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Results for “Cell Surface Extensions”

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PubMed2004

Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity.

Glutamate is the predominant excitatory neurotransmitter in the CNS, and it is removed from the synaptic cleft by sodium-dependent glutamate transport activity. Glutamate transporter-1 (GLT-1) is expressed predominantly in astroglial cells and is responsible for the largest proportion of glutamate transport in the adult forebrain. In the present study, we demonstrate the ability of endogenous and recombinant GLT-1 to form clusters in astrocytic processes and characterize the mobility and physiological importance of these clusters in the regulation of GLT-1 activity in the presence or absence of neurons. At the distal end of C6 glioma cell processes, GLT-1 clusters undergo rapid morphological changes in both shape and size, and these changes are inhibited by cytochalasin D treatment, suggesting that the morphogenesis of GLT-1 clusters is highly dependent on the actin network. Treatment of astrocytes with phorbol 12-myristate 13-acetate (PMA) quickly and preferentially decreases GLT-1 localization on the process membrane, leading to de novo generation of GLT-1 clusters along the process shaft. Pretreatment with the PKC inhibitor bisindolylmaleimide II (Bis II), with sucrose (0.4 m), or through the expression of a dominant-negative form of dynamin prevents PMA-induced GLT-1 internalization and cluster formation. In terms of glutamate transporter function, PMA treatment elicits a significant decrease in GLT-1 activity that is prevented by preexposure to either Bis II or hypertonic treatment. Together, these data indicate that GLT-1 trafficking and cluster formation in glial cell processes are dynamic events that play important roles in regulating glutamate uptake in astrocytes and glioma cells.

Actin Cytoskeleton↗

PubMed2004

Stalk phase formation: effects of dehydration and saddle splay modulus.

One of the earliest lipid intermediates forming in the course of membrane fusion is the lipid stalk. Although many aspects of the stalk hypothesis were elaborated theoretically and confirmed by experiments it remained unresolved whether stalk formation is always an energy consuming process or if there are conditions where the stalks are energetically favorable and form spontaneously resulting in an equilibrium stalk phase. Motivated by a recent breakthrough experiments we analyze the physical factors determining the spontaneous stalk formation. We show that this process can be driven by interplay between two factors: the elastic energy of lipid monolayers including a contribution of the saddle splay deformation and the energy of hydration repulsion acting between apposing membranes. We analyze the dependence of stalk formation on the saddle splay (Gaussian) modulus of the lipid monolayers and estimate the values of this modulus based on the experimentally established phase boundary between the lamellar and the stalk phases. We suggest that fusion proteins can induce stalk formation just by bringing the membranes into close contact, and accumulating, at least locally, a sufficiently large energy of the hydration repulsion.

Cell Surface Extensions↗

PubMed2005

Fluctuation analysis of Caulobacter crescentus adhesion.

The aquatic bacterium Caulobacter crescentus divides asymmetrically to a flagellated swarmer cell and a cell with a stalk. At the end of the stalk is an adhesive organelle known as the holdfast, which the stalked cell uses to attach to a solid surface. Often there are two or more cells with their stalks attached to the same holdfast. By analyzing the fluctuations in the stalk angle for a pair of cells attached to a single holdfast, we determine the elastic stiffness of the holdfast. We model the holdfast as three torsional springs in series and find that the effective torsional spring constant for the holdfast is of the order of (10(-17)-10(-18)) Nm, with unequal spring constants. The asymmetry suggests the sequence in which the cells attach to each other, and in some cases suggests that strong crosslinks form between the stalks as they make a shared holdfast.

Bacterial Adhesion↗

PubMed2005

Differential stiffness and lipid mobility in the leaflets of purple membranes.

Purple membranes (PM) are two-dimensional crystals formed by bacteriorhodopsin and a variety of lipids. The lipid composition and density in the cytoplasmic (CP) leaflet differ from those of the extracellular (EC) leaflet. A new way of differentiating the two sides of such asymmetric membranes using the phase signal in alternate contact atomic force microscopy is presented. This method does not require molecular resolution and is applied to study the stiffness and intertrimer lipid mobility in both leaflets of the PM independently over a broad range of pH and salt concentrations. PM stiffens with increasing salt concentration according to two different regimes. At low salt concentration, the membrane Young's normal modulus grows quickly but differentially for the EC and CP leaflets. At higher salt concentration, both leaflets behave similarly and their stiffness converges toward the native environment value. Changes in pH do not affect PM stiffness; however, the crystal assembly is less pronounced at pH > or = 10. Lipid mobility is high in the CP leaflet, especially at low salt concentration, but negligible in the EC leaflet regardless of pH or salt concentration. An independent lipid mobility study by solid-state NMR confirms and quantifies the atomic force microscopy qualitative observations.

Cell Surface Extensions↗

PubMed2006

Sialic acid and the central nervous system: perspectives on biological functions, detection, imaging methods and manipulation.

Glycobiology, broadly defined as the study of sugars in living systems, is becoming increasingly important for understanding the basic biology of the central nervous system (CNS) and diagnosing and devising new treatments for neurological disorders. Decades of research have uncovered many roles for both glycolipids and glycoproteins in the proper functioning of the brain; moreover many diseases are characterized by abnormalities in either the biosynthesis or catabolism of these cellular components. In many cases, however, only a rudimentary understanding of the basic biological roles of sugars in neural function exists. Similarly, methods to detect and diagnose glycosylation disorders are far from state-of-the-art compared to many facets of modern medicine. This review focuses on sialic acid, arguably the most important monosaccharide in CNS, and describes how recent advances in its manipulation by chemical and metabolic methods hold the possibility to converge with advanced instrumentation such as magnetic resonance imaging, positron emission tomography, diffusion tensor imaging, and single photon emission computerized tomography now used for imaging of the CNS in human subjects. Specifically, methods are under development for tagging sialic acids in living systems with contrast agents suitable for magnetic resonance imaging, in essence allowing for the functional imaging of sugars at a molecular level. One of these methods, biochemical engineering of sialic acids by use of small molecule metabolic substrates, also holds promise for the manipulation of sialic acids for the development of novel therapies for neurological disorders.

Animals↗

PubMed2000

Observation of the abluminal surface of the atrioventricular endothelium which undergoes transition into cardiac cushion mesenchymal cells in the chick embryo.

In the developing chick heart, endothelial cells in the atrioventricular canal (AV) undergo a series of morphological changes and transform into cushion mesenchymal cells. In the present scanning electron microscopic study, we examined the abluminal surface features of the AV endothelium through an artificial window in the myocardial wall. The AV endothelial cell at stages 12 or earlier had a smooth, flattened basal surface with only a few blebs. In the successive stages, the abluminal surface exhibited remarkable changes; 1) the number of blebs increased, 2) elongated microvillous projections emerged, and 3) a thick filopodium, or a migratory appendage developed. It appeared, however, that these changes do not occur synchronously within the entire AV endothelium but were initially observed mostly in the proximity of the endothelial "crease" which was a limited invagination of the endothelial sheet towards the underlying acellular matrix. In addition, even in the proximity of the crease, endothelial cells with flattened basal surfaces were also observed next to endothelial cells that showed apparent morphological indications of transition into mesenchymal cells. These findings suggest that AV endothelial cells are possibly heterogeneous in the competency of transformation into mesenchymal cells and such heterogeneity would be important for maintaining the continuity of the AV endothelium.

Animals↗

PubMed2005

Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

CX3CL1 (fractalkine), the only member of the delta subclass of chemokines, is a known chemotactic factor for monocytes/macrophages as well as NK cells and T lymphocytes. In several pathologies, excessive production of CX3CL1 at specific sites leads primarily to monocyte/macrophage recruitment, which causes tissue and vascular damage. Despite their clinical relevance, the mechanisms underlying monocyte/macrophage chemotaxis to CX3CL1 remain poorly documented. The present report addresses this issue and identifies cell signaling crucial for this process. Using the murine monocyte/macrophage RAW cell line, we show that CX3CL1 treatment elicits a rapid and transient increase in F-actin and the formation of F-actin-enriched cell protrusions. CX3CL1 also triggers tyrosine phosphorylation of proteins localized in those protrusions. The protein tyrosine kinase Syk is activated upon CX3CL1 treatment, and reduction of Syk expression using RNA-mediated interference results in a specific and massive impairment of RAW cell migration to CX3CL1. Similar results are obtained using the Syk inhibitor, piceatannol. Cells with reduced Syk expression also exhibit a major defect in CX3CL1-induced cytoskeletal remodeling. These data suggest that in monocytes/macrophages, Syk is essential for proper reorganization of the actin cytoskeleton in response to CX3CL1 and is therefore required for cell chemotaxis to CX3CL1.

Actins↗

PubMed2002

Remodeling of hippocampal synaptic networks by a brief anoxia-hypoglycemia.

Cerebral ischemia is a major cause of brain dysfunction. Using a model of delayed death induced by a brief, transient oxygen and glucose deprivation, we studied here how this affected the structural organization of hippocampal synaptic networks. We report that brief anoxic-hypoglycemic episodes rapidly modified the structure of synapses. This was characterized, at the electron microscopic level, by a transient increase in the proportion of perforated synapses, followed after 2 hr by an increase in images of multiple synapse boutons. These changes were considerable because 10-20% of all synapses were affected. This structural remodeling was correlated by three kinds of modifications observed using two-photon confocal microscopy: the growth of filopodia, occurring shortly (5-20 min) after anoxia-hypoglycemia, enlargements of existing spines, and formation of new spines, both seen mainly 20-60 min after the insult. All of these structural changes were calcium and NMDA receptor dependent and thus reproduced, to a larger scale, those associated with synaptic plasticity. Concomitantly and related to the severity of anoxia-hypoglycemia, we could also observe spine loss and images of spine, dendrite, or presynaptic terminal swellings that evolved up to membrane disruption. These changes were also calcium dependent and reduced by NMDA receptor antagonists. Thus, short anoxic-hypoglycemic episodes, through NMDA receptor activation and calcium influx, resulted in a profound structural remodeling of synaptic networks, through growth, formation, and elimination of spines and synapses.

Animals↗

PubMed2002

The projections of early enteric neurons are influenced by the direction of neural crest cell migration.

The enteric nervous system arises from the neural crest. In embryonic mice, vagal neural crest cells enter the developing foregut at approximately embryonic day 9.5 (E9.5) and then migrate rostrocaudally to colonize the entire gastrointestinal tract by E14.5. This study showed that a subpopulation of vagal crest-derived cells, very close to the migratory wavefront, starts to differentiate into neurons early, as shown by the expression of neuron-specific proteins and the absence of Sox10. Many of the early differentiating neurons transiently exhibited tyrosine hydroxylase (TH) immunoreactivity. The TH cells were demonstrated to be the progenitors of nitric oxide synthase (NOS) neurons. Immunohistochemistry, lesions, and DiI tracing were used to examine the projections of developing enteric neurons. The axons of first neurons in the gut (the TH-NOS neurons) projected in the same direction (caudally), and traversed the same pathways through the mesenchyme, as the migrating, undifferentiated, vagal crest-derived cells. To examine if the direction of migration and direction of axon projection are linked, coculture experiments were set up in which vagal crest-derived cells migrated either rostrocaudally (as they do in vivo), or caudorostrally (which they do not normally do), to colonize explants of embryonic aneural hindgut. The direction in which neurons projected was correlated with the direction of cell migration, but migration direction appears to be not the only mechanism influencing axon projection. Peristaltic reflexes involve both orally (rostrally) projecting neurons and anally (caudally) projecting neurons. Because few rostrally projecting neurons could be detected before birth, the full circuitry for peristaltic reflexes appears to develop after birth.

Animals↗

PubMed2001

Synaptically driven calcium transients via nicotinic receptors on somatic spines.

Dendritic spines commonly receive glutamatergic innervation at postsynaptic densities and compartmentalize calcium influx arising from synaptic signaling. Recently, it was shown that a class of nicotinic acetylcholine receptors containing alpha7 subunits is concentrated on somatic spines emanating from chick ciliary ganglion neurons. The receptors have a high relative calcium permeability and contribute importantly to synaptic currents, although they appear to be excluded from postsynaptic densities. Here we show that low-frequency synaptic stimulation of the alpha7-containing receptors induces calcium transients confined to the spines. High-frequency stimulation induces a transient calcium elevation in the spines and a more sustained cell-wide elevation. The high-frequency transient elevation again depends on alpha7-containing receptors, whereas the sustained elevation can be triggered by other nicotinic receptors and depends on calcium release from internal stores and probably influx through voltage-gated L-type calcium channels as well. Retrograde axonal stimulation of the neurons at high frequency mimics synaptic stimulation in producing sustained cell-wide calcium increases that depend on L-type channels and release from internal stores, but it does not produce calcium transients in the spines. Thus frequent action potentials are sufficient to generate the cell-wide increases, but alpha7-containing receptors are needed for spine-specific effects. Patch-clamp recording indicates that alpha7-containing receptors preferentially desensitize at high-frequency stimulation, accounting for the inability of the stimulation to sustain high calcium levels in the spines. The spatial and temporal differences in the patterns of calcium elevation could enable the neurons to monitor their own firing histories for regulatory purposes.

Action Potentials↗

PubMed2001

Abnormal development of dendritic spines in FMR1 knock-out mice.

Fragile X syndrome is caused by a mutation in the FMR1 gene leading to absence of the fragile X mental retardation protein (FMRP). Reports that patients and adult FMR1 knock-out mice have abnormally long dendritic spines of increased density suggested that the disorder might involve abnormal spine development. Because spine length, density, and motility change dramatically in the first postnatal weeks, we analyzed these properties in mutant mice and littermate controls at 1, 2, and 4 weeks of age. To label neurons, a viral vector carrying the enhanced green fluorescent protein gene was injected into the barrel cortex. Layer V neurons were imaged on a two-photon laser scanning microscope in fixed tissue sections. Analysis of >16,000 spines showed clear developmental patterns. Between 1 and 4 weeks of age, spine density increased 2.5-fold, and mean spine length decreased by 17% in normal animals. Early during cortical synaptogenesis, pyramidal cells in mutant mice had longer spines than controls. At 1 week, spine length was 28% greater in mutants than in controls. At 2 weeks, this difference was 10%, and at 4 weeks only 3%. Similarly, spine density was 33% greater in mutants than in controls at 1 week of age. At 2 or 4 weeks of age, differences were not detectable. The spine abnormality was not detected in neocortical organotypic cultures. The transient nature of the spine abnormality in the intact animal suggests that FMRP might play a role in the normal process of dendritic spine growth in coordination with the experience-dependent development of cortical circuits.

Aging↗

PubMed2001

Remodeling of synaptic membranes after induction of long-term potentiation.

Several morphological changes of synapses have been reported to be associated with the induction of long-term potentiation (LTP) in the CA1 hippocampus, including an transient increase in the proportion of synapses with perforated postsynaptic densities (PSDs) and a later occurrence of multiple spine boutons (MSBs) in which the two spines arise from the same dendrite. To investigate the functional significance of these modifications, we analyzed single sections and reconstructed 134 synapses labeled via activity using a calcium precipitation approach. Analyses of labeled spine profiles showed changes of the spine head area, PSD length, and proportion of spine profiles containing a coated vesicle that reflected variations in the relative proportion of different types of synapses. Three-dimensional reconstruction indicated that the increase of perforated spine profiles observed 30 min after LTP induction essentially resulted from synapses exhibiting segmented, completely partitioned PSDs. These synapses had spine head and PSD areas approximately three times larger than those of simple synapses. They contained coated vesicles in a much higher proportion than that of any other type of synapse and exhibited large spinules associated with the PSD. Also the MSBs with two spines arising from the same dendrite that were observed 1-2 hr after LTP induction included a spine that was smaller and a PSD that was smaller than those of simple synapses. These results support the idea that LTP induction is associated with an enhanced recycling of synaptic membrane and that this process could underlie the formation of synapses with segmented PSDs and eventually result in the formation of a new, immature spine.

Animals↗

PubMed2001

Experience- and age-related outgrowth of intrinsic neurons in the mushroom bodies of the adult worker honeybee.

A worker honeybee performs tasks within the hive for approximately the first 3 weeks of adult life. After this time, it becomes a forager, flying repeatedly to collect food outside of the hive for the remainder of its 5-6 week life. Previous studies have shown that foragers have an increased volume of neuropil associated with the mushroom bodies, a brain region involved in learning, memory, and sensory integration. We report here that growth of the mushroom body neuropil in adult bees occurs throughout adult life and continues after bees begin to forage. Studies using Golgi impregnation asked whether the growth of the collar region of the mushroom body neuropil was a result of growth of the dendritic processes of the mushroom body intrinsic neurons, the Kenyon cells. Branching and length of dendrites in the collar region of the calyces were strongly correlated with worker age, but when age-matched bees were directly compared, those with foraging experience had longer, more branched dendrites than bees that had foraged less or not at all. The density of Kenyon cell dendritic spines remained constant regardless of age or behavioral state. Older and more experienced foragers therefore have a greater total number of dendritic spines in the mushroom body neuropil. Our findings indicate that, under natural conditions, the cytoarchitectural complexity of neurons in the mushroom bodies of adult honeybees increases as a function of increasing age, but that foraging experience promotes additional dendritic branching and growth.

Aging↗

PubMed2001

Role of root hairs and lateral roots in silicon uptake by rice.

The rice plant (Oryza sativa L. cv Oochikara) is known to be a Si accumulator, but the mechanism responsible for the high uptake of Si by the roots is not well understood. We investigated the role of root hairs and lateral roots in the Si uptake using two mutants of rice, one defective in the formation of root hairs (RH2) and another in that of lateral roots (RM109). Uptake experiments with nutrient solution during both a short term (up to 12 h) and relatively long term (26 d) showed that there was no significant difference in Si uptake between RH2 and the wild type (WT), whereas the Si uptake of RM109 was much less than that of WT. The number of silica bodies formed on the third leaf in RH2 was similar to that in WT, but the number of silica bodies in RM109 was only 40% of that in WT, when grown in soil amended with Si under flooded conditions. There was also no difference in the shoot Si concentration between WT and RH2 when grown in soil under upland conditions. Using a multi-compartment transport box, the Si uptake at the root tip (0-1 cm, without lateral roots and root hairs) was found to be similar in WT, RH2, and RM109. However, the Si uptake in the mature zone (1-4 cm from root tip) was significantly lower in RM109 than in WT, whereas no difference was found in Si uptake between WT and RH2. All these results clearly indicate that lateral roots contribute to the Si uptake in rice plant, whereas root hairs do not. Analysis of F(2) populations between RM109 and WT showed that Si uptake was correlated with the presence of lateral roots and that the gene controlling formation of lateral roots and Si uptake is a dominant gene.

Cell Surface Extensions↗

PubMed2001

Phenotypical variation in a toxic strain of the phytoplankter, Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) during batch culture.

A nonaxenic strain of Cylindrospermopsis raciborskii Woloszynska (AWT 205) was grown in batch culture, with and without nitrate as the primary N source. Rapid log-phase growth with nitrate was 1.0 doubling/day versus 0.3 doubling/day without nitrate. Cylindrospermopsin (CYN) production was measured by HPLC. The rate of intracellular CYN production matched cell division rate for both the diazotrophies at cell densities less than 10(7) cell/ml. At cell density > 10(7) cell/ml, additional resource limitation in batch culture slowed log-phase growth to 0.04 division/day and cell division and CYN production decoupled. Intracellular CYN concentration increased at a rate of 0.08 doubling/day, twice the cell division rate. Extracellular CYN as a proportion of the total CYN increased from 20% during the rapid growth phase, to 50% during the slow growth phase. The total CYN yield from cultures grown out to stationary phase (55 days) exceeded 2 mg CYN/I. C. raciborskii cells in log-phase growth, exposed to 1 ppm copper (as copper sulphate), lysed within 24 hours. After copper treatment, all CYN was in the filterable fraction. These findings imply that in naturally occurring blooms of C. raciborskii, the movement of intracellular CYN into solution will be the greatest during stationary phase, when intracellular concentrations are highest and cell lysis is more frequent. The application of algicides that promote cell lysis will exacerbate this effect.

Alkaloids↗

PubMed2002

Ultrastructure of a somatic spine mat for nicotinic signaling in neurons.

Chick ciliary neurons have somatic spines grouped in discrete clumps or mats tightly folded against the soma and enriched in nicotinic receptors containing alpha7 subunits. An embryonic ciliary neuron has one to two dozen such spine mats, all overlaid by a large presynaptic calyx engulfing the cell. Three-dimensional tomographic reconstruction from serial thick sections revealed 13 somatic spines in one complete spine mat on a ciliary neuron late in embryogenesis. The spines varied in morphology and usually were branched but had numerous similarities to dendritic spines, including mean length, volume, surface area, presence of endoplasmic reticulum, and occasional multivesicular bodies. The spines invariably were connected to the soma via a narrow neck of approximately 0.2 micrometer in diameter as found for dendritic spines, suggesting restricted access from spine lumen to soma. A prominent difference between dendritic and somatic spines is the absence of postsynaptic densities from most somatic spines both on embryonic and adult ciliary neurons. Transmitter access to receptors on the spines may occur either by lateral diffusion from release sites over nearby postsynaptic densities or by release directly onto spines from the overlying calyx lined with vesicles. The latter is less likely in the adult, where some spines are adjacent to but not overlaid by vesicle-enriched presynaptic structures. The anatomical configuration of spine mats suggests coordinate spine activation by transmitter release into a confined volume while spine morphology is used to control the chemical consequences of synaptic signaling.

Aging↗

PubMed2002

Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth.

The ability of neurons to regenerate an axon after injury is determined by both the surrounding environment and factors intrinsic to the damaged neuron. We have used cDNA microarrays to survey those genes induced during successful sciatic nerve regeneration. The small proline-rich repeat protein 1A (SPRR1A) is not detectable in uninjured neurons but is induced by >60-fold after peripheral axonal damage. The protein is localized to injured neurons and axons. sprr1a is one of a group of epithelial differentiation genes, including s100c and p21/waf, that are coinduced in neurons by axotomy. Overexpressed SPRR1A colocalizes with F-actin in membrane ruffles and augments axonal outgrowth on a range of substrates. In axotomized sensory neurons, reduction of SPRR1A function restricts axonal outgrowth. Neuronal SPRR1A may be a significant contributor to successful nerve regeneration.

Actins↗

PubMedDate not supplied

[Structure of the cerebral cortex. Intrinsic organization and comparative analysis of the neocortex].

We review our present knowledge on the intrinsic organization of the neocortex based on studies carried out with the Golgi method in several mammalian species. An outline is presented on certain general aspects of the termination of specific afferent fibers in layer IV in insectivora, rodents, carnivora and primates. The principal components of the cerebral cortex have been classified in two broad types: pyramidal cells, which account for nearly 70% of the total population, and intrinsic neurons. We review different morphological characteristics of pyramidal cells and intrinsic neurons with a full description of cell varieties, dendritic morphology and several aspects of synaptic connectivity of intrinsic neurons in comparison between different species. Special attention has been paid to the description of spinous stellate cells which seems unique for a given animal species. We have reviewed intrinsic cortical circuits linking several neocortical layers, the distribution of dendritic plexuses, and the excitatory and inhibitory synaptic connections established between various neuronal categories. We finally review modern concepts of functional neocortical architecture based on the modular organization of the cerebral cortex, concluding that the cerebral cortex is rather uniform at en elementary level but the differences appear substantial when the comparison is made between different species.

Animals↗