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PubMed1999

Immunolocalization of CRHSP28 in exocrine digestive glands and gastrointestinal tissues of the rat.

The 28-kDa (on SDS-PAGE) Ca2+-regulated heat stable protein (CRHSP28) was recently purified as novel phosphoprotein in exocrine pancreas, since it undergoes an immediate increase in serine phosphorylation when acini are stimulated with Ca2+-mobilizing agonists. Examination of CRHSP28 protein expression in rat revealed that most was highly expressed in pancreas and other morphologically related exocrine tissues, including the parotid, lacrimal, and submandibular glands. Immunofluorescence staining in pancreas indicated that CRHSP28 was specifically concentrated in zymogen granule-rich areas in the apical cytoplasm of acinar cells. Lack of colocalization with pancreatic lipase in dual immunofluorescence studies confirmed localization of CRHSP28 to the area immediately surrounding the granules. Western analysis of pancreatic zymogen granule membrane proteins indicated CRHSP28 was not associated with the granules following their purification. A similar pattern of apical cytoplasmic secretory granule staining was noted in lacrimal and submandibular glands. CRHSP28 protein was also expressed at relatively high levels in mucosal epithelial cells of the stomach and small intestine. CRHSP28 was found in the supranuclear apical cytoplasm of cells lining the small intestinal crypts, including Paneth cells, and was abundant in the cytoplasm of goblet cells. In the stomach, strong CRHSP28 staining was seen in mucus-secreting cells in the upper portion of the gastric glands and in the apical, granule-rich cytoplasm of chief cells located in the lower portions of the glands. Dual labeling with anti-H+-K+-ATPase demonstrated a comparatively lower expression of CRHSP28 in parietal cells. Collectively, the high relative expression of CRHSP28 in various secretory cell types within the digestive system, together with its intracellular localization surrounding the acinar cell secretory granules, strongly supports a role for CRHSP28 in Ca2+-mediated exocrine secretion.

Animals↗
PubMed1996

Localization of gamma-aminobutyric acid and glutamic acid decarboxylase in the pancreas of the nonobese diabetic mouse.

Glutamic acid decarboxylase (GAD), among other potential autoantigens, is thought to play a crucial role in type I diabetes, particularly in a spontaneous model of the disease, the nonobese diabetic (NOD) mouse. In the pancreas, the presence of GAD and gamma-aminobutyric acid (GABA), the decarboxylation product of GAD and a putative neurotransmitter in the islets of Langerhans, is well documented in the beta-cells. This is particularly true in rats, in which another GABAergic structure exists near the islets, the neuronal bodies. In this study, first the GABA content was measured in isolated islets from NOD and C57BL/6 mice (controls), and a decrease was found in NOD females as their insulitis progressed. Second, for the first time in mice, confocal analysis of immunofluorescent-labeled pancreatic sections revealed near the islets neuronal structures in which GAD and neuropeptide Y were colocalized, as they are in the brain. These structures were always observed in the pancreata of both sexes of C57BL/6 mice at the various ages investigated. In NOD mice, however, these neuronal structures were only detected in young females ( < 10 weeks old) and in males until an intermediate age. Moreover, patches of T cells surrounding GAD-containing fibers were seen in the vicinity of the islets with incipient periinsulitis.

Animals↗
PubMed1999

Placental Fas ligand expression is a mechanism for maternal immune tolerance to the fetus.

Fas ligand (FasL) is a peptide that plays an important immunoregulatory role in limiting the host immune response. Several studies have shown that the expression of FasL in the anterior chamber of the eye and the testis allows these tissues to be immunoprivileged sites. Immunotolerance is achieved by binding of FasL to its receptor (Fas) on activated immune cells, which results in cell apoptosis. To determine whether FasL has a role in maternal immune tolerance to the fetus, we looked for the expression of FasL in the human placenta. Immunoperoxidase staining localized FasL to both syncytiotrophoblast and cytotrophoblast in placental villi and chorionic extravillous trophoblast. Western analysis demonstrated FasL in placental villi and a human first-trimester trophoblast cell line (ED27). In contrast, Fas was colocalized to CD45 (leukocyte common antigen) positive cells found in maternal decidua. When isolated peripheral blood lymphocytes were induced to express Fas with phytohemagglutinin (PHA) and interleukin-2 (IL-2) and then cocultured with trophoblast, 30% of the lymphocytes underwent apoptosis, as determined by the in situ death (TUNEL) assay. Neutralizing antibodies to FasL inhibited apoptosis by 40% in these studies. In contrast, activated lymphocytes cocultured with non-FasL-expressing fibroblasts or unactivated non-Fas-expressing lymphocytes cocultured with ED27 trophoblast showed little evidence of apoptosis. These findings suggest that FasL expressed by fetal trophoblast cells can induce apoptosis in activated lymphocytes there by providing a mechanism for maternal immune tolerance to the fetus.

Apoptosis↗
PubMed2005

Cbl-mediated ubiquitination of alpha5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation.

Fibroblast growth factor receptor signaling is an important mechanism regulating osteoblast function. To gain an insight into the regulatory role of FGF receptor-2 (FGFR2) signaling in osteoblasts, we investigated integrin-mediated attachment and cell survival in human calvarial osteoblasts expressing activated FGFR2. FGFR2 activation reduced osteoblast attachment on fibronectin. This was associated with reduced expression of the alpha5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restored alpha5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis showed that alpha5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that alpha5 integrin colocalizes with FGFR2 and Cbl at the leading edge in membrane ruffle regions. Transfection with the 70Z-Cbl mutant lacking the RING domain required for Cbl-ubiquitin interaction, or with the G306E Cbl mutant that abolishes the binding ability of Cbl phosphotyrosine-binding domain restored alpha5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in alpha5 integrin proteasome degradation induced by FGFR2 activation. Reduced alpha5 integrin expression was associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of alpha5 integrin rescued cell attachment and corrected both the Bax/Bcl-2 ratio and caspase-3 and caspase-9 activities in FGFR2 mutant osteoblasts. We show that Cbl recruitment induced by FGFR2 activation triggers alpha5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the alpha5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation in osteoblasts, and reveals that a Cbl-dependent mechanism is involved in the coordinated regulation of cell apoptosis induced by alpha5 integrin degradation.

Acetylcysteine↗
PubMed2007

C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix.

Cip1-interacting zinc finger protein 1 (Ciz1) stimulates DNA replication in vitro and is required for mammalian cells to enter S phase. Here, we show that a significant proportion of Ciz1 is retained in nuclear foci following extraction with nuclease and high salt. This suggests that Ciz1 is normally immobilized by interaction with non-chromatin nuclear structures, consistent with the nuclear matrix. Furthermore, matrix-associated Ciz1 foci strikingly colocalize with sites of newly synthesized DNA in S phase nuclei, suggesting that Ciz1 is present in DNA replication factories. Analysis of green fluorescent protein-tagged fragments indicates that nuclear immobilization of Ciz1 is mediated by sequences in its C-terminal third, encoded within amino acids 708-830. Immobilization occurs in a cell-cycle-dependent manner, most probably during late G1 or early S phase, to coincide with its reported point of action. Although C-terminal domains are sufficient for immobilization, N-terminal domains are also required to specify focal organization. Combined with previous work, which showed that the DNA replication activity of Ciz1 is encoded by N-terminal sequences, we suggest that Ciz1 is composed of two functionally distinct domains: an N-terminal replication domain and a C-terminal nuclear matrix anchor. This could contribute to the formation or function of DNA replication factories in mammalian cells.

Animals↗
PubMed2002

Complementary adhesion molecules promote neutrophil-Kupffer cell interaction and the elimination of bacteria taken up by the liver.

Most bacteria that enter the bloodstream are taken up by the liver. Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature. Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface. In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v. was reduced from approximately 60% of the inoculum in normal mice to approximately 15% in mice rendered Kupffer cell deficient. Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells. Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells. Complementary adhesion molecules promoted the interaction between these two cell populations. Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae. Complement was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils.

Animals↗
PubMed1999

Mouse transporter protein, a membrane protein that regulates cellular multidrug resistance, is localized to lysosomes.

Mouse transporter protein (MTP), a small, highly conserved mammalian intracellular membrane protein with four putative transmembrane domains, has been implicated in the transport of nucleosides and/or related molecules across intracellular membranes. The production of recombinant MTP in Saccharomyces cerevisiae alters sensitivity of yeast cells to a heterogeneous group of compounds (e.g., antimetabolites, antibiotics, anthracyclines, ionophores, and steroid hormones) by changing the subcellular compartmentalization of these drugs, suggesting that MTP functions similarly in higher organisms. The present study was undertaken to define the intracellular location of MTP in mammalian cells. Native MTP was not detected by indirect immunofluorescence in cell types that expressed MTP mRNA; therefore, a hemagglutinin (HA) epitope-tagged version of MTP was produced in cultured BHK21 cells by transient transfection, and its distribution within cells was determined by confocal microscopy using antibodies directed against the HA epitope and various organellar proteins. Antibodies directed against HA-MTP colocalized with antibodies against late endosomal and lysosomal proteins but not with antibodies against either Golgi or early endosomal proteins. Analysis of subcellular fractions from rat liver by immunoblotting with antibodies directed against MTP demonstrated the presence of a MTP-like protein in Golgi- and lysosome-enriched membranes but not in mitochondria. These results indicate that MTP resides in late endosomes and lysosomes, a finding that is consistent with the proposed role for MTP in the movement of a variety of small molecules across endosomal and lysosomal membranes. MTP shares a number of characteristics with other lysosome-associated proteins. We, therefore, propose that it be redesignated murine lysosome-associated protein transmembrane 4.

Amino Acid Sequence↗
PubMed2000

Expression of ciliary neurotrophic factor activated by retinal Müller cells in eyes with NMDA- and kainic acid-induced neuronal death.

PURPOSE: To elucidate the role of retinal Muller cells in N-methyl-D-aspartate (NMDA)- or kainic acid (KA)induced retinal damage. METHODS: In experimental eyes, NMDA or KA was injected into the vitreous of rat eyes. Immunohistochemistry and western blot analysis were conducted to elucidate expression and localization of glial fibrillary acidic protein (GFAP) and ciliary neurotrophic factor (CNTF). In addition, the neuroprotective effects of CNTF were calculated by counting cells in the ganglion cell layer (GCL) and by measuring the thickness of the various retinal layers. RESULTS: Morphometric analysis of retinal damage in NMDA- and KA-injected eyes showed significant cell loss in the GCL and thinning of the inner plexiform layer (IPL) of the retina, but not of other retinal layers. Immunohistochemistry demonstrated disappearance and/or decrease in immunoreactivities of calbindin- and calretinin- positive cells and their neurites and upregulated expression of both GFAP and CNTF in experimental eyes. Western blot analysis showed an increase in protein expression for CNTF in retinas of experimental eyes. Confocal images and sequential localization demonstrated colocalization of CNTF and GFAP in the inner retinal layer and possibly in Muller cells. In addition, pretreatment with CNTF (1 microg) before the intravitreal injection of NMDA (or KA) demonstrated that CNTF has neuroprotective effects against NMDA- or KA-induced neuronal death in the retina. CONCLUSIONS: These studies revealed the upregulated expression of CNTF and GFAP in Muller cells in response to NMDA- and KA-induced neuronal death, suggesting that production of CNTF in Muller cells may be a part of the endogenous neuroprotective system in the retina.

Animals↗
PubMed2001

Widespread expression of netrin-1 by neurons and oligodendrocytes in the adult mammalian spinal cord.

Netrins are a family of secreted proteins that function as chemotropic axon guidance cues during neural development. Here we demonstrate that netrin-1 continues to be expressed in the adult rat spinal cord at a level similar to that in the embryonic CNS. In contrast, netrin-3, which is also expressed in the embryonic spinal cord, was not detected in the adult. In situ hybridization analysis demonstrated that cells in the white matter and the gray matter of the adult spinal cord express netrin-1. Colocalization studies using the neuronal marker NeuN revealed that netrin-1 is expressed by multiple classes of spinal interneurons and motoneurons. Markers identifying glial cell types indicated that netrin-1 is expressed by most, if not all, oligodendrocytes but not by astrocytes. During neural development, netrin-1 has been proposed to function as a diffusible long-range cue for growing axons. We show that in the adult spinal cord the majority of netrin-1 protein is not freely soluble but is associated with membranes or the extracellular matrix. Fractionation of adult spinal cord white matter demonstrated that netrin-1 was absent from fractions enriched for compact myelin but was enriched in fractions containing periaxonal myelin and axolemma, indicating that netrin-1 protein may be localized to the periaxonal space. These findings suggest that in addition to its role as a long-range guidance cue for developing axons, netrin may have a short-range function associated with the cell surface that contributes to the maintenance of appropriate neuronal and axon-oligodendroglial interactions in the mature nervous system.

Aging↗
PubMed1999

Frequent deletions within FRA7G at 7q31.2 in invasive epithelial ovarian cancer.

We previously showed that FRA7G, an aphidicolin-inducible common fragile site at 7q31.2, colocalized with the common region of loss of heterozygosity (LOH) in a number of different tumors. Based on the sequence analysis of 150 Kb in the FRA7G region, we identified four new polymorphic microsatellite markers. In this article, we have used these four microsatellite markers and eight additional markers from 7q22-32 to analyze the breakage and loss of the region surrounding FRA7G in 49 invasive epithelial ovarian cancers and three borderline ovarian tumors. No allelic loss was detected in the ovarian tumors of borderline malignancy, but 71% (35/49) of the invasive tumors showed LOH at one or more loci in the region analyzed. Of the 12 markers analyzed, most of the markers exhibiting a high frequency of LOH were within FRA7G, and the highest frequency of LOH was seen with the new marker 7G14 (37%, 15/41). Breakpoint analysis in tumors with LOH demonstrated that the frequent loss of DNA sequences seen within the FRA7G region was due to frequent small interstitial deletions and not a result of loss of the whole fragile site region. These findings indicate that FRA7G does play a role in the breakage and loss of 7q sequences in invasive ovarian cancer. In addition, the newly identified markers enable us to further delineate a smallest common region of loss in invasive ovarian tumors to a 150-Kb region flanked by markers D7S486 and 7G14.

Carcinoma↗
PubMed2002

Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4(+) T cells.

In this report, we describe a crucial role of lipid raft-colocalized receptors in the entry of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T cells. We show that biochemically isolated detergent-resistant fractions have characteristics of lipid rafts. Lipid raft integrity was required for productive HIV-1 entry as determined by (i) semiquantitative PCR analysis and (ii) single-cycle infectivity assay using HIV-1 expressing the luciferase reporter gene and pseudotyped with HIV-1 HXB2 envelope or vesicular stomatitis virus envelope glycoprotein (VSV-G). Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) relocalized raft-resident markers to a nonraft environment but did not significantly change the surface expression of HIV-1 receptors. MbetaCD treatment inhibited productive infection of HIV-1 by 95% as determined by luciferase activity in cells infected with HXB2 envelope-pseudotyped virus. In contrast, infection with VSV-G-pseudotyped virus, which enters the cells through an endocytic pathway, was not suppressed. Biochemical fractionation and confocal imaging of HIV-1 receptor distribution in live cells demonstrated that CD4, CCR5, and CXCR4 colocalized with raft-resident markers, ganglioside GM1, and glycosylphosphatidylinositol-anchored CD48. While confocal microscopy analysis revealed that HIV-1 receptors localized most likely to the same lipid microdomains, sucrose gradient analysis of the receptor localization showed that, in contrast to CD4 and CCR5, CXCR4 was associated preferentially with the nonraft membrane fraction. The binding of HIV-1 envelope gp120 to lipid rafts in the presence, but not in the absence, of cholesterol strongly supports our hypothesis that raft-colocalized receptors are directly involved in virus entry. Dramatic changes in lipid raft and HIV-1 receptor redistribution were observed upon binding of HIV-1 NL4-3 to PM1 T cells. Colocalization of CCR5 with GM1 and gp120 upon engagement of CD4 and CXCR4 by HIV-1 further supports our observation that HIV-1 receptors localize to the same lipid rafts in PM1 T cells.

CD4 Antigens↗
PubMed2001

Zinc-enriched GABAergic terminals in mouse spinal cord.

Electrophysiological experiments have shown that zinc ions modulate glutamate and GABA receptors in brain slices. All the zinc-enriched neuronal pathways in the brain analyzed up until now have been found to be glutaminergic. Many years ago, zinc-enriched terminals with flat vesicles and symmetric synapses were found to be present in rat spinal cord by Henrik Daa Schrøder, and recently these findings have been supported by immunohistochemical and electron microscopical data in lamprey, mouse and rat. In the present study we expanded these observations by revealing a colocalization of zinc ions, zinc transporter-3 (ZnT3) and glutamic acid decarboxylase (GAD) in synaptic vesicles of zinc-enriched terminals throughout the mouse spinal cord. Confocal analysis of ZnT3 and GAD immunofluorescence was used at light microscopical levels, and a combination of zinc selenium autometallography and GAD immunocytochemistry at electron microscopic levels. Zinc-enriched/GABAergic terminals were observed in all laminae of the spinal gray matter, but most densely populated were laminae I and III in the dorsal horn. In the lateral and ventral funiculi of the white matter, rows of inhibitory zinc-enriched boutons were seen radiating from the gray matter. Ultrastructurally, colocalization of zinc ions and GAD immunoreactivity was seen in a pool of presynaptic terminals in the above locations. Some zinc-enriched terminals were not GAD-positive and some GAD-positive terminals were void of zinc ions. The majority of the zinc-enriched, not GABAergic terminals could be classified as excitatory based on their morphology, i.e. round clear vesicles and symmetric synapses. We conclude that a majority of the spinal cord zinc-enriched terminals are GABAergic. The zinc-enriched terminals with excitatory morphology are most likely glutaminergic, a few have an inhibitory morphology but are not GABAergic. These are most likely glycinergic.

Animals↗
PubMed1996

Coordinated expression of dopamine receptors in neostriatal medium spiny neurons.

In recent years, the distribution of dopamine receptor subtypes among the principal neurons of the neostriatum has been the subject of debate. Conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D1 and D2 class dopamine receptors are colocalized. One plausible explanation for the discrepancy is that some dopamine receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, we examined the expression of DA receptors in individual neostriatal neurons by patch-clamp and RT-PCR techniques. Because of the strong correlation between peptide expression and projection site, medium spiny neurons were divided into three groups on the basis of expression of mRNA for enkephalin (ENK) and substance P (SP). Neurons expressing detectable levels of SP but not ENK had abundant mRNA for the D1a receptor. A subset of these cells (approximately 50%) coexpressed D3 or D4 receptor mRNA. Neurons expressing detectable levels of ENK but not SP had abundant mRNA for D2 receptor isoforms (short and long). A subset (10-25%) of these neurons coexpressed D1a or D1b mRNAs. Neurons coexpressing ENK and SP mRNAs consistently coexpressed D1a and D2 mRNAs in relatively high abundance. Functional analysis of neurons expressing lower abundance mRNAs revealed clear physiological consequences that could be attributed to these receptors. These results suggest that, although colocalization of D1a and D2 receptors is limited, functional D1 and D2 class receptors are colocalized in nearly one-half of all medium spiny projection neurons.

Animals↗
PubMed2001

Structural analysis of emerin, an inner nuclear membrane protein mutated in X-linked Emery-Dreifuss muscular dystrophy.

Like Duchenne and Becker muscular dystrophies, Emery-Dreifuss muscular dystrophy (EDMD) is characterized by myopathic and cardiomyopathic abnormalities. EDMD has the particularity of being linked to mutations in nuclear proteins. The X-linked form of EDMD is caused by mutations in the emerin gene, whereas autosomal dominant EDMD is caused by mutations in the lamin A/C gene. Emerin colocalizes with lamin A/C in interphase cells, and binds in vitro to lamin A/C. Recent work suggests that lamin A/C might serve as a receptor for emerin. We have undertaken a structural analysis of emerin, and in particular of its N-terminal domain, which is comprised in the emerin segment critical for binding to lamin A/C. We show that region 2-54 of emerin adopts the LEM fold. This fold was originally described in the two N-terminal domains of another inner nuclear membrane protein called lamina-associated protein 2 (LAP2). The existence of a conserved solvent-exposed surface on the LEM domains of LAP2 and emerin is discussed, as well as the nature of a possible common target.

Amino Acid Sequence↗
PubMed2005

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance.

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.

Apoptosis↗
PubMed1993

Dystrophin-glycoprotein complex and laminin colocalize to the sarcolemma and transverse tubules of cardiac muscle.

The expression and subcellular distribution of the dystrophin-glycoprotein complex and laminin were examined in cardiac muscle by immunoblot and immunofluorescence analysis of rabbit and sheep papillary muscle. The five dystrophin-associated proteins (DAPs), 156-DAG, 59-DAP, 50-DAG, 43-DAG, and 35-DAG, were identified in rabbit ventricular muscle and found to codistribute with dystrophin in both papillary myofibers and Purkinje fibers. The DAPs and dystrophin codistributed not only in the free surface sarcolemma but also in interior regions of the myofibers where T tubules are present. Neither the DAPs nor dystrophin were detected in intercalated discs, a specialized region of cardiac sarcolemma where neighboring myocardial cells are physically joined by cell-cell junctions. Similarly, in bundles of Purkinje fibers, which lack T tubules, DAPs and dystrophin were also found to codistribute at the free surface sarcolemma but were not detected either in the region of surface sarcolemma closely apposed to a neighboring Purkinje fiber or in interior regions of these myofibers. Comparison between the distribution of the dystrophin-glycoprotein complex and laminin showed that laminin codistributes with the components of this complex in both papillary myofibers and Purkinje fibers. These results are consistent with previous findings demonstrating that the extracellularly exposed 156-DAG (dystroglycan) of the skeletal muscle dystrophin-glycoprotein complex binds laminin, a component of the basement membrane. Although we demonstrate that DAPs, dystrophin, and laminin colocalize to the sarcolemma in rabbit and sheep papillary myofibers as they do in skeletal myofibers, the most striking difference between the subcellular distribution of these proteins in cardiac and skeletal muscle is that the dystrophin-glycoprotein complex and laminin also localize to regions of the fibers where T tubules are distributed in cardiac but not in skeletal muscle. These results imply that the protein composition and thus possibly some functions of T tubules in cardiac muscle are distinct from those of skeletal muscle.

Animals↗
PubMed2005

Light and electron microscopic analysis of KChIP and Kv4 localization in rat cerebellar granule cells.

Potassium channels are key determinants of neuronal excitability. We recently identified KChIPs as a family of calcium binding proteins that coassociate and colocalize with Kv4 family potassium channels in mammalian brain (An et al. [2000] Nature 403:553). Here, we used light microscopic immunohistochemistry and multilabel immunofluorescence labeling, together with transmission electron microscopic immunohistochemistry, to examine the subcellular distribution of KChIPs and Kv4 channels in adult rat cerebellum. Light microscopic immunohistochemistry was performed on 40-microm free-floating sections using a diaminobenzidine labeling procedure. Multilabel immunofluorescence staining was performed on free-floating sections and on 1-microm ultrathin cryosections. Electron microscopic immunohistochemistry was performed using an immunoperoxidase pre-embedding labeling procedure. By light microscopy, immunoperoxidase labeling showed that Kv4.2, Kv4.3, and KChIPs 1, 3, and 4 (but not KChIP2) were expressed at high levels in cerebellar granule cells (GCs). Kv4.2 and KChIP1 were highly expressed in GCs in rostral cerebellum, whereas Kv4.3 was more highly expressed in GCs in caudal cerebellum. Immunofluorescence labeling revealed that KChIP1 and Kv4.2 are concentrated in somata of cerebellar granule cells and in synaptic glomeruli that surround synaptophysin-positive mossy fiber axon terminals. Electron microscopic analysis revealed that KChIP1 and Kv4.2 immunoreactivity is concentrated along the plasma membrane of cerebellar granule cell somata and dendrites. In synaptic glomeruli, KChIP1 and Kv4.2 immunoreactivity is concentrated along the granule cell dendritic membrane, but is not concentrated at postsynaptic densities. Taken together, these data suggest that A-type potassium channels containing Kv4.2 and KChIP1, and perhaps also KChIP3 and 4, play a critical role in regulating postsynaptic excitability at the cerebellar mossy-fiber/granule cell synapse.

Animals↗
PubMed1994

Colocalization of nestin and vimentin/desmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy.

During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggest that charged amino acid residues in the alpha-helical rod domain may play a role in the interaction.

Amino Acid Sequence↗