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PubMed1993

Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes.

Mice that bear the X-linked immunodeficiency (xid) mutation have a B lymphocyte-specific defect resulting in an inability to make antibody responses to polysaccharide antigens. A backcross of 1114 progeny revealed the colocalization of xid with Bruton's agammaglobulinemia tyrosine kinase (btk) gene, which is implicated in the human immune deficiency, X-linked agammaglobulinemia. Mice that carry xid have a missense mutation that alters a highly conserved arginine near the amino-terminus of the btk protein, Btk. Because this region of Btk lies outside any obvious kinase domain, the xid mutation may define another aspect of tyrosine kinase function.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1996

Cytochemical colocalization and quantitation of phenotypic and genotypic characteristics in individual bacterial cells.

The widely accepted view that most bacterial species have yet to be cultivated in vitro has gained support from recent ribosomal DNA-based environmental studies. To enable elucidation of the phenotypes of organisms recognized solely by molecular genetic techniques, we developed and evaluated cytochemical methods which colocalize phenotypic properties with in situ rRNA probe hybridization signals. Application of these methods to artificial mixtures of Pseudomonas putida and Escherichia coli or Vibrio vulnificus showed that biochemical properties, such as the cytochrome oxidase reaction and specific substrate-enhanced tetrazolium salt reduction, can be assigned to cells identified by signals from determinative fluorescent rRNA probe binding. By doing the reactions directly on the stage of an inverted microscope and monitoring reaction product formation with a charge-coupled device video camera, it was possible to determine the kinetics of oxidizable substrate utilization in single cells. Analysis of digitized images permitted quantitative study of the relationship between rRNA signal strength and the rate of tetrazolium salt reduction. The approach used in this study opens up new opportunities to investigate the biochemistry, physiology, and behavior of both culturable and nonculturable bacteria in their natural environments.

Bacteria↗

PubMed1997

IpaB, a Shigella flexneri invasin, colocalizes with interleukin-1 beta-converting enzyme in the cytoplasm of macrophages.

Shigellae are the most prevalent etiological agents of dysentery. A crucial step in shigella pathogenesis is the induction of macrophage apoptosis. The invasion plasmid antigen B (IpaB) is necessary and sufficient to induce macrophage programmed cell death. IpaB activates apoptosis by binding to interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) or a highly homologous protease. Here, we show that IpaB is disseminated throughout the cytoplasm of shigella-infected macrophages as detected by both immunofluorescence and immunoelectron microscopy. The cytoplasmic distribution of IpaB requires phagosome escape, and it is specific to IpaB, since lipopolysaccharide, used here as a bacterial marker, remains closely associated with the bacteria. In double-labeling experiments, we show that IpaB and ICE colocalize in the cytoplasm of the macrophage, suggesting that soon after secretion, IpaB binds to ICE to initiate apoptosis and to promote the cleavage of IL-1 beta.

Adhesins, Bacterial↗

PubMed2001

Trichinella spiralis-infected muscle cells: abundant RNA polymerase II in nuclear speckle domains colocalizes with nuclear antigens.

Infection of mammalian skeletal muscle cells by Trichinella spiralis causes host nuclei to become polyploid (ca. 4N) and abnormally enlarged. It has been postulated that this enlargement reflects an infection-induced elevation of host transcription. Anthelmintic treatment of T. spiralis-infected rodents with mebendazole (MBZ) causes a reduction in the size of infected cell nuclei and a significant reduction in the total RNA content of individual infected muscle cells. A monoclonal antibody to the large subunit of RNA polymerase II (Pol II) was used here to assess the effects of infection on Pol II levels in isolated infected cell nuclei. Pol II was localized to speckle domains in isolated infected cell nuclei. Similar domains have been previously localized to sites of RNA synthesis or processing. When compared to the levels in nuclei from other, uninfected host cells, speckle-localized Pol II (SL-Pol II) levels were significantly elevated in infected cell nuclei by a mean of 3.9- to 6.8-fold. Nuclear antigens (NA) recognized by antibodies against T. spiralis localized to infected cell nuclei. By use of confocal microscopy, a subpopulation of NA was found colocalized with most speckle domains defined by Pol II. MBZ treatment of chronically infected mice, which depletes NA from infected cell nuclei, caused a significant depletion of SL-Pol II from infected cell nuclei. Control nuclei had a mean of 70% more SL-Pol II than MBZ-treated nuclei. The mean residual level of Pol II in these polyploid nuclei remained elevated by 120% over the level in 2N control nuclei. These observations may indicate two distinct effects of infection on Pol II levels in host cells.

Animals↗

PubMed2002

Chlamydial antigens colocalize within IncA-laden fibers extending from the inclusion membrane into the host cytosol.

Chlamydial IncA localizes to the inclusion membrane and to vesicular fibers extending away from the inclusion. Chlamydial outer membrane components, in the absence of developmental forms, are found within these fibers. This colocalization may explain how chlamydial developmental form antigens are localized outside of the inclusion within infected cells.

Antigens, Bacterial↗

PubMed1997

The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus.

The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain. Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.

Cell Compartmentation↗

PubMed2001

Green fluorescent protein-Dal80p illuminates up to 16 distinct foci that colocalize with and exhibit the same behavior as chromosomal DNA proceeding through the cell cycle of Saccharomyces cerevisiae.

Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in Saccharomyces cerevisiae. Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA. Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization. Many Dal80p binding site-homologous sequences are relatively evenly distributed across the S. cerevisiae genome, raising the possibility that Dal80p might be able to "stain" DNA. We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4',6'-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.

Cell Cycle↗

PubMed2006

Mitotic kinesin-like protein 2 binds and colocalizes with papillomavirus E2 during mitosis.

MKlp2 is a kinesin-like motor protein of the central mitotic spindle required for completion of cytokinesis. Papillomavirus E2 is a sequence specific DNA binding protein that regulates viral transcription and replication and is responsible for partitioning viral episomes into daughter cells during cell division. We demonstrate that MKlp2 specifically associates with the E2 protein during mitosis. Using chromatin immunoprecipitation, we show viral genomes are in complex with MKlp2 only within this stage of cell cycle. By immunofluorescence, a subpopulation of papillomavirus E2 colocalizes with MKlp2 in the midbody/midplate during late mitosis. We conclude that during specific stages of mitosis, the papillomavirus E2 protein binds to MKlp2, and infer that association with this motor protein ensures viral genome partitioning during cytokinesis.

Cell Line, Tumor↗

PubMed1991

Nuclear colocalization of cellular and viral myc proteins with HSP70 in myc-overexpressing cells.

The c-myc oncogene and its viral counterpart v-myc encode phosphoproteins which have been located within cell nuclei, excluding nucleoli. We have expressed the c-myc gene under the simian virus 40 early promoter and studied the distribution of its protein product in transient expression assays in COS, HeLa, and 293 cells. We found three distinct patterns of c-myc immunofluorescence in the transfected cells: one-third of the c-myc-positive cells displayed a diffuse nuclear distribution, and in two-thirds of the cells the c-myc fluorescence was accumulated either in small amorphous or in large multilobed phase-dense nuclear structures. Unexpectedly, these structures also stained for the HSP70 heat shock protein in both heat-shocked and untreated cells. Our results indicate that both transient and stable overexpression of either the c-myc or v-myc protein induces translocation of the endogenous HSP70 protein from the cytoplasm to the nucleus, where it becomes sequestered in structures containing the myc protein. Interestingly, the closely related N-myc protein does not stimulate substantial nuclear expression of the HSP70 protein. Studies with chimeric myc proteins revealed that polypeptide sequences encoded by the second exon of c-myc are involved in colocalization with HSP70.

Animals↗

PubMed1992

Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells.

The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically. In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells. By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins. In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci. Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells. These results suggest that replication centers similar to those established by other viruses exist for AAV. These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro.

Antibodies, Monoclonal↗

PubMed1994

Subcellular distribution of human immunodeficiency virus type 1 Rev and colocalization of Rev with RNA splicing factors in a speckled pattern in the nucleoplasm.

The human immunodeficiency virus type 1 (HIV-1) Rev (regulator of virion protein expression) protein exemplifies a new type of posttranscriptional regulation. One main function of Rev is to increase the cytoplasmic expression of unspliced and incompletely spliced retroviral mRNAs from which viral structural proteins are made. In that way, Rev is essential in order to complete the retroviral life cycle. The biology of Rev in the host cell has remained elusive. In this study, a complex distribution of Rev in single cells was found. Rev was found in the cytoplasm, in a perinuclear zone, in the nucleoplasm, and in the nucleoli. In the nucleoplasm, Rev colocalized in a speckled pattern with host cell factors known to assemble on nascent transcripts. Those factors are involved in the processing of heterogeneous RNA to spliced mRNA in the nucleoplasm of all cells. The distribution of Rev was dependent only on Rev and host cell interactions, since neither the Rev target RNA nor other HIV proteins were expressed in the cells. Rev was found in the same subcellular compartments of cells treated for extended periods with cycloheximide, an inhibitor of protein synthesis. This finding implies that Rev shuttles continuously between cytoplasmic and nucleoplasmic compartments. The results suggest a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA.

Amino Acid Sequence↗

PubMed1995

Evaluation of colocalization interactions between the IE110, IE175, and IE63 transactivator proteins of herpes simplex virus within subcellular punctate structures.

A number of previous studies have implied that three herpes simplex virus-encoded nuclear transactivator proteins, IE175 (ICP4), IE110 (ICP0), and IE63 (ICP27), may cooperate in transcriptional and posttranscriptional stimulation of viral gene expression. Using double-label immunofluorescence assays (IFA) in transient expression assays, we have examined the intracellular localization of these three proteins in DNA-transfected cells. The IE110 protein on its own forms spherical punctate domains within the nucleus, whereas the IE175 and IE63 proteins alone give uniform and speckled diffuse patterns, respectively. In infected cells, the IE110 punctate granules have been shown to correspond to novel preexisting subnuclear structures referred to as ND10 domains or PODs that contain a variety of cellular proteins, including SP100 and the PML proto-oncogene product. Cotransfection experiments with wild-type nuclear forms of both IE175 and IE110 provided direct evidence for partial redistribution of IE175 into the same punctate granules that contained IE110. Surprisingly, nuclear forms of IE110 were found to move a cytoplasmic form of IE175 into nuclear punctate structures, and a cytoplasmic form of IE110 was able to retain nuclear forms of IE175 in cytoplasmic punctate structures. Therefore, the punctate characteristic of IE110 appeared to both dominate the interactions and override the normal nuclear localization signals. The domains responsible for the interaction mapped to between codons 518 and 768 in 1E110 and to between codons 835 and 1029 in IE175. Importantly, a truncated nuclear form of the 1,298-amino-acid IE175 protein, which lacked the C-terminal domain beyond codon 834, was found to be excluded from the IE110 punctate granules. Cotransfection of nuclear or cytoplasmic IE110 with a truncated nuclear form of IE63 also led to partial redistribution of IE63 into either nuclear or cytoplasmic punctate granules containing IE110. Both the IE63-IE110 and IE175-IE110 colocalization interactions were demonstrated in Vero cells but not in 293 cells. Consequently, they differ from IE110 self-interactions, which correlate with in vitro dimerization and occur efficiently in both cell types. These interactions may help to explain the altered promoter target specificity and synergism observed when IE175 is cotransfected with IE110 in transactivation studies.

Amino Acid Sequence↗

PubMed1996

Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis.

The helicase-like 1a and polymerase-like 2a proteins of brome mosaic virus (BMV) are required for viral RNA replication in vivo, are present in membrane-bound viral RNA polymerase extracts, and share conservation with the many other members of the alphavirus-like superfamily. To better understand BMV RNA replication and BMV-host interactions, we used confocal microscopy and double-label immunofluorescence to determine and compare the sites of 1a, 2a, and nascent viral RNA accumulation in BMV-infected barley protoplasts. 1a and 2a showed nearly complete colocalization throughout infection, accumulating in defined cytoplasmic spots usually adjacent to or surrounding the nucleus. These spots grew throughout infection and by 16 h postinoculation often assumed a vesicle-like appearance. The BMV RNA replication complex incorporated 5-bromouridine 5'-triphosphate into RNA in vitro and in vivo, allowing immunofluorescent detection of nascent RNA. The cytoplasmic sites of BMV-specific RNA synthesis coincided with the sites of 1a and 2a accumulation, and at the resolution of confocal microscopy, all sites of 1a and 2a accumulation were sites of BMV RNA synthesis. Double-label immunofluorescence detection of selected subcellular markers and 1a or 2a showed that BMV replication complexes were tightly associated with markers for the endoplasmic reticulum but not the medial Golgi or later compartments of the cellular secretory pathway. Defining this association of BMV RNA replication complexes with endoplasmic reticulum markers should assist in identifying and characterizing host factors involved in BMV RNA replication.

Bromovirus↗

PubMed1996

The primary target cells of the high-risk cottontail rabbit papillomavirus colocalize with hair follicle stem cells.

Papillomaviruses are small DNA tumor viruses with a life cycle inseparably linked to the differentiation of the pluristratified epithelium. The infection of epithelial layers of the skin may remain latent or may result in the development of benign tumors. A certain number of distinct papillomavirus types, however, cause lesions which have a high risk of progression into carcinomas, and extensive efforts have been made to understand this process. comparatively little is known about the initial events during the establishment of a persistent infection and papilloma development. Although it is generally accepted that the growth of a papilloma requires the infection of cells in the basal layer of the epithelium, it remains unknown which cells perform this task. We have analyzed by in situ hybridization biopsy samples taken at various time points after infection of domestic rabbits with cottontail rabbit papillomavirus. The positive cells detected at a low frequency in biopsy samples taken after 11 days predominantly expressed high levels of E6 and E7 mRNA and were localized in the outer epithelial root sheath and in the bulbs of hair follicles. A clonal analysis of keratinocytes isolated from different subfragments of individual rabbit hair follicles demonstrated a clear colocalization of cottontail rabbit papillomavirus mRNA-positive cells with clonogenic cells in hair follicles. These data suggest that the cells competent to establish papillomatous growth represent a subpopulation of keratinocytes in hair follicles with properties expected of epithelial stem cells.

3T3 Cells↗

PubMed1997

The human T-cell leukemia virus type 1 transactivator protein Tax colocalizes in unique nuclear structures with NF-kappaB proteins.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent activator of viral transcription. Tax also activates the expression of specific cellular genes involved in the control of T-lymphocyte growth via effects on cellular transcription factors, including members of the NF-kappaB/cRel family. Immunocytochemistry and electron microscopy were used to characterize the intracellular localization of Tax and identify cellular factors which are the potential targets for its transcriptional activity. These studies indicated that Tax localizes in discrete nuclear foci in T lymphocytes transformed by HTLV-1 and in cells transduced with Tax expression vectors. The Tax-containing foci are complex nuclear structures comprising a central core in which Tax colocalizes with splicing factor Sm. In addition to splicing factors Sm and SC-35, the Tax-containing nuclear structures also contain transcriptional components, including the largest subunit of RNA polymerase II and cyclin-dependent kinase CDK8. The inclusion of the two subunits of NF-kappaB, p50 and RelA, and the presence of the mRNA from a gene specifically activated by Tax through NF-kappaB binding sites suggest that these unique nuclear structures participate in Tax-mediated activation of gene expression via the NF-kappaB pathway.

Animals↗

PubMed1998

A novel human cytomegalovirus glycoprotein, gpUS9, which promotes cell-to-cell spread in polarized epithelial cells, colocalizes with the cytoskeletal proteins E-cadherin and F-actin.

Processes by which human herpesviruses penetrate and are released from polarized epithelial cells, which have distinct apical and basolateral membrane domains differing in protein and lipid content, are poorly understood. We recently reported that human cytomegalovirus (CMV) mutants with deletions of the gene US9 formed wild-type plaques in cultures of human fibroblasts but were impaired in the capacity for cell-to-cell spread in polarized human retinal pigment epithelial cells. Unlike the glycoproteins that are required for infection, the protein encoded by CMV US9 plays an accessory role by promoting dissemination of virus across cell-cell junctions of polarized epithelial cells. To identify the product and investigate its specialized functions, we selected Madine-Darby canine kidney II (MDCK) epithelial cells that constitutively express CMV US9 or, as a control, US8. The gene products, designated gpUS9 and gpUS8, were glycosylated proteins of comparable molecular masses but differed considerably in intracellular distribution and solubility. Immunofluorescence laser scanning confocal microscopy indicated that, like gpUS8, gpUS9 was present in the endoplasmic reticulum and Golgi compartments of nonpolarized cells. In polarized epithelial cells, gpUS9 also accumulated along lateral membranes, colocalizing with cadherin and actin, and was insoluble in Triton X-100, a property shared with proteins that associate with the cytoskeleton. We hypothesize that gpUS9 may enhance the dissemination of CMV in infected epithelial tissues by associating with the cytoskeletal matrix.

Actins↗

PubMed1999

Vif and the p55(Gag) polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane-free cytoplasmic complexes.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259-5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55(Gag) polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.

Cell Line↗

PubMed2000

ICP0 induces the accumulation of colocalizing conjugated ubiquitin.

Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 is a general activator of viral gene expression which stimulates the initiation of lytic infection and reactivation from quiescence and latency. The importance of ICP0 to the biology of HSV-1 infection has stimulated interest in its mode of action. Previous studies have reported its interactions with other viral regulatory molecules, with the translation apparatus, with cyclin D3, and with a ubiquitin-specific protease. It has been demonstrated that ICP0 is able to induce the proteasome-dependent degradation of a number of cellular proteins, including components of centromeres and small nuclear substructures known as ND10 or PML nuclear bodies. ICP0 has a RING finger zinc-binding domain which is essential for its functions. In view of several recent examples of other RING finger proteins which modulate the stability of specific target proteins by acting as components of E3 ubiquitin ligase complexes, this study has explored whether ICP0 might operate via a similar mechanism. Evidence that the foci of accumulated ICP0 in transfected and infected cells contain enhanced levels of conjugated ubiquitin is presented. This effect was dependent on the RING finger region of ICP0, and comparison of the properties of a number of ICP0 mutants revealed an excellent correlation between previously established functions of ICP0 and its ability to induce concentrations of colocalizing conjugated ubiquitin. These results strongly support the hypothesis that a major factor in the mechanism by which ICP0 influences virus infection is its ability to induce the degradation of specific cellular targets by interaction with the ubiquitin-proteasome pathway.

Amino Acid Motifs↗