Biomedical subjects

M E Martone

Publications and source records attributed to M E Martone.

At least 19 recordsLinked to original sources

PubMed1999

Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C.

We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.

Actinin↗

PubMed1999

Modification of postsynaptic densities after transient cerebral ischemia: a quantitative and three-dimensional ultrastructural study.

Abnormal synaptic transmission has been hypothesized to be a cause of neuronal death resulting from transient ischemia, although the mechanisms are not fully understood. Here, we present evidence that synapses are markedly modified in the hippocampus after transient cerebral ischemia. Using both conventional and high-voltage electron microscopy, we performed two- and three-dimensional analyses of synapses selectively stained with ethanolic phosphotungstic acid in the hippocampus of rats subjected to 15 min of ischemia followed by various periods of reperfusion. Postsynaptic densities (PSDs) from both area CA1 and the dentate gyrus were thicker and fluffier in postischemic hippocampus than in controls. Three-dimensional reconstructions of selectively stained PSDs created using electron tomography indicated that postsynaptic densities became more irregular and loosely configured in postischemic brains compared with those in controls. A quantitative study based on thin sections of the time course of PSD modification indicated that the increase in thickness was both greater and more long-lived in area CA1 than in dentate gyrus. Whereas the magnitude of morphological change in dentate gyrus peaked at 4 hr of reperfusion (140% of control values) and declined thereafter, changes in area CA1 persisted and increased at 24 hr of reperfusion (191% of control values). We hypothesize that the degenerative ultrastructural alteration of PSDs may produce a toxic signal such as a greater calcium influx, which is integrated from the thousands of excitatory synapses onto dendrites, and is propagated to the neuronal somata where it causes or contributes to neuronal damage during the postischemic phase.

Animals↗

PubMed1999

Neuronal acetylcholine receptors with alpha7 subunits are concentrated on somatic spines for synaptic signaling in embryonic chick ciliary ganglia.

Nicotinic acetylcholine receptors containing alpha7 subunits are widely distributed in the vertebrate nervous system. In the chick ciliary ganglion such receptors generate large synaptic currents but appear to be excluded from postsynaptic densities on the cells. We show here that alpha7-containing receptors are concentrated on somatic spines in close proximity to putative sites of presynaptic transmitter release. Intermediate voltage electron microscopy on thick sections, together with tomographic reconstruction, permitted three-dimensional analysis of finger-like projections emanating from cell bodies. The projections were identified as spines based on their morphology, cytoskeletal content, and proximity to presynaptic elements. Both in situ and after ganglionic dissociation, the spines were grouped on the cell surface and tightly folded into mats. Immunogold labeling of receptors containing alpha7 subunits showed them to be preferentially concentrated on the somatic spines. Postsynaptic densities were present in vivo both on the soma near spines and occasionally on the spines themselves. Synaptic vesicle-filled projections from the presynaptic calyx were interdigitated among the spines. Moreover, the synaptic vesicles often abutted the membrane and sometimes included Omega profiles as if caught in an exocytotic event, even when no postsynaptic densities were juxtaposed on the spine. The results suggest several mechanisms for delivering transmitter to alpha7-containing receptors, and they support new ideas about synaptic signaling via spines. They also indicate that neurons must have specific mechanisms for targeting alpha7-containing receptors to desired locations.

Animals↗

PubMed1999

Cellular and subcellular distribution of the calcium-binding protein NCS-1 in the central nervous system of the rat.

NCS-1 (neuronal calcium sensor) is a recently characterized member of a highly conserved neuron-specific family of calcium-binding proteins, which also includes frequenin and recoverin. The cellular and subcellular distributions of NCS-1 in the rat nervous system were investigated using light- and electron-microscopic immunohistochemistry. NCS-1 immunoreactivity was localized to neuronal cell bodies and axons throughout the brain and spinal cord but not to glial cells. The most intense labeling was observed in myelinated axons, the axonal ramifications of the basket cell in the cerebellar cortex, and large neurons in the brainstem and pons. These same structures were also characterized by heavy labeling for neurofilament protein, as determined by double-labeling experiments. Most axon terminals were unlabeled or only lightly labeled. The most remarkable subcellular staining occurred in the perikarya where intense labeling was associated with the membranes of the trans saccules of the Golgi apparatus. The widespread distribution of NCS-1 indicates that it may be active in a variety of calcium-dependent neuronal functions, whereas the specific subcellular localization to the Golgi apparatus and neurofilament-rich structures suggests a specialized role in calcium regulated protein trafficking and cytoskeletal interactions.

Animals↗

PubMed1999

Persistent phosphorylation of cyclic AMP responsive element-binding protein and activating transcription factor-2 transcription factors following transient cerebral ischemia in rat brain.

The transcription factors cyclic AMP responsive element-binding protein (CREB) and activating transcription factor-2 were studied in rat brains subjected to 15 min ischemia followed by varied periods of reperfusion using western blot and immunocytochemical analyses. The total amounts of both CREB and activating transcription factor-2 were not altered in the hippocampus after ischemia. In contrast, levels of the phosphorylated forms of both transcription factors decreased during ischemia but rebounded following reperfusion. The phospho-forms of CREB and activating transcription factor-2 showed regional and temporal differences in their expression. Phospho-CREB was increased relative to control levels at 30 min, and continued to increase for at least three days postischemia, mainly in dentate granule cells. The level of phospho-activating transcription factor-2 appeared to be higher in CAI pyramidal cells than in dentate granule cells after ischemia. The present findings suggest that the signaling pathways for phosphorylation of CREB may be neuroprotective for dentate cells, which are relatively resistant to ischemic insults. The increased phospho-activating transcription factor-2 may reflect increased stresses in these neurons. The more modest activation of CREB pathways in CA1 neurons may not be enough to overcome the increased stresses in these neurons, contributing to delayed neuronal death.

Activating Transcription Factor 2↗

PubMed1999

Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy.

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy, but the mechanism of this pathology is unknown. Mutations in cytoskeletal proteins such as dystrophin cause hereditary dilated cardiomyopathy, but it is unclear if similar mechanisms underlie acquired forms of heart failure. We demonstrate here that purified Coxsackievirus protease 2A cleaves dystrophin in vitro as predicted by computer analysis. Dystrophin is also cleaved during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function. In vivo, dystrophin and the dystrophin-associated glycoproteins alpha-sarcoglycan and beta-dystroglycan are morphologically disrupted in infected myocytes. We suggest a molecular mechanism through which enteroviral infection contributes to the pathogenesis of acquired forms of dilated cardiomyopathy.

Animals↗

PubMed1998

Assembly of proteins to postsynaptic densities after transient cerebral ischemia.

Transient ischemia leads to changes in synaptic efficacy and results in selective neuronal damage during the postischemic phase, although the mechanisms are not fully understood. The protein composition and ultrastructure of postsynaptic densities (PSDs) were studied by using a rat transient ischemic model. We found that a brief ischemic episode induced a marked accumulation in PSDs of the protein assembly ATPases, N-ethylmaleimide-sensitive fusion protein, and heat-shock cognate protein-70 as well as the BDNF receptor (trkB) and protein kinases, as determined by protein microsequencing. The changes in PSD composition were accompanied by a 2.5-fold increase in the yield of PSD protein relative to controls. Biochemical modification of PSDs correlated well with an increase in PSD thickness observed in vivo by electron microscopy. We conclude that a brief ischemic episode modifies the molecular composition and ultrastructure of synapses by assembly of proteins to the postsynaptic density, which may underlie observed changes in synaptic function and selective neuronal damage.

Adenosine Triphosphatases↗

PubMed1998

Subcellular localization of mRNA in neuronal cells. Contributions of high-resolution in situ hybridization techniques.

The development of technologies for high-resolution nucleic acid localization in cells and tissues has contributed significantly to our understanding of transcriptional and translational regulation in eukaryotic cells. These methods include nonisotopic in situ hybridization methods for light and electron microscopy, and fluorescent tagging for the study of nucleic acid behavior in living cells. In situ hybridization to detect messenger RNA has led to the discovery that individual transcripts may be selectively targeted to particular subcellular domains. In the nervous system, certain species of mRNA have been localized in distal processes in nerve cells and glia. Direct visualization of mRNA and its interactions with subcellular features, such as synaptic specializations, cytoskeletal elements, and nuclear pores, have been achieved. Of particular interest is the presence of mRNA and ribosomes in dendrites, beneath synaptic contacts, suggesting the possibility of synaptic regulation of protein synthesis. The following article will describe the application of high-resolution in situ hybridization and live imaging techniques to the study of mRNA targeting in neurons.

Animals↗

PubMed1997

Differential distribution and subcellular localization of ryanodine receptor isoforms in the chicken cerebellum during development.

The distribution of ryanodine receptor (RyR) isoforms was examined using isoform-specific monoclonal antibodies in the developing chicken brain, from E18 through adulthood, using light and electron microscopic immunocytochemistry. Monoclonal antibody 110F is specific for the alpha-skeletal muscle form of RyR, while monoclonal antibody 110E recognizes both the beta-skeletal muscle and cardiac isoforms, but does not distinguish between the two. Significant differences in the distribution of the alpha- and beta/cardiac forms were observed. Labeling for the alpha-form was restricted to cerebellar Purkinje neurons while the beta/cardiac form was observed in neurons throughout the brain. A major finding was the presence of labeling for the beta/cardiac in presynaptic terminals of the parallel fibers in the molecular layer and the mossy fiber terminals in the granular layer glomeruli in late development and during adulthood. Labeling for the beta/cardiac, but not the alpha-form, underwent a major redistribution in the cerebellum during the course of development. At 1 day of age, beta/cardiac labeling was present mainly in Purkinje neurons. From 1 day to 4 weeks, immunolabeling for the beta/cardiac form gradually disappeared from Purkinje neurons, but increased in granule cells. Within the molecular layer, the labeling pattern changed from being primarily within Purkinje dendrites to a more diffuse pattern. Electron microscopic examination of the cerebellar molecular layer of 2-week-old chicks revealed that beta/cardiac-labeling was mainly present in the axons and presynaptic processes of the parallel fibers. No developmental changes were observed in other brain regions. This study represents the first demonstration of ryanodine receptor immunoreactivity in presynaptic boutons and suggests that the ryanodine receptor may modulate neurotransmitter release through local regulation of intracellular calcium in the parallel fiber synapse.

Animals↗

PubMed1997

Immunolocalization of the receptor tyrosine kinase EphA4 in the adult rat central nervous system.

EphA4 is a receptor tyrosine kinase of the Eph family previously designated Cek8 in chicken, Tyro1 in rat, and Sek1 in mouse, which is preferentially expressed in the embryonic and adult nervous system. We have mapped the distribution of EphA4 in the adult rat brain and spinal cord using a polyclonal antibody raised against a synthetic carboxy-terminal peptide. Immunoblotting experiments revealed that EphA4 is widely distributed in various regions of the adult rat brain. At the light microscopic level, intense immunoreactivity was apparent in the cerebral cortex, hippocampus, matrix compartment of the neostriatum, cholinergic neurons in the basal forebrain, cerebellar Purkinje cells, and substantia gelatinosa of the spinal cord. Among white matter tracts, EphA4 expression was detected in the corpus callosum, fornix, and posterior portion of the anterior commissure, but not in the lateral olfactory tract, mammillothalamic tract, or optic chiasm. Interestingly, expression in the optic chiasm is high at postnatal day 6, but decreases with the maturation of this structure. While in some regions of the neuropil neuronal cell bodies were prominently labeled, in others EphA4 immunoreactivity was detected in a punctate pattern. This punctuate staining did not coincide with synaptophysin localization. At the electron microscopic level, EphA4 immunoreactivity was observed in dendrites in the gray matter, particularly associated with dendritic spines, and in myelinated axons, but not their myelin sheaths in the white matter. The widespread distribution and diverse subcellular compartmentalization of EphA4 suggest that this receptor is important for the maintenance of multiple structures in the adult nervous system.

Animals↗

PubMed1997

Subcellular localization of the K+ channel subunit Kv3.1b in selected rat CNS neurons.

Voltage-gated potassium channels constitute the largest group of heteromeric ion channels discovered to date. Over 20 genes have been isolated, encoding different channel subunit proteins which form functional tetrameric K+ channels. We have analyzed the subcellular localization of subunit Kv3.1b, a member of the Kv3 (Shaw-like) subfamily, in rat brain at the light and electron microscopic level, using immunocytochemical detection. Detailed localization was carried out in specific neurons of the neocortex, hippocampus and cerebellum. The identity of Kv3.1b-positive neurons was established using double labeling with markers for specific neuronal populations. In the neocortex, the Kv3.1b subunit was expressed in most parvalbumin-containing bipolar, basket or chandelier cells, and in some bipolar or double bouquet neurons containing calbindin. In the hippocampus, Kv3.1b was expressed in many parvalbumin-containing basket cells, as well as in calbindin-positive neurons in the stratum oriens, and in a small number of interneurons that did not stain for either parvalbumin or calbindin. Kv3.1b protein was not present in pyramidal cells in the neocortex and the hippocampus, but these cells were outlined by labeled presynaptic terminals from interneuron axons that surround the postsynaptic cell. In the cerebellar cortex, granule cells were the only population expressing the channel protein. Careful examination of individual granule cells revealed a non-uniform distribution of Kv3.1 staining on the somata: circular bands of labeling were present in the vicinity of the axon hillock. In cortical and hippocampal interneurons, as well as in cerebellar granule cells, the Kv3.1b subunit was present in somatic and unmyelinated axonal membranes and adjacent cytoplasm, as well as in the most proximal portion of dendritic processes, but not throughout most of the dendrite. Labeling was also seen in the terminals of labeled axons, but not at a higher concentration than in other parts of the axon. The distribution in the cells analyzed supports a role in action potential transmission by regulating action potential duration.

Animals↗

PubMed1997

Distribution of inositol-1,4,5-trisphosphate and ryanodine receptors in rat neostriatum.

The distribution of the inositol-1,4,5-trisphosphate (IP3) and the cardiac form of the ryanodine receptor, two intracellular calcium channels, was examined in the rat neostriatum. Both IP3 and ryanodine receptor labeling occurred within striatal medium spiny cells but only ryanodine receptor labeling was present in choline acetyltransferase- and parvalbumin-positive interneurons. IP3 receptor labeling was observed within cell bodies, dendrites and spines of spiny striatal neurons, as seen at both the light and electron microscopic levels. Subcellular labeling for the ryanodine receptor was restricted to cell bodies and proximal dendrites when a polyclonal antibody raised against a peptide sequence from the dog cardiac ryanodine receptor was employed. More extensive dendritic labeling was seen using monoclonal antibody MA3-916, also raised against the canine cardiac ryanodine receptor. At the ultrastructural level, labeled dendritic spines were observed frequently with the monoclonal but not the polyclonal antibody. Ryanodine receptor labeling also was present within astrocytic processes surrounding blood vessels and within the neuropil, regardless of the antibody used. The results of these studies suggest that the ryanodine receptor plays a general role in intracellular calcium regulation within striatal cells while the IP3 receptor plays a specialized role within spiny neurons.

Animals↗

PubMed1997

Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts.

The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in approximately 500 nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1 degree or 2 degrees ranging from -60 degrees to +60 degrees. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each approximately 7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of approximately 22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, approximately 14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes approximately 14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion.

Animals↗

PubMed1997

Electron tomography of large, multicomponent biological structures.

Electron tomography is an extremely useful method for deriving three-dimensional structure from electron microscope images. The application of this technique to the reconstruction of large, complex structures such as mitochondria is described in conjunction with several tools for segmentation, measurement, classification, and visualization. In addition, the use of massively parallel computers to perform the tomographic reconstruction efficiently using R-weighted backprojection or iterative techniques is described.

Algorithms↗

PubMed1996

Translocation of RNA granules in living neurons.

Sorting of RNAs to specific subcellular loci occurs in diverse settings from fly oocytes to mammalian neurons. Using the membrane-permeable nucleic acid stain SYTO 14, we directly visualized the translocation of endogenous RNA in living cells. Labeled RNA was distributed nonrandomly as discrete granules in neuronal processes. The labeled granules colocalized with poly(A+) mRNA, with the 60S ribosomal subunit, and with elongation factor 1alpha, suggesting that granules represent a translational unit. A subset of labeled granules colocalized with beta-actin mRNA. Correlative light and electron microscopy indicated that the fluorescent granules corresponded to clusters of ribosomes at the ultrastructural level. Poststaining of sections with heavy metals confirmed the presence of ribosomes within these granules. In living neurons, a subpopulation of RNA granules was motile during the observation period. They moved at an average rate of 0.1 microm/sec. In young cultures their movements were exclusively anterograde, but after 7 d in culture, one-half of the motile granules moved in the retrograde direction. Granules in neurites were delocalized after treatment with microtubule-disrupting drugs. These results raise the possibility of a cellular trafficking system for the targeting of RNA in neurons.

Animals↗

PubMed1996

Ultrastructural localization of dendritic messenger RNA in adult rat hippocampus.

An ultrastructural examination of mRNA within adult rat CA1 hippocampal dendrites was conducted using two different methods. The messages for the alpha and beta forms of the calcium-calmodulin-dependent protein kinase II were localized in ultracryosections using silver-intensified gold detection of isoform-specific oligonucleotide probes. Labeling for both isoforms was observed within the cell bodies and proximal dendrites of pyramidal neurons, but only the alpha form was observed in more distal dendrites. Unfortunately, the morphological preservation of the tissue was not sufficient to determine the localization of labeling relative to subcellular features such as dendritic spines. To address this issue, a preembedding peroxidase-based method was developed, resulting in better preservation of the neuropil. The total population of polyadenylated [poly(A)] mRNA was localized in hippocampus using a biotinylated poly(dT) probe. Poly(A) mRNA was present in the nucleus and throughout the cell body of all hippocampal cells and within isolated dendrites and glial processes within the neuropil. Within pyramidal neurons, labeling was distributed in a longitudinal pattern in proximal apical dendrites. More distally, the amount of labeling diminished, and smaller foci of labeling were observed, particularly near the plasma membrane. Concentrated labeling was present at the base of dendritic spines and, less frequently, near synapses onto the dendritic shaft. These results suggest that dendritic mRNA is found in the vicinity of postsynaptic sites and provide additional evidence that local protein synthesis may play an important role in establishing and maintaining synaptic specializations.

Animals↗

PubMed1996

Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1.

Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM) are required in the nervous system, among a host of cellular responses, for the modulation of several important enzymes and ion channels involved in synaptic efficacy and neuronal plasticity. Here, we report that CaM can be replaced by the neuronal calcium sensor NCS-1 both in vitro and in vivo. NCS-1 is a calcium binding protein with two Ca(2+)-binding domains that shares only 21% of homology with CaM. We observe that NCS-1 directly activates two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide phosphodiesterase and protein phosphatase calcineurin). Co-activation of nitric oxide synthase by NCS-1 and CaM results in a higher activity than with CaM alone. Moreover, NCS-1 is coexpressed with calcineurin and nitric oxide synthase in several neuron populations. Finally, injections of NCS-1 into calmodulin-defective cam1 Paramecium partially restore wildtype behavioral responses. With this highly purified preparation of NCS-1, we have obtained crystals suitable for crystallographic structure studies. NCS-1, despite its very different structure, distribution, and Ca(2+)-binding affinity as compared with CaM, can substitute for or potentiate CaM functions. Therefore, NCS-1 represents a novel protein capable of mediating multiple Ca(2+)-signaling pathways in the nervous system.

Animals↗

PubMed1995

Localization of human immunodeficiency virus Rev in transfected and virus-infected cells.

The rev gene product of human immunodeficiency virus (HIV) is obligatory for viral replication. Rev interacts specifically with a structured RNA sequence within the viral genome termed the REV response element (RRE). Although the importance of Rev for the expression of viral proteins is well documented, its functional mechanism remains unresolved. Previous studies identified Rev in the absence of RRE to be a nuclear protein localized primarily within the nucleoli. To extend our understanding of the role of Rev in viral replication, immunolocalization studies of Rev and other nuclear components were carried out in transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV. In both types of cells, Rev-like immunoreactivity was distributed both in the nucleoplasm and cytoplasm. Within the nucleus, Rev immunoreactivity was not evenly distributed but was present within focal concentrations. In transfected cells that were double labeled for Rev and SC-35, which labels a known component of spliceosomes, the foci of Rev labeling were distinct from the "speckles" labeled by SC-35, although Rev foci and speckles were often juxtaposed. In addition, morphological changes in the three-dimensional network of speckles were observed in both transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV-1 and HIV-2. Our observations are consistent with the proposed dual role of Rev in mRNA transport and splicing.

Base Sequence↗