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Results for “Cell Surface Extensions”

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At least 145 records · Page 8Linked to original sources

PubMed2005

Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival.

Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.

Amino Acid Sequence↗

PubMed2005

Multiple membrane tethers probed by atomic force microscopy.

Using the atomic force microscope to locally probe the cell membrane, we observed the formation of multiple tethers (thin nanotubes, each requiring a similar pulling force) as reproducible features within force profiles recorded on individual cells. Forces obtained with Chinese hamster ovary cells, a malignant human brain tumor cell line, and human endothelial cells (EA hy926) were found to be 28 +/- 10 pN, 29 +/- 9 pN, and 29 +/- 10 pN, respectively, independent of the nature of attachment to the cantilever. The rather large variation of the tether pulling forces measured at several locations on individual cells points to the existence of heterogeneity in the membrane properties of a morphologically homogeneous cell. Measurement of the summary lengths of the simultaneously extracted tethers provides a measure of the size of the available membrane reservoir through which co-existing tethers are associated. As expected, partial disruption of the actin cytoskeleton and removal of the hyaluronan backbone of the glycocalyx were observed to result in a marked decrease (30-50%) in the magnitude and a significant sharpening of the force distribution indicating reduced heterogeneity of membrane properties. Taken together, our results demonstrate the ability of the plasma membrane to locally produce multiple interdependent tethers-a process that could play an important role in the mechanical association of cells with their environment.

Animals↗

PubMed2006

Double-tether extraction from human umbilical vein and dermal microvascular endothelial cells.

Multiple tethers are very likely extracted when leukocytes roll on the endothelium under high shear stress. Endothelial cells have been predicted to contribute more significantly to simultaneous tethers and thus to the overall rolling stabilization. We therefore extracted and quantified double tethers from endothelial cells with the micropipette aspiration technique. We show that the constitutive parameters (threshold force (F0) and effective viscosity (etaeff)) for double-tether extraction are twice those for single-tether extraction and are remarkably similar for human neonatal (F0=105+/-5 pN; etaeff=1.0+/-0.1 pN.s/microm) and adult (F0=118+/-13 pN; etaeff=1.3+/-0.2 pN.s/microm) dermal microvascular, and human umbilical vein (F0=99+/-3 pN; etaeff=1.0+/-0.1 pN.s/microm) endothelial cells. Additionally, these parameters are also independent of surface receptor type, cytokine stimulation, and attachment state of the endothelial cell. We also introduce a novel correlation between the cell-substrate contact stress and gap width, with which we can predict the apparent cell-substrate separation range to be 0.01-0.1 microm during leukocyte rolling. With a biomechanical model of leukocyte rolling, we calculate the force history on the receptor-ligand bond during tether extraction and predict maximum stabilization for the double simultaneous tether extraction case.

Cell Adhesion↗

PubMed2006

Interaction of toposome from sea-urchin yolk granules with dimyristoyl phosphatidylserine model membranes: a 2H-NMR study.

The yolk granule is the most abundant membrane-bound organelle present in sea urchin eggs and embryos. The major protein component of this organelle, toposome, accounts for approximately 50% of the total yolk protein and has been shown to be localized to the embryonic cell surface. Extensive characterization in several laboratories has defined a role for toposome in mediating membrane-membrane interactions. The current study expands the analysis of toposome-membrane interaction by defining toposome-induced changes to the lipid bilayer. The effect of toposome on the biophysical properties of phosphatidyl serine (PS) multibilayers was investigated using deuterium nuclear magnetic resonance and perdeuterated dimyristoyl PS (DMPS-d(54)). Toposome was found to have little effect on DMPS-d(54) chain orientational order in both the gel and liquid-crystalline phases. Timescales for DMPS-d(54) reorientation were investigated using quadropole echo decay. Echo decay times were sensitive to toposome in the liquid-crystalline phase but not in the gel phase. Additional information about the perturbation of bilayer motions by toposome was obtained by analyzing its effect on the decay of Carr-Purcell-Meiboom-Gill echo trains. Collectively, these results suggest that toposome interacts peripherally with DMPS bilayers and that it increases the amplitude of lipid reorientation, possibly through local enhancement of bilayer curvature.

Animals↗

PubMed2003

Subcellular localization of components of the dystrophin glycoprotein complex in cultured retinal muller glial cells.

The dystrophin glycoprotein complex (DGC) is a membrane-associated protein complex binding extracellular matrix (ECM) molecules, such as laminin and forming a bridge towards the cytoskeleton. The molecular composition of the DGC is cell type dependent and it is involved in cell adhesion and motility. Here we present immunocytochemical localization of beta-dystroglycan, the central member of the DGC, utrophin and Dp71f, the spliced 71 kDa dystrophin protein product of the DMD gene, in cultured retinal Muller glial cells. It is shown that beta-dystroglycan and utrophin are colocalized in clusters in all parts of Muller cells including the lamellipodium and leading edge of migrating cells. As a contrast, Dp71f labels are distinct from beta-dystroglycan and confined to the perinuclear cytoplasm of Muller cells indicating that Dp71f is not a member of the DGC in cultured Muller cells.

Actins↗

PubMed2001

Development and maintenance of a B220- memory B cell compartment.

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.

Amino Acid Sequence↗

PubMed2002

Intrinsic program for migration of cerebellar granule cells in vitro.

Cerebellar granule cells exhibit distinct modes of migration in different cortical layers. The role of external cues in controlling these alterations has been suggested, but the significance of internal programs is not well understood. In the present study, we examined autonomous changes of migratory behavior of isolated granule cells in microexplant cultures of the postnatal mouse cerebellum. We found that isolated granule cells sequentially go through three characteristic phases of migration without cell-cell contact. In the first phase (0-20 hr in vitro) granule cells exhibit the highest rate of turning behavior and have multiple short processes. The length of the movement cycle is shortest. In the second phase (20-40 hr in vitro), granule cells extend a long and thick process and exhibit an elongated cycle of movement. Their speed is fastest, whereas the rate of turning is lowest. In the third phase (40-60 hr in vitro), granule cells slow down their movement and slightly increase their turnings. The length of the movement cycle further increases. At the end, the cells become permanently stationary, extend a lamellipodium around the soma, and emit several thin processes. Interestingly, granule cells sequentially develop four different modes of turning. These results indicate that internal (intrinsic) programs control alterations of granule cell behavior in a stage-dependent manner, suggesting that such programs independent of local cell-cell contacts may be essential for granule cell translocation in the developing cerebellum.

Animals↗

PubMed2001

Spine formation and correlated assembly of presynaptic and postsynaptic molecules.

Hippocampal pyramidal neurons in culture showed a developmental shift in synapse distribution from dendritic shafts to spines. Using dual wavelength time-lapse fluorescence microscopy, we analyzed the morphogenesis of three synaptic components: dendritic spines, postsynaptic densities (PSDs), and presynaptic vesicles. Local assembly of a major PSD protein, PSD-95, was spatially and temporally correlated with spine morphogenesis. Clustering of postsynaptic PSD-95 and that of a predominant synaptic vesicle protein, synaptophysin, were also correlated. In contrast, pre-existing PSD-95 clusters in dendritic shafts were preferentially eliminated without promoting spine formation. The local and stepwise assembly of synaptic components at the contact sites between dendritic protrusions and axons explains the developmental remodeling of excitatory synapses.

Adenoviridae↗

PubMed2001

Regulation of dendritic spine motility in cultured hippocampal neurons.

Regulation of dendritic spine motility was studied in dissociated cultures of the rat and mouse hippocampus, using green fluorescent protein-labeled neurons or neurons loaded with the calcium-sensitive dye Oregon Green-1. Cells were time-lapse-photographed on a confocal laser-scanning microscope at high resolution to detect movements as well as spontaneous fluctuations of intracellular calcium concentrations in their dendritic spines. Active presynaptic terminals attached to the spines were labeled with FM4-64, which marks a subset of synaptophysin-labeled terminals. Dendritic spines were highly motile in young, 4- to 7-d-old cells. At this age, neurons had little spontaneous calcium fluctuation or FM4-64 labeling. Within 2-3 weeks in culture, dendritic spines were much less motile, they were associated with active presynaptic terminals, and they expressed high rates of spontaneous calcium fluctuations. Irrespective of age, and even on the same dendrite, there was an inverse relationship between spine motility and presence of FM4-64-labeled terminals in contact with the imaged spines. Spine motility was blocked by latrunculin, which prevents actin polymerization, and was disinhibited by blockade of action potential discharges with tetrodotoxin. It is proposed that an active presynaptic terminal restricts motility of dendritic spines.

Action Potentials↗

PubMedDate not supplied

Theiler virus-GDVII strain (TMEV-GDVII) infection of cultured astrocytes. An image analysis of its effects on cell activation.

Our original aim was to determine whether dBcAMP-induced activation of cultured astrocytes affected the course of subsequent viral infection. After 2 h exposure of 2-day-old first subculture of mouse astrocytes to dBcAMP 1 mM, cell monolayers grown in glass coverslips of Leighton tubes were inoculated with 10(3) PFU of Theiler virus-GDVII strain (TMEV-GDVII). At 9 days post-infection (pi), viral infectivity persisted in supernatants from dBcAMP-treated cultures, but was no longer detectable in non-stimulated controls. The relatively spared astroglial monolayer at day 1 pi, hardly affected by progressive viral cytolytic effect, was chosen for immunolabeled cell count, whether by viral antigen or GFAP. To this end, 20 fields for each coverslip were digitalized at 250x final magnification. In dBcAMP treated cultures, viral antigen(+) cells were fewer and lower in percentage versus infected cultures lacking stimulation. As regards GFAP staining, stimulation or infection per se induced a greater number and percentage of labeled astrocytes. According to morphometric characterization, such increase was due to a greater number of process-bearing astrocytes. It may be concluded that, regardless of previous dBcAMP treatment, early TMEV-GDVII infection enhanced immunocytochemical and morphological differentiation in cultured astrocytes.

Animals↗

PubMed2001

Responses of epithelial and fibroblast-like cells to discontinuous configuration of the culture substrate.

The behaviour of epitheliocytes, their transformed analogues, and fibroblasts was studied on special culture substrates--lattices with large square openings (the area of an opening was 2000 microm2). It was shown that normal epithelocytes and fibroblasts initially attached to and spread on the lattice bars, were soon displaced into the lattice openings and appeared to be "sagged" in the substrate-free spaces. The cells remained attached to the bars only by their edges (epitheliocytes) or lateral processes (fibroblasts), whereas basal surfaces of the cells had no contacts with the substrate. Displacement of the cells from the bars into the lattice openings was observed only if during spreading the cell body was located on two perpendicular bars. In this position the cell body underwent bending which presumably induced stretching of the cell and its displacement into the opening. Unlike epitheliocytes, which gradually "covered" the lattice openings completely, the fibroblasts were retracted and elongated upon their displacement, "crossing" the openings by their bodies and processes. The epitheliocytes transformed by the ras oncogene and displaying a fibroblast-like shape, most often remained on the bars and were not displaced into the lattice openings. Induction of the epithelioid phenotype in fibroblasts by the agents, depolymerizing (colcemid) or disintegrating (taxol) the cytoskeletal system of microtubuli, was accompanied by a change in the behaviour of the cells: the treated fibroblasts, like epitheliocytes, acquired the ability to "cover" the lattice openings. Possible mechanisms of the cell reactions to the substrate having discontinuous configuration are discussed. It is supposed that these distinctions in reactions of epitheliocytes and fibroblast-like cells may result from different bending ability of the cells and/or differences between forces responsible for the cell adhesion to the lattice bars and forces stretching the cells over the lattice openings.

Animals↗

PubMed2001

Modular transport of postsynaptic density-95 clusters and association with stable spine precursors during early development of cortical neurons.

The properties of filopodia and spines and their association with the postsynaptic density (PSD) protein PSD-95 were studied during early development of cultured cortical neurons using time-lapse confocal microscopy. Neurons were transfected with recombinant PSD-95 constructs fused to green fluorescent protein (GFP) for, on average, either 8 d in vitro (DIV) or 14 DIV. We find that, during 1 hr of imaging, filopodia and spines bearing PSD-95/GFP clusters are significantly more stable (i.e., do not turnover) than those lacking clusters. When present within a spine precursor, a PSD-95/GFP cluster appeared to nucleate a relatively stable structure around which filopodium-spine membranes can move. Although processes bearing clusters were generally stable, in 8 DIV neurons, we observed that a subset ( approximately 10%) of PSD-95/GFP clusters underwent rapid modular translocation between filopodia-spines and dendritic shafts. We conclude that, during early synaptic maturation, prefabricated PSD-95 clusters are trafficked in a developmentally regulated process that is associated with filopodial stabilization and synapse formation.

Animals↗

PubMed2001

Synapse-forming axons and recombinant agrin induce microprocess formation on myotubes.

We examined cell-surface behavior at nerve-muscle contacts during synaptogenesis in cocultures of rat ventral spinal cord (VSC) neurons and myotubes. Developing synapses in 1-d-old cocultures were identified by the presence of axon-induced acetylcholine receptor (AChR) aggregation. Identified regions were then examined by transmission and scanning electron microscopy. The myotube surface near contacts with axons that induced AChR aggregation typically displayed ruffles, microvilli, and filopodia (microprocesses), indicating motility of the myotube surface. At some of these contact sites microprocesses were wrapped around the axon, resulting in the partial or total "submersion" of the axon within the myotube contours. Sites of myotube contact with somata and dendrites of the same neurons showed much less evidence of motility and surface interaction than sites of contact with axons. Moreover, the distance between opposed membranes of axons and myotubes was smaller than between dendrites or somata and myotubes, suggesting stronger adhesion of axons. These results suggest polarized expression of molecules involved in the induction of microprocess formation and adhesion in developing VSC neurons. We therefore tested the ability of agrin, which is preferentially secreted by axons, to induce microprocess formation in myotubes. Addition of recombinant C-terminal agrin to culture medium resulted in formation of microprocesses within 3 hr. Myotubes transfected with full-length rat agrin constructs displayed numerous filopodia, as revealed by fluorescence microscopy. The results suggest that the induction of muscle cell surface motility may be linked to the signaling processes that trigger the initial formation of the neuromuscular junction.

Agrin↗

PubMed2002

Interaction between metabotropic and NMDA subtypes of glutamate receptors in sprout suppression at young synapses.

Recently, NMDA receptors (NMDARs) have been implicated in a cell contact-dependent suppression of sprouting in cultured Xenopus tectal neurons during an early period when neither AMPA/kainate (KA) receptors nor action potentials play a prominent role in cell-cell communication. We asked how the NMDA receptors function in the absence of the depolarizing effect of AMPA/KA receptor activity. We show that type II metabotropic glutamate receptors (mGluRs) can operate synergistically with NMDA receptors in the absence of AMPA/KA receptor function to suppress an early neurite sprouting response of the tectal neurons. Calcium imaging with fluo-3 AM and morphological analyses after exposure to glutamate receptor antagonists show that a combination of AMPA/KA receptor and type II mGluR blockers mimics the decrease in intracellular free calcium in response to glutamate and the structural effects produced by NMDA receptor antagonists in these cultures. Patch-clamp analyses confirmed a decrease in NMDA receptor-mediated currents with type II mGluR blockade, and 8-bromo cAMP application produced a decrease in NMDA receptor-mediated calcium influx. These data suggest that type II mGluRs potentiate NMDA receptor function by decreasing cAMP levels in tectal neurons. We also show that NMDARs exhibit low magnesium sensitivity in tectal neurons during the first few days in culture. Thus both metabotropic and ionotropic glutamate receptors can play a role in the contact-mediated suppression of ongoing sprouting at early neuron-neuron contacts before action potential activity.

Animals↗

PubMed2002

Process outgrowth of oligodendrocytes is promoted by interaction of fyn kinase with the cytoskeletal protein tau.

Fyn kinase plays an important role during myelination and has been shown to promote morphological differentiation of cultured oligodendrocytes. We analyzed the downstream targets of Fyn kinase in oligodendrocytes. Because process outgrowth and wrapping of axons involve cytoskeletal rearrangement, we focused on cytoskeletal proteins linked to Fyn. Here we demonstrate that Fyn binds to the cytoskeletal proteins Tau and alpha-Tubulin in oligodendrocytes. Tau interacts with the Fyn SH3 domain whereas alpha-Tubulin binds to the Fyn SH2 and SH3 domains. To study the function of the Fyn-Tau interaction in oligodendrocytes, we designed a Tau deletion mutant that would compete with endogenous Tau-Fyn binding in transfected cells. The mutant Tau protein binds to the Fyn SH3 domain but lacks the microtubuli interaction domain and thus cannot bind to microtubuli. In the presence of the mutant Tau protein, a reduction of the process number and process length in oligodendroglial cells was observed. This effect is likely to be caused by interference with the Fyn-Tau-microtubuli cascade rather than inactivation of the kinase, because Fyn bound to the mutant Tau retains activity. A similar inhibition of process outgrowth was observed when oliogodendroglial cells were cultured in the presence of Fumonisin B1, an inhibitor of sphingolipid synthesis that prevents the formation of rafts. Because ligation of the cell adhesion molecule F3 on oligodendrocytes leads to activation of Fyn kinase localized in rafts, these findings suggest that recruitment of Tau and Tubulin to activated Fyn kinase in rafts is an important step in the initiation of myelination.

Animals↗

PubMed2002

Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters.

Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

Adaptor Proteins, Signal Transducing↗

PubMed2002

A novel mechanism of dendritic spine plasticity involving estradiol induction of prostaglandin-E2.

The mechanisms establishing and maintaining dendritic spines in the mammalian CNS remain primarily unknown. We report a novel mechanism of neuronal spine plasticity in the developing preoptic area in which estradiol induces prostaglandin-E2 (PGE2) synthesis that in turn increases the density of spine-like processes. Estradiol requires PGE2 synthesis, in vivo and in vitro, and upregulates the dendritic spine protein spinophilin, an effect attenuated by antagonism of the AMPA-kainate receptor. This signaling pathway may involve cross talk between neighboring neurons and astrocytes and appears specific to the preoptic area in that hippocampal neurons responded with an increase in spinophilin to estradiol but not PGE2. Regionally specific mechanisms of estradiol-mediated synaptic plasticity allow for epigenetic control of complex sex-typic behaviors.

Animals↗

PubMed2003

Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and beta-actin mRNA in dendrites and spines of hippocampal neurons.

RNA binding proteins may be important mediators of the activity-dependent transport of mRNAs to dendritic spines of activated synapses. We used fluorescence microscopy and digital imaging techniques applied to both fixed and live cultured hippocampal neurons to visualize the localization of the mRNA binding protein, zipcode binding protein 1 (ZBP1), and its dynamic movements in response to KCl-induced depolarization at high spatial and temporal resolution. With the use of immunofluorescence, image deconvolution, and three-dimensional reconstruction, ZBP1 was localized in the form of granules that were distributed in dendrites, spines, and subsynaptic sites. KCl depolarization increased the dendritic localization of ZBP1 that was not attributed to an increase in ZBP1 expression. Live cell imaging of single cells before and after perfusion of KCl revealed the rapid and directed efflux of ZBP1 granules from the cell body into dendrites in a proximo-distal gradient. High-speed imaging of enhanced green fluorescence protein-ZBP1 granules revealed rapid anterograde and retrograde movements in dendrites as well as dynamic movements in dendritic spines. A population of ZBP1 granules colocalized with beta-actin mRNA, and their spatial association in dendrites was increased by KCl depolarization. The NMDA receptor antagonist AP-5 impaired the dendritic localization of ZBP1 and beta-actin mRNA and inhibited the KCl-induced transport of ZBP1. The activity-dependent trafficking of ZBP1 and its dynamic movements within dendritic spines provide new evidence to implicate RNA binding proteins as regulators of mRNA transport to activated synapses in response to synaptic activity.

Actins↗