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PubMed1996

Immunocytochemical demonstration of a new vimentin-associated protein in 3T3 fibroblasts.

Using a xanthophore cytoskeletal preparation as immunogen, we have produced a monoclonal antibody, A2, which recognized a 160 kDa protein in 3T3 fibroblasts. This protein makes up a cytoplasmic filamentous system, which colocalizes with vimentin filaments. When microtubules and actin filaments are dissolved by high salt extraction, staining with antibody A2 is unaffected. Immunoblot analysis confirms that the 160 kDa protein is co-isolated with vimentin during in vivo high salt extraction. Following vinblastine treatment, both the 160 kDa protein and vimentin become localized to perinuclear caps, as do other intermediate filaments and their associated proteins; after vinblastine removal, the immunostaining produced by A2 becomes filamentous. Immunoelectron microscopy demonstrates that antibody A2 stains a filament system with a diameter of about 10 nm. Our observations suggest that the 160 kDa protein may be a new vimentin-associated protein which differs from the intermediate filament-associated proteins previously reported, and is widely distributed in several cell types.

3T3 Cells↗
PubMed2004

Molecular genetic characterization and subcellular localization of a putative Theileria annulata membrane protein.

A Theileria annulata protein (TaD) exhibiting an N-terminal signal sequence for endoplasmic reticulum membrane translocation and a conserved cysteine-rich region was isolated by screening the mRNA of a T. annulata-infected bovine lymphoblastoid cell line with degenerated primers directed against T. annulata-targeting sequences. The TaD-coding sequence was found to be most closely related to the genomic DNA sequence of T. parva (TIGR database, 72%) and the amino acid sequence of Plasmodium falciparum (41%), P. yoelii yoelii (38%) and Cryptosporidium parvum (36%). The TaD mRNA is expressed within the sporozoite, schizont and merozoite stages of the parasite, implying that it is constitutively transcribed throughout the parasite's life cycle. Allelic variants were found between isolates originating from different geographical regions, however not affecting conserved cysteines. The open reading frame encoded a protein of 19.5 kDa and non-reducing SDS-PAGE analysis demonstrated a homodimeric protein. Using confocal microscopy, the protein was found to be both located in the parasite cytoplasm and to colocalize with a transmembrane protein of the schizonts within infected cells.

Amino Acid Sequence↗
PubMed2002

Suprachiasmatic nucleus organization.

The suprachiasmatic nucleus (SCN) of the hypothalamus is a dominant circadian pacemaker in the mammalian brain controlling the rest-activity cycle and a series of physiological and endocrine functions to provide a foundation for the successful elaboration of adaptive sleep and waking behavior. The SCN is anatomically and functionally organized into two subdivisions: (1) a core that lies adjacent to the optic chiasm, comprises predominantly neurons producing vasoactive intestinal polypeptide (VIP) or gastrin-releasing peptide (GRP) colocalized with GABA and receives dense visual and midbrain raphe afferents, and (2) a shell that surrounds the core, contains a large population of arginine vasopressin (AVP)-producing neurons in its dorsomedial portion, and a smaller population of calretinin (CAR)-producing neurons dorsally and laterally, colocalized with GABA, and receives input from non-visual cortical and subcortical regions. In this paper, we present a detailed quantitative analysis of the organization of the SCN core and shell in the rat and place this in the context of the functional significance of the subdivisions in the circadian control of regulatory systems.

Animals↗
PubMed1999

Nspl1, a new Z-band-associated protein.

Molecular characterization of a novel gene designated Neuroendocrine-Specific Protein-Like-1 (Nspl1) had revealed that this gene is expressed as two transcripts, a 1.2 kb transcript found predominantly in skeletal muscle and a 2.1 kb transcript expressed in the brain. The exceptionally high level of skeletal muscle expression prompted us to determine where the protein is localized to skeletal muscle. In vitro studies were performed using two plasmid constructs that generate full-length Nspl1 muscle-specific protein fused to the green fluorescent protein (GFP). In one construct, the GFP cDNA was fused to the N-terminus of the Nspl1 cDNA while in the second construct, the GFP cDNA was fused to the C-terminus of the Nspl1 cDNA. Transfection of either plasmid into mononucleated myoblasts showed that the Nspl1-GFP chimeric protein was associated with intermediate filaments. This was confirmed by using an antibody to stain desmin and finding that GFP-Nspl1 colocalizes with desmin. Chick primary myoblasts were transfected with the chimeric cDNAs and allowed to differentiate into mature myotubes. Results from this analysis and the use of monoclonal antibody to stain alpha-actinin, further localized the Nspl1 protein to the Z-band of mature myotubes. Confocal microscopy of the myotubes containing Nspl1-GFP demonstrates that Nspl1 is distributed continuously throughout the Z-disks.

Animals↗
PubMed2006

Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts.

GABAA receptors mediate the majority of fast synaptic inhibition in the brain. The accumulation of these ligand-gated ion channels at synaptic sites is a prerequisite for neuronal inhibition, but the molecular mechanisms underlying this phenomenon remain obscure. To further understand these processes, we have examined the cellular origins of synaptic GABAA receptors. To do so, we have created fluorescent GABAA receptors that are capable of binding -bungarotoxin (Bgt), facilitating the visualization of receptor endocytosis, exocytosis and delivery to synaptic sites. Imaging with Bgt in hippocampal neurons revealed that GABAA receptor endocytosis occurred exclusively at extrasynaptic sites, consistent with the preferential colocalization of extrasynaptic receptors with the AP2 adaptin. Receptor insertion into the plasma membrane was also predominantly extrasynaptic, and pulse-chase analysis revealed that these newly inserted receptors were then able to access directly synaptic sites. Therefore, our results demonstrate that synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. Moreover, they illustrate a dynamic mechanism for neurons to modulate GABAA receptor number at inhibitory synapses by controlling the stability of extrasynaptic receptors.

Animals↗
PubMed2005

An in vitro model of hepatitis C virion production.

The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5' and 3' ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5' and 3' ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of approximately 50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.

Base Sequence↗
PubMed2006

Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation.

L-type Ca(2+) channels play a critical role in regulating Ca(2+)-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca(2+) channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca(2+) channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for beta(2)-adrenergic receptor (AR) regulation of I(Ca,L). Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Ca(v)1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Ca(v)1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Ca(v)1.2, beta(2)-AR (not beta(1)-AR), G protein alpha(s), adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized beta(2)-AR/Ca(v)1.2 signaling complex, beta(2)-AR stimulation (salbutamol plus atenolol) of I(Ca,L) was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I(Ca,L) in response to beta(2)-AR activation was eliminated by disruption of caveolae with 10 mM methyl beta-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas beta(1)-AR stimulation (norepinephrine plus prazosin) of I(Ca,L) was not altered. These findings demonstrate that subcellular localization of L-type Ca(2+) channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways.

Adenylyl Cyclases↗
PubMed1996

Heat shock protein 84 forms a complex with mutant p53 protein predominantly within a cytoplasmic compartment of the cell.

Cellular DNA damage results in the increased expression and accumulation of the p53 tumor suppressor protein within the nucleus which leads to cell cycle arrest or apoptosis. In some cases, however, wild-type p53 and some mutant forms of p53 reside in the cytoplasm of cancer cells. To understand the mechanism responsible for its cytoplasmic retention, studies were undertaken to determine if unique proteins form a complex with mutant p53 within the cytoplasm of transformed cells. One protein, with an apparent molecular mass of 92 kDa (p92), was observed to form a complex with a temperature-sensitive mutant p53 (TSp53(Val-135)) in the cytoplasm of transformed rat embryo fibroblasts at the non-permissive temperature. p92 copurified with TSp53(Val-135) on a p53-specific immunoaffinity column and a gel filtration column. The protein was purified to homogeneity and identified as hsp84 by partial amino acid sequence analysis. hsp84 is a member of the hsp90 class of proteins. At the non-permissive temperature, TSp53(Val-135) and hsp84 colocalized in the cytoplasm near the nuclear envelope. At the permissive temperature, TSp53(Val-135) resides in the nucleus and expresses a "wild-type like" conformation. Under these conditions hsp84 continued to reside in the cytoplasm and little or no hsp84 formed a complex with p53. The results suggest that hsp84 binds mutant p53 in a spatial and/or conformation dependent manner.

Amino Acid Sequence↗
PubMed1993

Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions.

The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.

Amino Acid Sequence↗
PubMed1996

Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin.

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5-kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210-kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin-binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."

Amino Acid Sequence↗
PubMed2001

ZNF265--a novel spliceosomal protein able to induce alternative splicing.

The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-beta1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF(35). Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.

Active Transport, Cell Nucleus↗
PubMed2025

Panduratin A Induces Autophagy Through AMPK Activation Independent of mTOR Inhibition and Restricts Mycobacterium tuberculosis in Host Macrophages.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health burden, especially with the increasing prevalence of drug-resistant strains. There is an urgent need for new therapeutics that act via alternative mechanisms. Autophagy, a vital cell-autonomous defense process, allows macrophages to degrade intracellular pathogens such as Mtb and has gained attention as a potential target for host-directed therapy. In this study, we conducted a high-content imaging screen of herb-derived compounds to identify autophagy inducers in RAW264.7 macrophages. Panduratin A (NPA), a natural compound from Boesenbergia rotunda, was found to potently induce autophagy. NPA promoted autophagic vacuole formation in a dose-dependent fashion at low micromolar levels. Its autophagy-inducing effect was validated using RFP-GFP-LC3 dual fluorescence assays and immunoblotting in the presence of bafilomycin A1. Further mechanistic analysis revealed that NPA activates autophagy through AMPK activation, independent of mTOR inhibition. Importantly, NPA significantly promoted intracellular Mtb clearance and increased colocalization of Mtb with autophagosomes and lysosomes, in a manner dependent on Beclin-1. These findings highlight NPA as a potent enhancer of macrophage antimicrobial responses via autophagy, supporting its potential as a candidate for host-directed adjunctive therapy against TB.

Autophagy↗
PubMed2006

Sleeping beauty transposase has an affinity for heterochromatin conformation.

The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4',6'-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.

Animals↗
PubMed2005

The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins.

A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.

Cell Nucleus↗
PubMed1996

Circumferential stress and matrix metalloproteinase 1 in human coronary atherosclerosis. Implications for plaque rupture.

Atherosclerotic plaque rupture may occur when regions of weakened extracellular matrix are subjected to increased mechanical stresses. Since collagen is a major determinant of extracellular matrix strength, enzymes that degrade collagen may play an important role in destabilizing the atherosclerotic lesion. To test the hypothesis that matrix metalloproteinase 1 (interstitial collagenase, or MMP-1), which initiates degradation of fibrillar collagens, colocalizes with increased stress in the fibrous cap of the atherosclerotic lesion, 12 unruptured human coronary lesions were studied. Finite-element analysis was used to determine the distribution of stress in the lesion, with estimates of material properties from previous measurements of human tissues. A computerized image analysis system was used to determine the distribution of immunoreactive MMP-1 within the fibrous tissue of the lesion. There was a significant correlation between immunoreactive MMP-1 and circumferential tensile stress in the fibrous cap within a given lesion (median Spearman rank correlation coefficient, .36; interquartile range, -.02 to .81; P < .02). Within a given lesion, the highest-stress region had twofold greater MMP-1 expression than the lowest-stress regions. In unruptured human atherosclerotic coronary lesions, overexpression of MMP-1 is associated with increased circumferential stress in the fibrous plaque. Degradation and weakening of the collagenous extracellular matrix at these critical high-stress regions may play a role in the pathogenesis of plaque rupture and acute ischemic syndromes.

Collagenases↗
PubMed1996

Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy.

Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.

Adult↗
PubMed2004

Differential expression of steroidogenic factor-1/adrenal 4 binding protein and liver receptor homolog-1 (LRH-1)/fetoprotein transcription factor in the rat testis: LRH-1 as a potential regulator of testicular aromatase expression.

Aromatase converts testicular androgens to estrogens, which are essential for male fertility. Aromatase expression in testis occurs via transcription from promoter II, and requires the presence of a nuclear receptor half-site that binds the orphan receptor steroidogenic factor-1 [SF-1 (nuclear receptor 5A1)] to mediate basal and (in part) cAMP-induced transcription. We hypothesized that liver receptor homolog-1 (LRH-1) (nuclear receptor 5A2), a receptor closely related to SF-1, could also play a role in regulating aromatase expression in the testis. We demonstrate expression of LRH-1 in adult rat and immature mouse Leydig cells (LHR-1 > SF-1) as well as in pachytene spermatocytes and round spermatids but not in Sertoli cells, which in contrast, express high levels of SF-1. In transient transfection assays using TM3 Leydig cells and TM4 Sertoli cells, a rat promoter II luciferase reporter construct was stimulated by cotransfection of LRH-1 expression vector. Mutation analysis showed that induction by LRH-1 in TM3 and TM4 cells requires an AGGTCA motif at position -90, to which LRH-1 bound in gel shift analysis. We therefore provide evidence that LRH-1 plays an important role in the regulation of aromatase expression in Leydig cells. The colocalization of LRH-1 and aromatase to multiple testis cell types suggests that LRH-1 may have important effects on estrogen production, testis development, spermatogenesis, and testicular carcinogenesis.

Animals↗
PubMed2002

Genetic identity of Marinesco-Sjögren/myoglobinuria and CCFDN syndromes.

OBJECTIVE AND BACKGROUND: To describe three Gypsy families with Marinesco-Sjögren syndrome (MSS), demyelinating neuropathy, and recurrent episodes of myoglobinuria in five of the six affected subjects. Because these families originated from the same genetically isolated founder population as did patients with congenital cataracts facial dysmorphism neuropathy (CCFDN) syndrome, and because the two syndromes have clinical manifestations in common, we hypothesized that the two related, albeit distinct, syndromes may represent clinical variants of a single genetic disorder. METHODS: Clinical studies were conducted and linkage and haplotype analyses were performed for the three families. A total of 16 individuals, including the 6 with MSS and 10 unaffected relatives, were genotyped for six polymorphic microsatellite markers from the CCFDN region on 18qter. RESULTS: Linkage analysis of markers in the 18qter region, where we previously had located the CCFDN gene, produced a lod score of 3.55, demonstrating colocalization of the gene responsible for MSS with demyelinating neuropathy and myoglobinuria with the CCFDN gene. Moreover, the patients with MSS shared the conserved marker haplotype found in CCFDN chromosomes. CONCLUSIONS: These data suggest that Marinesco-Sjögren syndrome with peripheral neuropathy and myoglobinuria, and congenital cataracts facial dysmorphism neuropathy syndrome are genetically identical and are caused by a single founder mutation.

Adolescent↗