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PubMed2007

Polarized distribution of ion channels within microdomains of the axon initial segment.

Voltage-gated sodium (Na(v)) channels accumulate at the axon initial segment (IS), where their high density supports spike initiation. Maintenance of this high density of Na(v) channels involves a macromolecular complex that includes the cytoskeletal linker protein ankyrin-G, the only protein known to bind Na(v) channels and localize them at the IS. We found previously that Na(v)1.6 is the predominant Na(v) channel isoform at IS of adult rodent retinal ganglion cells. However, here we report that Na(v)1.6 immunostaining is consistently reduced or absent in short regions of the IS proximal to the soma, although both ankyrin-G and pan-Na(v) antibodies stain this region. We show that this proximal IS subregion is a unique axonal microdomain, containing an accumulation of Na(v)1.1 channels that are spatially segregated from the Na(v)1.6 channels of the distal IS. Additionally, we find that axonal K(v)1.2 potassium channels are present within the distal IS, but are also excluded from the Na(v)1.1-enriched proximal IS microdomain. Because ankyrin-G was prominent in both proximal and distal subcompartments of the IS, where it colocalized with either Na(v)1.1 or Na(v)1.6, respectively, mechanisms other than association with ankyrin-G must mediate differential targeting of Na(v) channel subtypes to achieve the spatial precision observed within the IS. This precise arrangement of ion channels within the axon initial segment is likely an important determinant of the firing properties of ganglion cells and other mammalian neurons.

Action Potentials↗
PubMed1981

3H-thymidine autoradiographic analysis of telencephalic histogenesis in the chick embryo: I. Neuronal birthdates of telencephalic compartments in situ.

The birthdates of neuronal populations comprising the chick telencephalon were determined by 3H-thymidine labeling and were mapped with respect to their terminal positions in the 16-day embryo. Essentially all neurons were generated between four and nine days of embryonic development. Each telencephalic structure (based on terminology used by Karten and Hodos, '67) was characterized by a specific range of birthdates: some regions such as the core of the ectostriatum or the paleostriatum primitivum, were generated within a single day, while others, such as the hyperstriatum accessorium, required up to five days for generation of the complete population. Spatial-temporal gradients of neuronal birthdates, lateromedial and ventrodorsal, were seen in the telencephalon as a whole and within individual subcompartments as well. An "outside-in" pattern of histogenesis predominated throughout the entire telencephalon, including the dorsolateral cortex. However, notable exceptions pertaining to the paleostriatum augmentatum, hyperstriatum intercalatus and field "L" were observed. Glial cells, generated for the most part after day ten, were found to be distributed homogeneously throughout all areas of the telencephalon. These data provide the first birthdating data for an avian telencephalon and bring greater resolution to previous analyses of the histogenesis of this brain region. Further, the compartmentalization of the proliferative neuroepithelium is revealed by these data, and the possibility of a common time of origin in the neuroepithelium for neurons of related function is discussed.

Animals↗
PubMed1985

Organization of thalamic projections in the nucleus accumbens and the caudate nucleus in cats and its relation with hippocampal and other subcortical afferents.

The organization of thalamic projections in the nucleus accumbens (NA) and the caudate nucleus of cats and its relation to other subcortical striatal afferents were studied with a retrograde tracing technique by use of lectin-conjugated horseradish peroxidase. The study showed that the paraventricular and medial parafascicular nuclei (PF) of the thalamus project to the medial NA and the parataenial and medial PF project to the lateral NA. The ventral tegmental area and substantia nigra pars dorsalis (SNpd) project to medial and lateral NA. The midline thalamic nuclei, rostral intralaminar nuclei, ventroanterior nucleus, medial and lateral PF, lateral posterior complex, and nucleus limitans project to medial caudate nucleus. The most medial substantia nigra pars compacta (SNpc) and rostral SNpd project to medial caudate nucleus. The center median, ventrolateral, and the central lateral nuclei of thalamus, SNpc, and SNpd project to lateral caudate nucleus. These results suggest that the thalamic and subcortical nuclei known to connect with the limbic and frontal cortices project to NA and medial caudate nucleus. Those thalamic nuclei connected with the motor system project to lateral caudate nucleus. The hippocampus projects selectively to medial NA. The amygdala, raphe, and other mesencephalic nuclei project only to NA and medial caudate nucleus. The organization of hippocampal, amygdala, and other subcortical afferents suggests that NA and caudate nucleus can be separated into medial "limbic" and lateral nonlimbic "sensory-motor" compartments. A brief review of the distribution pattern of some neurotransmitters, neuropeptides, and their receptors and behavior studies provides additional support to the concept that the striatum can be divided into several subcompartments.

Amygdala↗
PubMed1989

Compartmental and topographical distributions of axons in nerves to the amphibian (Bufo marinus) glutaeus muscle.

The present work seeks to determine if axons to an amphibian muscle are segregated in nerve trunks between the spinal cord and muscle according to their primary nerve destination or their topographical projection in the muscle. The distribution of axons to different compartments and subcompartments of the amphibian (Bufo marinus) glutaeus muscle has been determined in transverse sections of spinal and limb nerves after retrogradely labelling the axons with horseradish peroxidase. Glutaeus axons were dispersed widely through spinal nerves 8 and 9 but loosely gathered together in one quadrant of the sciatic nerve after passing through the lumbar plexus. Glutaeus axons became tightly clustered to the exclusion of other axons along the length of the triceps femoris nerve after it divides from the sciatic nerve. Furthermore, axons destined for one of the two glutaeus primary nerve branches segregate from those of the other branch at the level of the triceps femoris nerve before the glutaeus nerve forms. On the other hand, motoneurones that subserve a primary branch are not segregated, but are found throughout the rostrocaudal extent of the glutaeus motoneurone pool. Injection of horseradish peroxidase under the epimysium of either the ventral or the dorsal surfaces of the glutaeus muscle labelled motoneurones preferentially in either the rostral or caudal part of the motoneurone pool, respectively. This confirms studies that have shown a topographical projection from the spinal motoneurone pool onto the glutaeus muscle. However, there was no segregation of dorsally projecting axons in the glutaeus and primary nerve branches. Thus, glutaeus axons segregate according to their muscle compartmental projections well before entering the muscle, but they show no organization in nerves with respect to their topographical projections within a compartment.

Animals↗
PubMed1993

Anatomy of the mushroom bodies in the honey bee brain: the neuronal connections of the alpha-lobe.

Neural connections between the mushroom body (MB) and other protocerebral areas of the honeybee's brain were studied with the help of cobalt chloride and Golgi staining methods. Focal injections of cobalt ions into the alpha-lobe neuropil of the MB reveal seven clusters of somata located in the protocerebrum and deutocerebrum of each brain hemisphere. These neurons connect the mushroom body neuropil with protocerebral areas and number approximately 400. They contact the layered organization of the alpha-lobe at different locations. Some project not only into the alpha-lobe, but also into the beta-lobe and pedunculus neuropils. Fifteen cell types which form intraprotocerebral circuits are morphologically described. They can be divided into three categories: 1) unilateral neurons, with projection fields restricted to the ipsilateral protocerebrum; these neurons connect the alpha-lobe with areas in the protocerebral lobe and ramify with densely layered arborisations arranged perpendicularly to the longitudinal axis of the alpha-lobe; 2) recurrent neurons, which interconnect subcompartments of the MB, forming loops at different levels of the neuropil; their arborisations are mainly restricted to the alpha-lobe, beta-lobe, pedunculus, and calyces of the ipsilateral MB; they also ramify sparsely around the neuropil of the alpha-lobe; and 3) bilateral neurons, which either interconnect both alpha-lobes or connect the ipsilateral alpha-lobe and protocerebral lobe with the dorsolateral protocerebral lobe of the contralateral hemisphere. The connections of different compartments of the MB with other parts of the protocerebrum as revealed in this study are discussed in the context of hypotheses about the functional role of MBs in the honeybee brain.

Animals↗
PubMed1994

A topographically organized gamma-aminobutyric acid projection from the ventral pallidum to the nucleus accumbens in the rat.

Anatomical and electrophysiological studies have indicated that a reciprocal projection from the ventral pallidum back to the nucleus accumbens exists and has functional relevance. In this study, the topographical projection from the ventral pallidum to the nucleus accumbens was examined by using retrograde tracing with fluoro-gold iontophoresed in subcompartments of the nucleus accumbens in rats combined with either in situ hybridization for glutamic acid decarboxylase and preproenkephalin mRNA or substance P immunoreactivity. Deposits made into the medial nucleus accumbens preferentially labeled neurons in the medial ventral pallidum, while deposits into the dorsolateral nucleus accumbens, at or lateral to the anterior commissure, labeled primarily cells in the dorsal and lateral ventral pallidum. A mediolateral to rostrocaudal topography was also observed, with the medial deposits preferentially labeling cells in rostral ventral pallidum and the lateral deposits resulting in retrogradely labeled cells in the ventral pallidum below the crossing of the posterior anterior commissure (subcommissural) as well as below the globus pallidus (sublenticular). The majority of cells retrogradely labeled with fluoro-gold were double-labeled for glutamic acid decarboxylase mRNA. In contrast, very few retrogradely labeled neurons in the ventral pallidum were double labeled for mRNA for preproenkephalin. These data demonstrate a topographically organized projection from the ventral pallidum to the nucleus accumbens that is primarily gamma-aminobutyric acid (GABA)-ergic and reciprocal to the GABAergic projection from the nucleus accumbens to the ventral pallidum.

Animals↗
PubMed1993

Quantitative analysis of mitotic and early-G1 cells using monoclonal antibodies against the AF-2 protein.

We have recently described a novel protein (AF-2), conserved between fission yeast and man, and we have shown by flow cytometry (FCM) that AF-2 is highly accessible to specific monoclonal antibodies (MoAbs) in mitotic and postmitotic early-G1 phase cells. The aim of the present study was to optimize the FCM methodology using MoAbs against AF-2 and to show that the evaluation of the mitotic cells, using different cell lines, was quantitative and reproducible. We found that a method based on fixation with ethanol, instead of formalin, resulted in improved DNA histogram coefficients of variation and implemented separation of early-G1 cells from late-G1 cells. In addition, by eliminating several cell permeabilization and protein salt extraction steps, the method became straightforward, conserved a clear-cut separation of the green fluorescence of M- with respect to G2-phase cells, and did not significantly affect cellular integrity. The coefficient of correlation among the mitotic index values evaluated by this FCM method using MoAbs against AF-2 and by microscopic visual counting was R = 0.94. When the FCM/AF-2 method was tested against an independent FCM method, which allows clear separation of M- and G2-phase cells according to 90 degrees scattering, we found R = 0.93. We conclude that MoAbs against the AF-2 protein may be used in FCM for quantitative analysis and for isolation of M-phase cells, providing as well, the identification of the early-G1 cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF-2 antigen.

Antibodies, Monoclonal↗
PubMed1998

Fluid-phase marker transport in rat liver: free-flow electrophoresis separates distinct endosome subpopulations.

Free-flow electrophoresis (FFE) was used to investigate the intracellular compartments involved in fluid-phase marker, fluoresceine isothiocyanate (FITC)-dextran, transport in the isolated perfused rat liver. One to 2 min after uptake at 37 degrees C, FITC-dextran was found in endosomes with the same electrophoretic mobility as early sorting endosomes labeled either by the hepatocyte-specific marker asialoorosomucoid (ASOR) or by transferrin that enters all liver cells. Labeling at low temperature (16 degrees C) blocked transport of ASOR and dextran in early endosomes. With increasing internalization time (3-13 min) at 37 degrees C, FITC-dextran-labeled compartments co-localized with late, ASOR-containing endosomes. Since localization of FITC-dextran in late transcytotic compartments was not observed upon FFE separation, it is concluded that the majority of internalized markers is directed to lysosomes. The FITC-label did not account for the predominant lysosomal targeting of the dextran, since [3H]dextran-labeled endosomes exhibited an identical FFE pattern. Taken together, these data indicate that the fluid-phase marker dextran is transported through intracellular compartments with identical characteristics as endosome subcompartments of the receptor-mediated lysosomal route.

Animals↗
PubMed1990

Pretranslational and posttranslational regulation of the EGF receptor during the prereplicative phase of liver regeneration.

We studied the regulation of the epidermal growth factor receptor mRNA and the number of epidermal growth factor binding sites in subcellular compartments involved in the biosynthesis and endocytosis of the epidermal growth factor receptor during the prereplicative phase of liver regeneration. The epidermal growth factor receptor mRNA, quantified by solution hybridization, decreased after partial hepatectomy, with a nadir of about 35% 18 hr after hepatectomy. An even stronger decrease in the number of epidermal growth factor binding sites after partial hepatectomy was observed in a Golgi-enriched low-density membrane fraction, reflecting available newly synthesized epidermal growth factor receptors. It is suggested that this decrease in newly synthesized available epidermal growth factor receptors is caused primarily, but not entirely, by decreased epidermal growth factor receptor mRNA levels and the additional down-regulation of epidermal growth factor binding sites may involve posttranslational mechanisms such as intracellular occupation by transforming growth factor-alpha. The observation that the number of specific epidermal growth factor binding sites after partial hepatectomy was only moderately reduced in prelysosomal endosomes and in lysosomes, compared with the newly synthesized receptors, may indicate that a pool of receptors targeted for lysosomes exists and these receptors are regulated in a different manner than the receptor pool targeted for the cell surface. Furthermore, at least two separable endocytic subcompartments are involved in the transport of the epidermal growth factor/epidermal growth factor receptor complex in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

The polymeric immunoglobulin receptor is the major calmodulin-binding protein in an endosome fraction from rat liver enriched in recycling receptors.

Rat liver endosomes contain one major high-affinity calmodulin-binding protein (CaMBP) that now has been identified as the polymeric immunoglobulin receptor (pIgR). In isolated endosomes pIgR was enriched in the receptor-recycling compartment (RRC); lesser enrichment was found in 'early' endosome (CURL) and much less in 'late' endosome fractions (multivesicular bodies, MVB). The distribution of the major CaMBP, shown by Western blotting or by overlay with I125-calmodulin in the isolated fractions, was consistent with rapid accumulation of I125-immunoglobulin A (IgA) in RRC and CURL after intravenous injection into rats. The receptor was also found in sinusoidal plasma membranes but not in cell fractions containing apical (bile canalicular) or lateral plasma membrane domains of the hepatocyte. The interaction of pIgR with calmodulin was shown by direct binding assays and by affinity chromatography. Thus, calmodulin is the first cytoplasmic protein shown to interact with the pIgR. We postulate that calmodulin regulates pIgA trafficking in rat liver. In addition, the receptor recycling fraction emerges as an endosomal subcompartment involved in pIgA transport via pIgR.

Adult↗
PubMed1990

Purification and characterization of tyrosylprotein sulfotransferase.

Tyrosylprotein sulfotransferase (TPST) is a Golgi membrane enzyme involved in the post-translational modification of secretory and membrane proteins. Here we describe the 140,000-fold purification of this enzyme from bovine adrenal medulla to apparent homogeneity and determine its substrate specificity. The key step in the purification was affinity chromatography on a substrate peptide to which the enzyme bound in the presence of nucleotide cosubstrate. TPST is a 54-50 kd integral membrane glycoprotein. The presence of sialic acid strongly suggests that within the Golgi complex, TPST is localized in the trans-most subcompartment. TPST was found to specifically sulfate tyrosine residues adjacent to acidic amino acids. These results define a major determinant for the specificity of protein sulfation in the trans Golgi.

Adrenal Medulla↗
PubMed1991

DNA template effect on RNA splicing: two copies of the same gene in the same nucleus are processed differently.

Many cellular and viral genes are parts of complex transcription units containing multiple splicing choices. During the course of an adenoviral replicative cycle, different spliced versions of a single gene predominate, depending on the stage of infection. This is true for several adenoviral genes. In this paper we show for the viral E1B transcription unit that splice site usage regulates this process. The change in alternative splicing in this system does not depend on the sequence of the transcribed genes. Non-adenoviral genes, such as the SV40 early region and the polyoma early region, which normally show little or no regulation of spliced RNA product formation, become regulated for mRNA production after insertion into the adenoviral genome. Additional studies show that E1B splicing regulation in adenovirus is a cis effect. Staggered infections using two discernable viral genomes resulted in a situation where both early and late genomes exist in the same nucleus. Neither genome was able to impose its regulated splicing pattern on the other, indicating that the cue for the switch in viral gene splicing is not directly dependent on global changes in trans-acting splicing factors. This suggests a model where the signal for changes in RNA processing for the E1B gene is linked to the state of the DNA template or its localization within nuclear subcompartments.

Adenoviridae↗
PubMed1995

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex.

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.

Biological Transport↗
PubMed1993

Double immunofluorescent staining of alpha 2,6 sialyltransferase and beta 1,4 galactosyltransferase in monensin-treated cells: evidence for different Golgi compartments?

Beta 1,4 galactosyl- and alpha 2,6 sialyltransferase (gal-T EC 2.4.1.22 and sialyl-T EC 2.4.99.1) sequentially elongate and terminate complex N-glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi-disturbing agents. Polyclonal, peptide-specific antibodies to human sialyl-T expressed as a beta-galactosidase-sialyl-T fusion protein in E. coli were developed and applied together with mABs to human milk gal-T. In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl-T and gal-T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal-T followed by sialyl-T; in the reassembled Golgi apparatus sialyl-T and gal-T were co-localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal-T positive Golgi elements while sialyl-T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal-T and sialyl-T remained co-localized. Treatment with chloroquine affected Golgi structures less than monensin and led to condensation of gal-T positive and to slight enlargement of sialyl-T positive structures. Sequential recovery from BFA of gal-T and sialyl-T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.

Antibodies, Monoclonal↗
PubMed1984

Changes in nuclear antigens during reactivation of chick erythrocyte nuclei in heterokaryons.

Reactivation of dormant chick erythrocyte (CE) nuclei was studied by fusing chick red cells with rat myoblasts, HeLa cells, and chick fibroblasts. Heterokaryons representing different stages of nuclear reactivation were fixed and examined for nuclear antigens using polyclonal patient autoantisera reacting with mammalian (human, mouse, and rat) nuclear envelope, nucleoplasm, and chromatin (DNA) antigens. Reactivation of CE nuclei was associated with marked changes in nuclear antigenicity. In rat myoblast and HeLa heterokaryons the CE nuclei acquired mammalian nucleoplasmic and nuclear envelope antigens in the corresponding nuclear subcompartments. Drastic changes in nuclear antigenicity were noted also in heterokaryons stained with DNA antisera. The compact chromatin of nuclei in mature chick erythrocytes showed little binding of DNA antibodies. Isolated nuclei on the other hand gave a strong immunofluorescence. CE nuclei in heterokaryons were strongly positive during the early stages of nuclear reactivation but then exhibited decreased reactivity. An unexpected finding was a marked reduction in the capacity of mammalian nuclei in heterokaryons to bind DNA-antibodies. This observation is discussed in relation to the previous finding that in CE-heterokaryons these nuclei often show reduced transcription and replication. The present results indicate that in heterokaryons both types of nuclei exchange macromolecules of regulatory importance via the common cytoplasm.

Animals↗
PubMed1991

Cell cycle dependent alterations of chromatin structure in situ as revealed by the accessibility of the nuclear protein AF-2 to monoclonal antibodies.

We have recently described a novel nuclear antigen, AF-2, which is related to cell cycle dependent alterations of chromatin structure. We show by two parameter flow cytometry on a cell by cell basis that the antigen is accessible to specific monoclonal antibodies only in mitotic and postmitotic early G1-phase cells. The evaluation of nuclease susceptibility and AF-2 antigen accessibility reveals different subcompartments of the G1-phase of the cell cycle with distinct chromatin conformations. Digestion with DNase I seems to alter the chromatin structure according to concentration and this is reflected by an increase of the antigen accessibility. Chromatin in the more condensed early G1-phase is specifically digested by lower concentrations of the enzyme than chromatin in later stages of interphase. Chromatin from cells in the late-G1, S-, and G2-phases shows a higher relative resistance to DNase I and a reduced accessibility of the AF-2 antigen to monoclonal antibodies. Nuclease S1 has a similar effect on chromatin topology, as revealed by the reaction with anti-AF-2 antibodies, without digestion of detectable amounts of DNA. The antigen becomes available to the antibodies in almost all cells by digestion with high concentrations of DNase I or Nuclease S1.

Antibodies, Monoclonal↗
PubMed1995

Immunogold studies on peroxisomes: review of the localization of specific proteins in vertebrate peroxisomes.

Peroxisomes, since their discovery as microbodies, have been studied mostly independently by electron microscopists and biochemists. The fine structure has been studied by electron microscopy, and the compositional enzymes and proteins by protein biochemistry. Electron microscopic histochemistry has been used to try to clarify the relationship between the fine structure and its constituents. The immunogold technique, a combination of electron microscopy and protein biochemistry, for the first time resolved this problem due to the high sensitivity and resolution power of the staining and the high reliability of the technique. The present paper reviews the way in which the immunogold techniques, especially the protein A-gold technique, revealed the localization of various enzymes or proteins in peroxisomes or peroxisomal subcompartments, and discusses why this technique should be employed in peroxisome research.

Animals↗
PubMed2001

Nerve fibers innervating the cranial and spinal meninges: morphology of nerve fiber terminals and their structural integration.

Pachymeninx and leptomeninx of cranial cavity and spine are considerably different in their collagenous fiber texture, cellular composition, vascularization, and innervation. The majority of meningeal nerve fibers terminate as free nerve endings whereas encapsulated and lamellated nerve terminals additionally occur in higher vertebrates including man. With respect to nerve fiber classification, arborization pattern, topography, and organization of the microenvironment at the termination site afferent and efferent nerve terminals are differentiated. Only the dura mater and the pial subcompartment of the leptomeninx possess the morphological prerequisites for neurogenic inflammation. In the current review, the results of morphological studies regarding the meningeal innervation including the sites of CSF (cerebrospinal fluid) production and absorption are discussed with emphasis on their structure-function relationships.

Arachnoid↗