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V Muresan

Publications and source records attributed to V Muresan.

17 recordsLinked to original sources
PubMed2001

Dynactin-dependent, dynein-driven vesicle transport in the absence of membrane proteins: a role for spectrin and acidic phospholipids.

We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.

Acids↗
PubMed1999

The kinesin motor KIF3A is a component of the presynaptic ribbon in vertebrate photoreceptors.

Kinesin motors are presumed to transport various membrane compartments within neurons, but their specific in vivo functions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.

Animals↗
PubMed1998

KIF3C and KIF3A form a novel neuronal heteromeric kinesin that associates with membrane vesicles.

We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.

Amino Acid Sequence↗
PubMed1997

Evidence for kinesin-related proteins associated with the axoneme of retinal photoreceptors.

Situated at the junction between inner and outer segment, the connecting cilium of retinal photoreceptors supports regulated transport of molecules that function distally, while restricting diffusion of membrane proteins from one plasmalemmal domain to the other. Both functions are thought to be performed by a group of proteins stably or transiently associated with the axoneme. We have identified two types of unique polypeptides which associated with the axoneme in a nucleotide-dependent manner: they bind to the axonemes in the presence of adenosine monophosphate (AMP)-PNP, and are solubilized in the presence of adenosine triphosphate (ATP). The first group contained glyconjugates, previously shown to be part of the axoneme-plasmalemma cross-linkers at the connecting cilium. The second group cross-reacted with antibodies to two different conserved peptide sequences (called LAGSE and HIPYR) of kinesin-related proteins, and included polypeptides of approximately 85-97 kDa. Immunofluorescence microscopy of whole-mounted axonemes with the two anti-kinesin antibodies showed labeling throughout the axoneme, including the connecting cilium-basal body region. These results suggest that the identified proteins may serve as motor molecules for transport of material to the outer segment via the connecting cilium.

Adenosine Triphosphate↗
PubMed1996

Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules.

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)-induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.

Adenylyl Imidodiphosphate↗
PubMed1994

Complex intermolecular interactions maintain a stable linkage between the photoreceptor connecting cilium axoneme and plasma membrane.

Microtubule-membrane cross-linkers in motile and nonmotile cilia are supramolecular structures, held together by strong interactions between the constituent molecules. We have characterized these interactions in the photoreceptor connecting cilium, where cross-linkers co-fractionate and maintain their in situ location after Triton X-100 extraction of axonemes. In bovine photoreceptor cells, the transmembrane assemblage that is cross-linked to the connecting cilium axoneme contains three high molecular mass glycoconjugates of 425, 600, and 700 kDa (Horst et al., 1987). The relative amounts of the three glycoconjugates, as judged from band intensity in electrophoretograms, depend strongly on sample treatment prior to electrophoresis. The electrophoretic pattern was reproducible after several weeks of storage of the axoneme fraction in extraction buffer containing 50% sucrose. Removal of sucrose from the buffer by dialysis eliminated the 600 kDa and 700 kDa, and decreased the detected amount of the 425 kDa glycoconjugate. When samples were incubated in Laemmli sample buffer at increasing temperatures (23 degrees, 60 degrees, 95 degrees C), a gradual reduction in the intensity of the three bands was observed. The quantitative reduction of high molecular mass glycoconjugates was accompanied by the appearance of novel protein species of lower molecular mass, as detected by lectin and antibody overlays of axonemal transblots. These results suggest that the previously characterized cross-linker glycoconjugates are complex, SDS-resistant multi-molecular conglomerates. We have further used fluorescent lectins to monitor the presence of glycoconjugates on whole-mounted axonemes, in conditions aimed to selectively solubilize the cross-linkers. The cross-linker complexes could not be dissociated from the axoneme by incubation with buffers containing 1 M of either Na2SO4 or NaI. The results indicate that the connecting cilium-specific cross-linker complexes are bound via high-affinity interactions to both axoneme and overlying plasma membrane.

Animals↗
PubMed1993

Gamma-tubulin in differentiated cell types: localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia.

gamma-Tubulin, a newly discovered member of the tubulin superfamily required for microtubule nucleation, is associated with the centrosome(s) throughout the vertebrate cell cycle. We have used a polyclonal antibody, generated against a highly conserved segment of gamma-tubulin, to localize this protein in postmitotic, ciliated cells, in which the major microtubule organizing centers are the basal bodies. Single-cilium photoreceptor cells from bovine retina contained a strongly immunoreactive species, with molecular characteristics of gamma-tubulin, in association with a detergent-resistant, cytoskeletal fraction devoid of cytoplasmic microtubules. gamma-Tubulin was discretely localized throughout the basal body region, extending opposite to the axonemal shaft, in mechanically detached rod outer segments and whole-mounted, connecting cilium-derived axonemes. In multiciliated epithelia from bovine trachea and oviduct, gamma-tubulin immunoreactivity was detected at the base of the cilia, where basal bodies are located. These results suggest that this key centrosomal protein of mitotically active cells is also an integral component of microtubule organizing centers, required for the generation of the microtubule network in terminally differentiated cells.

Animals↗
PubMed1987

Cell surface chemistry of arterial endothelium and blood monocytes in the normolipidemic rabbit.

Chemical mapping of the luminal surface of normal rabbit aortic and coronary endothelium was investigated cytochemically to establish a baseline for further comparison with the biochemical changes possibly induced by the experimental hypercholesterolemia. Morphometric analysis showed that in the aortic endothelium the plasma membrane exposes a large number of uniformly-distributed positively-charged groups of high pKa, and a heterogeneous pattern of dense anionic groups of low pKa. Among the latter, only a third was represented by neuraminidase-cleavable sialic acids. These are constituted by various classes of N-, and O-substituted sialyl residues in glycoconjugates, most frequent being those non-O-acetylated at C8 or C9. Among the oligosaccharides detected with lectins, very abundant were the glycoconjugates containing mannosyl and subterminal galactosyl, whereas N-acetyl-glucosamine, terminal galactosyl and N-acetyl-galactosaminyl moieties were rather poorly represented. The density of the latter two markedly increased after its unmasking by neuraminidase treatment. Coated pits contained both anionic and cationic sites, but only few sialic acids and saccharide residues in significantly lower amounts than plasma membrane. The membrane of plasmalemmal vesicles displayed a high number of cationic sites and mannosyl residues, but very few anionic groups, sialyl residues, and galactosyl and N-acetyl-galactosaminyl moieties. Coronary endothelium displayed a chemical pattern similar to aorta, with some differences, especially in the frequency of some oligosaccharides. Vena cava was low in acidic groups but rather rich in galactose. Plasmalemmal vesicles were only occasionally labeled by the probes used. Monocyte surface exhibited a high density of anionic sites, and binding sites for wheat germ agglutinin and Ricinus communis agglutinin. No mononuclear cells were observed adhering to endothelial surface.

Acetylgalactosamine↗
PubMed1987

Prelesional events in atherogenesis. Changes induced by hypercholesterolemia in the cell surface chemistry of arterial endothelium and blood monocytes, in rabbit.

We investigated the modifications that diet-induced hypercholesterolemia, in rabbit, can produce in the cell surface charge and chemistry of arterial endothelium (E) and blood monocytes (M). Weekly, up to 8 weeks, after blood samples were taken for lipid analysis and blood cell preparation, the vasculature was washed free of blood and the endothelial luminal surface (ES) exposed to cytochemical probes for detecting charged groups, sialoconjugates and oligosaccharides. After fixation in situ, specimens collected from lesion-prone regions (aortic arch and coronary artery) and vena cava, were processed for electron microscopy. Morphometric analysis of tracer distribution on endothelium of nonlesional and lesional areas occurring in various stages of structural alterations, showed a remarkable resistance of the cell coat to very high level of serum cholesterol. In nonlesional zones the E surface charge and glycoconjugates were not significantly changed. In lesional areas, including those with forming fatty streaks, while cationic sites, galactosyl-, and N-acetyl-galactosaminyl residues were not altered whereas mannosyl moieties increased in density. A reduction in anionic groups and sialoconjugates appeared only after advanced extracellular and intracellular accumulation of lipoprotein-derived material and stromal proliferation developed in the intima. Moreover, these ES changes were usually restricted to the relatively rare E cells heavily loaded with lipid inclusions. The modulations were generally paralleled by comparable variations in the M surface. Regardless the extent of surface charge reduction, monocytes continued to migrate and foam cells to egress from the vessel wall. The results suggest that the onset and progression of early intimal lesions are not preceded but followed by significant restricted alterations in cell surface charge and glycoconjugates of arterial endothelium and monocytes.

Animals↗
PubMed1985

Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas.

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.

Animals↗
PubMed1982

Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas.

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.

Animals↗
PubMed1982

Purification and use of limulin: a sialic acid-specific lectin.

A simple and rapid method for the isolation of the sialic acid-specific lectin, Limulus polyphemus hemagglutinin (LPA), from the hemolymph of Limulus polyphemus is described. Declotted hemolymph is adsorbed to an affinity chromatographic column consisting of hog gastric mucin glycopeptides coupled to agarose and LPA is eluted in a single step with a Ca2+-free buffer, giving an apparent purification of approximately 25,000-fold. The eluted material is homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing and consists of identical subunits each of 29,000 daltons. Hemagglutination inhibition studies with horse red blood cells indicate specificity of LPA for N-acetyl- and N-glycolylneuraminic acid; binding is Ca2+ -dependent and abolished by neuraminidase treatment. LPA was covalently coupled to rhodamine and to horseradish peroxidase for use in detection of sialoglycoconjugates on cells and tissues by light and electron microscopy. Examples of the use of LPA for detection of sialoglycoconjugates in rat renal tubules and glomeruli, blood vessels in rat pancreas, and on horse red blood cells are shown. The procedures described here should prove useful as a cytochemical probe for detection of sialoglycoconjugates in a variety of systems. An accompanying article utilizes these probes for the detection of sialoglycoconjugates on the plasmalemma of adult and differentiating rat pancreatic acinar cells.

Animals↗
PubMed1981

Cell surface properties of normal, differentiating, and neoplastic pancreatic acinar cells.

Acinar, centroacinar, and endocrine cells of the adult rat pancreas each exhibit distinctive cell-surface glycoconjugate patterns as detected by binding of a battery of lectin-ferritin conjugates. Acquisition of these unique glycoconjugate patterns appears to be developmentally regulated, as studies on embryonic rat pancreases at days 15, 17, and 19 of gestation indicate. Further, the three cell types appear to arise from a common stem cell(s) with surface glycoconjugate properties similar to those of the adult centroacinar cell. STudies on the cell-surface properties of a rat acinar cell tumor indicate that the neoplastic acinar cells are likely to be arrested at a developmental stage equivalent to acinar cells of the day 19 embryonic pancreas and are characterized by absence of detectable basal lamina. We hypothesize that pancreatic cancers may arise from the equivalent of the undifferentiated embryonic stem cell(s) and that the morphologic features of the tumor depend on the extent of cell differentiation, including expression of cell-surface glycoproteins and extracellular matrix, prior to neoplastic proliferation.

Animals↗
PubMedDate not supplied

One axon, many kinesins: What's the logic?

A large number of membrane-bounded organelles, protein complexes, and mRNAs are transported along microtubules to different locations within the neuronal axon. Axonal transport in the anterograde direction is carried out by members of a superfamily of specialized motor proteins, the kinesins. All kinesins contain a conserved motor domain that hydrolyses ATP to generate movement along microtubules. Regions outside the motor domain are responsible for cargo binding and regulation of motor activity. Present in a soluble, inactive form in the cytoplasm, kinesins are activated upon cargo binding. Selective targeting of different types of kinesin motors to specific cargoes is directed by amino acid sequences situated in their variable tails. Cargo proteins with specific function at their destination, bind directly to specific kinesins for transport. Whereas most kinesins move to microtubule plus-ends, a small number of them move to microtubule minus-ends, and may participate in retrograde axonal transport. Axonal transport by kinesins has a logic: Fully assembled, multisubunit, functional complexes (e.g., ion channel complexes, signaling complexes, RNA-protein complexes) are transported to their destination by kinesin motors that interact transiently (i.e., during transport only) with one of the complexes' subunits.

Animals↗
PubMedDate not supplied

Far-Eastern interpretation of cellular pathology: yang-type components of a pancreatic acinar tumor.

Conjugates of a sialic acid-binding lectin, limulin, with rhodamine and horseradish peroxidase, were used to detect yang-type components at the surface of pancreatic carcinoma cells. Fluorescence microscopy of frozen sections revealed an even distribution of yang residues over the surface of neoplastic cells, in contrast to the polarized one on normal acinar cells. Electron microscopy indicated a lower concentration of yang-type molecules on the tumor cell surface than on its normal counterpart. It was concluded that the neoplastic process induces an alteration of yin/yang ratio at cellular level.

Animals↗