 system components in the endoplasmic reticulum

P450s are membrane‐bound proteins found in the endoplasmic reticulum (ER). The ER is known to be heterogeneous, containing both ordered and disordered regions. Ordered microdomains are more tightly packed and not readily solubilized with non‐ionic detergents and are called detergent resistant membranes (DRM). The goal of this study is to determine if these microdomains affect P450 localization. The localization of P450 system proteins was examined in rabbit liver microsomes after incubation with 1% Brij 98, followed by sucrose gradient centrifugation. Unlike CYP1A2 (which localizes in the DRM), CYP2E1 proteins localized in non‐DRM and CYP2B4 was found to be equally distributed between both domains. Importantly, for P450 to function, it is necessary for this heme protein to form a complex with its electron donor CPR; however, CPR is found largely in the DRM. Consequently, for CYP2E1 to function, a mechanism for complex formation is required, such as translocation of either CYP2E1 or CPR between the lipid domains, or the interaction of CYP2E1 with the small amount of residual CPR localized in the disordered region, which would limit its activity. These results demonstrate that the components of the P450 system are organized in specific membrane regions, and not randomly distributed throughout in the ER membrane. Supported by ES004344

GROWTH AND DEVELOPMENT SYMPOSIUM: Endotoxin, inflammation, and intestinal function in livestock1,2

Endotoxin, also referred to as lipopolysaccharide (LPS), can stimulate localized or systemic inflammation via the activation of pattern recognition receptors. Additionally, endotoxin and inflammation can regulate intestinal epithelial function by altering integrity, nutrient transport, and utilization. The gastrointestinal tract is a large reservoir of both gram-positive and gram-negative bacteria, of which the gram-negative bacteria serve as a source of endotoxin. Luminal endotoxin can enter circulation via two routes: 1) nonspecific paracellular transport through epithelial cell tight junctions, and 2) transcellular transport through lipid raft membrane domains involving receptor-mediated endocytosis. Paracellular transport of endotoxin occurs through dissociation of tight junction protein complexes resulting in reduced intestinal barrier integrity, which can be a result of enteric disease, inflammation, or environmental and metabolic stress. Transcellular transport, via specialized membrane regions rich in glycolipids, sphingolipids, cholesterol, and saturated fatty acids, is a result of raft recruitment of endotoxin-related signaling proteins leading to endotoxin signaling and endocytosis. Both transport routes and sensitivity to endotoxin may be altered by diet and environmental and metabolic stresses. Intestinal-derived endotoxin and inflammation result in suppressed appetite, activation of the immune system, and partitioning of energy and nutrients away from growth toward supporting the immune system requirements. In livestock, this leads to the suppression of growth, particularly suppression of lean tissue accretion. In this paper, we summarize the evidence that intestinal transport of endotoxin and the subsequent inflammation leads to decrease in the production performance of agricultural animals and we present an overview of endotoxin detoxification mechanisms in livestock.

Bifunctional electro-optical nanoprobe to real-time detect local biochemical processes in single cells

A bifunctional electro-optical nanoprobe with integrated nanoring electrode and optical nanotip was fabricated and investigated to simultaneously detect both electrical and optical signals in real-time with high spatial resolution. Concurrent measurements of the oxidant generation and the intracellular antioxidant levels in single cells correlate the stronger oxidant generation with an altered initial antioxidant response in the breast cancer cells in comparison to the normal ones suggesting that the cell malignancy is associated with the strength of oxidative stress, and the higher antioxidant level may be the cause of the drug resistance. While the optical detection indicates the fluctuation of the intracellular redox homeostasis, the chronoamperometric signals allow quantitative real-time detection of the H 2O 2 release and decay. Furthermore, the nanoscale probe enables localized simultaneous detections thus discovering that activated enzymes responsible for the oxidative stress target at specific membrane regions. This method promises applications in study of the dynamics of important physiological processes, and provides the opportunity to unravel the interplay of various signaling pathways.

The platelet interior revisited: electron tomography reveals tubular α-granule subtypes

AbstractWe have used (cryo) electron tomography to provide a 3-dimensional (3D) map of the intracellular membrane organization of human platelets at high spatial resolution. Our study shows that the open canalicular system and dense tubular system are highly intertwined and form close associations in specialized membrane regions. 3D reconstructions of individual α-granules revealed large heterogeneity in their membrane organization. On the basis of their divergent morphology, we categorized α-granules into the following subtypes: spherical granules with electron-dense and electron-lucent zone containing 12-nm von Willebrand factor tubules, subtypes containing a multitude of luminal vesicles, 50-nm-wide tubular organelles, and a population with 18.4-nm crystalline cross-striations. Low-dose (cryo) electron tomography and 3D reconstruction of whole vitrified platelets confirmed the existence of long tubular granules with a remarkably curved architecture. Immunoelectron microscopy confirmed that these extended structures represent α-granule subtypes. Tubular α-granules represent approximately 16% of the total α-granule population and are detected in approximately half of the platelet population. They express membrane-bound proteins GLUT3 and αIIb-β3 integrin and contain abundant fibrinogen and albumin but low levels of β-thromboglobulin and no von Willebrand factor. Our 3D study demonstrates that, besides the existence of morphologically different α-granule subtypes, high spatial segregation of cargo exists within individual α-granules.

Generation of Dimers between Itk Molecules, Recruiting Receptors and Substrates (87.23)

Abstract Itk is a Tec family of tyrosine kinase that transduces signals from the T cell receptor and other receptors to modulate T cell development and activation. Itk is activated by receptors via a PI3 kinase mediated pathway, which results in recruitment of Itk to the plasma membrane via its PH domain. We show that membrane localization of Itk results in the formation of clusters of at least 2 molecules within 80 angstroms of each other. In addition, only the PH domain, but not the PRR, SH3 or SH2 domain, or kinase activity of Itk, is required for its formation of clusters at the plasma membrane. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane since only receptors that recruit PI3 kinase reside in the same membrane vicinity as the recruited Itk. Itk also forms dimers or clusters when associated with the adaptor protein SLP-76. Our results indicate that Itk forms membrane dimers with receptors in specific membrane regions. Furthermore, Itk forms dimers with adaptor proteins that assemble multiprotein complexes.

Tec Kinase Itk Forms Membrane Clusters Specifically in the Vicinity of Recruiting Receptors

The Tec family of tyrosine kinases transduces signals from antigen and other receptors in cells of the hematopoietic system. In particular, interleukin-2 inducible T cell kinase (Itk) plays an important role in modulating T cell development and activation. Itk is activated by receptors via a phosphatidylinositol 3-kinase-mediated pathway, which results in recruitment of Itk to the plasma membrane via its pleckstrin homology domain. We show here that membrane localization of Itk results in the formation of clusters of at least two molecules within 80 A of each other, which is dependent on the integrity of its pleckstrin homology domain. By contrast, the proline-rich region within the Tec homology domain, SH3 or SH2 domains, or kinase activity were not required for this event. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane as only receptors that recruit phosphatidylinositol 3-kinase reside in the same membrane vicinity as the recruited Itk. Our results indicate that Itk forms dimers in the membrane and that receptors that recruit Itk do so to specific membrane regions.

Association between the muscle-specific proteins desmin and caveolin-3 in muscle cells

The muscle-specific intermediate filament protein desmin is expressed in mononucleated myoblasts and in differentiated myotubes. Desmin has been shown to associate with the sarcolemma in specific structures, such as neuromuscular junctions and the dystrophin-associated protein complex. Since these are specialized membrane regions, the study of a possible association between desmin and liquid-ordered membrane microdomains is of particular interest. We have carried out an analysis of the association between desmin and the muscle-specific protein caveolin-3, a major component of caveolar microdomains. Our results demonstrate that (1) desmin precisely co-localizes with caveolin-3 in myoblasts and multinucleated myotubes, (2) caveolin-3 is up-regulated during in vitro chick muscle development, (3) desmin is detectable in caveolae-enriched membrane fractions prepared from skeletal muscle, and (4) caveolin-3 co-immunoprecipitates with desmin. We have thus shown, for the first time, an association between the intermediate filament protein desmin and caveolin-3 in myogenic cells.

Influenza virus assembly and budding in raft-derived microdomains: A quantitative analysis of the surface distribution of HA, NA and M2 proteins

Influenza virus hemagglutinin (HA) and neuraminidase (NA) are known to associate with lipid rafts, membrane microdomains comprised of densely packed cholesterol and sphingolipids. These specialized membrane regions are believed to be involved in the budding of many enveloped viruses including influenza virus. Quantitative analysis of HA distribution on the surface of virus-infected cells by immunogold staining shows an organization into clusters that grow in size as the expression level of HA increases with time post-infection (p.i.) (∼325–500 nm at 4 h p.i. and ∼425–600 nm at 6 h p.i.). These HA-containing clusters are likely derived from lipid rafts as they contain a high density of the raft marker ganglioside GM1 and are dependent upon the presence of cholesterol. The clustering of HA is an intrinsic property of the HA protein and occurs in the absence of expression of other viral proteins. NA is also found sequestered within the same microdomains as HA, whereas the M2 ion channel protein does not concentrate within the raft-like microdomains. Quantification of the distribution of surface expressed HA by examining serial sections of virus-infected cells suggests that the HA-containing microdomains give rise to regions of influenza assembly and budding.

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.

Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the gamma-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin. We then applied tcTPC to identify more than 20 proteins residing in the vicinity of the cellular prion protein (PrPC), suggesting that PrP is embedded in specialized membrane regions with a subset of molecules that, like PrP, use a glycosylphosphatidylinositol anchor for membrane attachment. Many of these proteins have been implicated in cell adhesion/neuritic outgrowth, and harbor immunoglobulin C2 and fibronectin type III-like motifs.

Les MAGUK : au-delà de l’accrochage des canaux ioniques

A family of anchoring proteins named MAGUK (for membrane associated guanylate kinase) has emerged as a key element in the organization of protein complexes in specialized membrane regions. These proteins are characterized by the presence of multipe protein-protein interaction domains including PDZ and SH3 domains. The MAGUK family comprises the post-synaptic density 95 (PSD-95) protein and closely related molecules such as chapsyn-110, synapse-associated protein 102 (SAP-102), and SAP-97. These are located either on the pre- and/or post-synaptic sides of synapses or at cell-cell adhesion sites of epithelial cells. MAGUK proteins interact with glutamate receptors and various ionic channels. For instance, an interaction has been reported between the first two PDZ domains of MAGUK proteins and several channels via a consensus sequence Thr/Ser-X-Val/Leu usually located at their carboxy terminus. The role of these anchoring proteins in channel function is not fully understood. MAGUK proteins enhance the current density by increasing the number of functional channels to the sarcolemma. They can also facilitate signaling between channels and several enzymes or G protein-dependent signaling pathways. In the heart also, MAGUK proteins are abundantly expressed and they interact with various channels including Shaker Kv1.5 and connexins.

Membrane lipid rafts: new targets for immunoregulation.

Engagement of immune receptors by antigen may lead to activation, cell proliferation, differentiation and effector functions. It has recently been proposed that the initiation and propagation of the signaling events taking place in immune cells occur in specialized membrane regions called lipid rafts. These detergent-insoluble glycolipid domains are specialized membrane compartments enriched in cholesterol and glycolipids. They also contain many lipid-modified signaling proteins such as tyrosine kinases of the Src family, GPI (glycosylphosphatidylinositol)-linked proteins as well as adaptor proteins. The confinement of signaling molecules in membrane subdomains suggests that lipid rafts function as platforms for the formation of multicomponent transduction complexes. Indeed, upon receptor binding, immune receptors become raft-associated and additional components of the signaling pathways are recruited to rafts in order to form signaling complexes. It has been speculated that the entry of immune receptors into rafts can regulate cell activation. Accordingly, numerous experiments have provided substantial evidence that raft integrity is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, Th1 versus Th2 lymphocytes) and sensitive to pharmacological agents. The aim of the present review is to summarize the current knowledge of immune receptor signal transduction with particular emphasis on the role of membrane compartments in immune activation. Finally, experimental evidences indicating that these membrane structures may represent clinically relevant potential targets for immune regulation, will be discussed.

Brain ischaemia transiently activates Ca2+/calmodulin-independent protein kinase II.

The activity of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) during short-term global ischaemia was investigated in the gerbil brain hippocampus and cortex. Ischaemia of 0.5 min duration significantly stimulated Ca(2+)-independent 'autonomous' activity, indicating activation of the first step of intramolecular enzyme phosphorylation just after ischaemia has developed. Prolongation of the ischaemic period up to 5 min inhibited both Ca(2+)-dependent and, to a lesser extent, Ca(2+)-independent activities of CaM-KII. These last events coincide with an extensive translocation of CaM-KII protein from the soluble to the membranous fraction. In effect, in spite of inhibition of total CaM-KII activity, its Ca(2+)-independent, persistently active component can still remain more abundant at specific membrane regions.

The topogenic fate of the polytopic transmembrane proteins, synaptophysin and connexin, is determined by their membrane-spanning domains

The synaptophysins and connexins are polytopic transmembrane proteins of similar secondary structure that accumulate as multiple homo-oligomers in specialized membrane regions, the presynaptic transmitter vesicles or gap junctions. Transfection and expression of the respective genes in cultured epithelial cells results in the de novo formation of either small cytoplasmic, synaptophysin-rich vesicles, or functional gap junctions consisting of clustered connexin molecules. To examine the molecular requirements for the specific enrichment and topogenesis of both types of molecule, chimeric cDNAs were constructed composed of different parts of the rat synaptophysin and rat liver connexin32 genes. Expression of the encoded chimeric polypeptides in hepatocellular carcinoma-derived cells showed that only chimeras with all four transmembrane domains from either parent molecule were delivered to their specific destination. In contrast, chimeras with transmembrane domains from both connexin32 and synaptophysin were always retained in the endoplasmic reticulum. The topogenic nature of the transmembrane domains was further demonstrated by deletion mutagenesis, indicating that removal of cytoplasmic end domains or intravesicular loops does not abolish targeting. On the other hand, excision of individual transmembrane domains or introduction of point mutations in transmembrane segments resulted in retention in the endoplasmic reticulum.

Innervation directs receptor synthesis and localization in Drosophila embryo synaptogenesis.

In the Drosophila embryo, motor neurons form stereotyped synapses (neuromuscular junctions) on identified muscles. We have used a mutant (prospero) that removes or delays innervation to assay the role of the presynaptic motor neuron in the development of the receptive field of the postsynaptic muscle. prospero (pros) is not expressed in the muscles or their precursors. Here we find that the muscle defines the correct synaptic zone in the absence of the motor neuron by restricting putative guidance molecules to this specialized membrane region. Furthermore, the muscle expresses functional transmitter receptors at the correct developmental time without innervation. On the other hand, the muscle does not localize receptors to the synapse without instruction from the motor neuron, nor does a second, much larger, synthesis of receptors occur in muscles deprived of innervation. In muscles receiving delayed innervation, or muscles innervated at aberrant synaptic sites, both receptor clustering and receptor synthesis are delayed or redirected, consistent with the new pattern of innervation. We conclude that the muscle autonomously defines the synaptic site, whereas the motor neuron directs the development of the muscle's receptive field by stimulating the synthesis and localization of transmitter receptors.

Immunolocalisation de la fodrine dans la rétine des vertébrés

Fodrin, a member of the spectrin family, is abundantly found in brain and lens. Based on its often observed peripheral cellular distribution, it is generally believed that fodrin - like other spectrins - participates in the linkage of membrane components to cytoskeletal elements providing their anchorage to distinct, highly specialized membrane regions. To investigate the role of fodrin in the vertebrate (amphibian) retina, we have produced polyclonal antibodies in rabbits against fodrin purified from veal lens. The distribution of fodrin within the retina of Xenopus and Rana has been assessed by indirect immunofluorescence on cryostat sections and on isolated epithelial cells. In general, the staining pattern reflects the structural and functional polarity of the retinal layers. Fodrin is found in axons, dendrites and synapses of neurons, in the inner segment of photoreceptors, and as a peripheral ring of the epithelial cells. This study represents the first step of an investigation on the development and differentiation of specific, fodrin-containing membrane regions in the amphibian retina.

Membrane domains and macromolecular transport in intestinal epithelial cells.

Epithelial cell plasma membranes are organized as biochemically and functionally distinct domains and subdomains. We have explored the composition and maintenance of specific membrane regions during endocytosis and transepithelial vesicular transport in specialized cells of the intestinal epithelium. A unique membrane glycoprotein has been identified in apical endocytic subdomains and endosomal tubules of absorptive cells in suckling rat ileum. This endocytic system is involved in sorting and transepithelial transport of peptide growth factors as well as delivery of milk macromolecules to lysosomes. In M cells of follicle-associated epithelium, we have shown that membrane-bound proteins are efficiently transported to a specialized subdomain of basolateral membrane. The apical membranes of these cells bear immunoglobulin binding sites and transport monoclonal IgA antibodies to the basolateral side, where they may interact with cells of the mucosal immune system.

Fluorescent localization of calcium at sites of cell attachment and spreading.

Chlorotetracycline (CTC), a calcium-chelating fluorescent probe identifies and localizes calcium when applied to cultured neural crest cells. The fluorescence occurs at specific membrane regions involved in cell attachment and spreading as well as with the formation of cytoskeletal stress fibers (actin microfilament bundles). The observed CTC reaction indicates that calcium sequestration and the development of these membrane-cytoskeletal features share a temporal and spatial pattern. Thus, the selective availability of calcium may be an important determinant of cell morphogenesis.

Gap junction formation between reaggregated Novikoff hepatoma cells.

We have combined freeze-fracture and electrophysiological methods in a study of gap junction formation between reaggregated Novikoff hepatoma cells. Cell clumps are dissociated with EDTA, and the resulting single cells are allowed to reaggregate (5-180 min) in loose pellets in the presence of calcium at 37 degrees . The earliest electron microscopic evidence for the genesis of new junctions is the appearance of flattened regions of the plasma membrane with a relative paucity of small intramembranous particles. These regions contain instead loosely organized groupings of 9- to 11-nm intramembranous particles, which are seen on the A face of the fractured plasma membrane, while corresponding pits occur on the membrane B face. We have termed the specialized membrane regions "formation plaques." They are seen as early as 5 min after reaggregation and are quite numerous by 30 min. Larger plaques are observed at later times. Plaques seen at 30 min are consistently matched with other plaques on apposed cells, although the extracellular space separating these structures still exceeds 10 nm. By 60 min, some matched plaques display a reduced extracellular space, resembling that of normal gap junctions. Between 30 and 60 min, aggregates of closely packed particles on A faces and hexagonally arranged pits on B faces frequently appear in the formation plaques. The aggregates, which are indistinguishable from small gap junctions, appear to enlarge over the subsequent 2-hr period as the number of unaggregated 9- to 11-nm particles declines. Microelectrode studies demonstrate progressive increases in the percent of interfaces containing lowresistance junctions and in the degree of elctrical coupling in preparations incubated up to 2 hr. Coupling is first detected at about the same time as particle aggregates (or formation plaques with reduced extracellular spaces), and increases as aggregate sizes increase.