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PubMed1992

Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies.

We have recently shown that discrete foci are present in the nuclei of mammalian cells in which each of the U1, U2, U4/U6, and U5 snRNPs involved in pre-mRNA splicing, and the non-snRNP-splicing factor U2AF, are concentrated (Carmo-Fonseca, M., D. Tollervey, R. Pepperkok, S. Barabino, A. Merdes, C. Brunner, P. D. Zamore, M. R. Green, E. Hurt, and A. I. Lamond. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:195-206; Carmo-Fonseca, M., R. Pepperkok, B. S. Sproat, W. Ansorge, M. S. Swanson, and A. I. Lamond. 1991 EMBO (Eur. Mol. Biol. Organ.) J. 10:1863-1873). Here, we identify these snRNP-rich organelles as coiled bodies. snRNPs no longer concentrate in coiled bodies after cells are treated with the transcription inhibitors alpha-amanitin or actinomycin D. snRNP association with coiled bodies is also disrupted by heat shock. This indicates that the association of snRNPs with coiled bodies may be connected with the metabolism of nascent transcripts. A novel labeling method is described which shows both the RNA and protein components of individual snRNPs colocalizing in situ. Using this procedure all spliceosomal snRNPs are seen distributed in a nonhomogeneous pattern throughout the nucleoplasm, excluding nucleoli. They are most concentrated in coiled bodies, but in addition are present in "speckled" structures which are distinct from coiled bodies and which contain the non-snRNP splicing factor SC-35. U1 snRNP shows a more widespread nucleoplasmic staining, outside of coiled bodies and "speckled" structures, relative to the other snRNPs. The association of snRNPs with "speckles" is disrupted by heat shock but enhanced when cells are treated with alpha-amanitin.

Amanitins↗

PubMed1994

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

Amino Acid Sequence↗

PubMed1996

The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae.

We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the three-dimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere-associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten foci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3- strain, and similarly, Sir3p staining is no longer punctate in a sir4- strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.

Antibody Specificity↗

PubMed1997

mRNAs for microtubule proteins are specifically colocalized during the sequential formation of basal body, flagella, and cytoskeletal microtubules in the differentiation of Naegleria gruberi.

We have examined the distribution of four mRNAs-alpha-tubulin, beta-tubulin, flagellar calmodulin, and Class I mRNA-during differentiation of Naegleria gruberi amebas into flagellates by in situ hybridization. Three of the four mRNAs-alpha-tubulin, beta-tubulin, and Class I mRNA-began to be colocalized at the periphery of the cells as soon as transcription of the respective genes was activated and before any microtubular structures were observable. At 70 min after the initiation of differentiation, these mRNAs were relocalized to the base of the growing flagella, adjacent to the basal bodies and microtubule organizing center for the cytoskeletal microtubules. Within an additional 15 min, the mRNAs were translocated to the posterior of the flagellated cells, and by the end of differentiation (120 min), very low levels of the mRNAs were observed. Cytochalasin D inhibited stage-specific localization of the mRNAs, demonstrating that RNA localization was actin dependent. Since cytochalasin D also blocked differentiation, this raises the possibility that actin-dependent RNA movement is an essential process for differentiation.

Actin Cytoskeleton↗

PubMed1998

An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3.

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCzeta and PKClambda have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype-specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKClambda to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.

3T3 Cells↗

PubMed2000

Nuclear eukaryotic initiation factor 4E (eIF4E) colocalizes with splicing factors in speckles.

The eukaryotic initiation factor 4E (eIF4E) plays a pivotal role in the control of protein synthesis. eIF4E binds to the mRNA 5' cap structure, m(7)GpppN (where N is any nucleotide) and promotes ribosome binding to the mRNA. It was previously shown that a fraction of eIF4E localizes to the nucleus (Lejbkowicz, F., C. Goyer, A. Darveau, S. Neron, R. Lemieux, and N. Sonenberg. 1992. Proc. Natl. Acad. Sci. USA. 89:9612-9616). Here, we show that the nuclear eIF4E is present throughout the nucleoplasm, but is concentrated in speckled regions. Double label immunofluorescence confocal microscopy shows that eIF4E colocalizes with Sm and U1snRNP. We also demonstrate that eIF4E is specifically released from the speckles by the cap analogue m(7)GpppG in a cell permeabilization assay. However, eIF4E is not released from the speckles by RNase A treatment, suggesting that retention of eIF4E in the speckles is not RNA-mediated. 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) treatment of cells causes the condensation of eIF4E nuclear speckles. In addition, overexpression of the dual specificity kinase, Clk/Sty, but not of the catalytically inactive form, results in the dispersion of eIF4E nuclear speckles.

Autoantigens↗

PubMed2000

Caenorhabditis elegans UNC-45 is a component of muscle thick filaments and colocalizes with myosin heavy chain B, but not myosin heavy chain A.

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.

Animals↗

PubMed2000

Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse.

Axonal growth cone collapse is accompanied by a reduction in filopodial F-actin. We demonstrate here that semaphorin 3A (Sema3A) induces a coordinated rearrangement of Sema3A receptors and F-actin during growth cone collapse. Differential interference contrast microscopy reveals that some sites of Sema3A-induced F-actin reorganization correlate with discrete vacuoles, structures involved in endocytosis. Endocytosis of FITC-dextran by the growth cone is enhanced during Sema3A treatment, and sites of dextran accumulation colocalize with actin-rich vacuoles and ridges of membrane. Furthermore, the Sema3A receptor proteins, neuropilin-1 and plexin, and the Sema3A signaling molecule, rac1, also reorganize to vacuoles and membrane ridges after Sema3A treatment. These data support a model whereby Sema3A stimulates endocytosis by focal and coordinated rearrangement of receptor and cytoskeletal elements. Dextran accumulation is also increased in retinal ganglion cell (RGC) growth cones, in response to ephrin A5, and in RGC and DRG growth cones, in response to myelin and phorbol-ester. Therefore, enhanced endocytosis may be a general principle of physiologic growth cone collapse. We suggest that growth cone collapse is mediated by both actin filament rearrangements and alterations in membrane dynamics.

Actins↗

PubMed2000

Colocalization and redistribution of dishevelled and actin during Wnt-induced mesenchymal morphogenesis.

Activation of the Wnt signaling pathway is important for induction of gene expression and cell morphogenesis throughout embryonic development. We examined the subcellular localization of dishevelled, the immediate downstream component from the Wnt receptor, in the embryonic mouse kidney. Using immunofluorescence staining, confocal microscopy, and coimmunoprecipitation experiments, we show that dishevelled associates with actin fibers and focal adhesion plaques in metanephric mesenchymal cells. Stimulation of Wnt signaling leads to profound changes in metanephric mesenchymal cell morphology, including disruption of the actin cytoskeleton, increased cell spreading, and increased karyokinesis. Upon activation of Wnt signaling, dishevelled also accumulates in and around the nucleus. Casein kinase Iepsilon colocalizes with dishevelled along actin fibers and in the perinuclear region, whereas axin and GSK-3 are only present around the nucleus. These data indicate a branched Wnt signaling pathway comprising a canonical signal that targets the nucleus and gene expression, and another signal that targets the cytoskeleton and regulates cell morphogenesis.

Actins↗

PubMed2000

CREB-binding protein (CBP)/p300 and RNA polymerase II colocalize in transcriptionally active domains in the nucleus.

The spatial organization of transcription- associated proteins is an important control mechanism of eukaryotic gene expression. Here we analyzed the nuclear distribution of the transcriptional coactivators CREB-binding protein (CBP)/p300 in situ by confocal laser scanning microscopy, and in vivo complex formation by coimmunoprecipitation. A subpopulation of CBP and p300 is targeted to active sites of transcription and partially colocalizes with hyper- and hypophosphorylated RNA polymerase II (pol II) in discrete regions of variable size throughout the nucleus. However, the coactivators were found in tight association with hypophosphorylated, but not hyperphosphorylated pol II. Transcriptional inhibition induced a relocation of CBP/p300 and pol II into speckles. Moreover, double and triple immunofluorescence analyses revealed the presence of CBP, p300, and pol II in a subset of promyelocytic leukemia (PML) bodies. Our results provide evidence for a dynamic spacial link between coactivators of transcription and the basal transcription machinery in discrete nuclear domains dependent upon the transcriptional activity of the cell. The identification of pol II in CBP/PML-containing nuclear bodies supports the idea that transcription takes place at PML bodies.

Acetyltransferases↗

PubMed2001

Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole.

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end-directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.

Animals↗

PubMed2003

Colocalization of synapsin and actin during synaptic vesicle recycling.

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

Actins↗

PubMed1990

Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages.

We have studied the distribution of talin in J774 cells and mouse peritoneal macrophages undergoing Fc receptor-mediated phagocytosis. At early stages of phagocytosis, talin accumulates in the cells' cortical cytoplasm adjacent to the forming phagosome and extends into pseudopods that are encircling the particle. Talin colocalizes with F-actin at these sites. After particle ingestion is completed, F-actin and talin are no longer concentrated adjacent to phagosomes. Thus, talin and F-actin undergo dynamic and coordinate changes in their cytoplasmic location during Fc receptor-mediated phagocytosis.

Actins↗

PubMed1999

The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes.

The natural resistance associated macrophage protein (Nramp) gene family is composed of two members in mammals, Nramp1 and Nramp2. Nramp1 is expressed primarily in macrophages and mutations at this locus cause susceptibility to infectious diseases. Nramp2 has a much broader range of tissue expression and mutations at Nramp2 result in iron deficiency, indicating a role for Nramp2 in iron metabolism. To get further insight into the function and mechanism of action of Nramp proteins, we have generated isoform specific anti-Nramp1 and anti-Nramp2 antisera. Immunoblotting experiments indicate that Nramp2 is present in a number of cell types, including hemopoietic precursors, and is coexpressed with Nramp1 in primary macrophages and macrophage cell lines. Nramp2 is expressed as a 90-100-kD integral membrane protein extensively modified by glycosylation (>40% of molecular mass). Subcellular localization studies by immunofluorescence and confocal microscopy indicate distinct and nonoverlapping localization for Nramp1 and Nramp2. Nramp1 is expressed in the lysosomal compartment, whereas Nramp2 is not detectable in the lysosomes but is expressed primarily in recycling endosomes and also, to a lower extent, at the plasma membrane, colocalizing with transferrin. These findings suggest that Nramp2 plays a key role in the metabolism of transferrin-bound iron by transporting free Fe2+ across the endosomal membrane and into the cytoplasm.

Animals↗

PubMed2002

Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells.

Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-IC(cl2) is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-IC(cl2) cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

Amino Acid Sequence↗

PubMed2000

Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion.

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca(2+), cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide-gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory alpha subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide-gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22-56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide-gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide-gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.

Adenoviridae↗

PubMed1998

Increased transduction efficiency of primary hematopoietic cells by physical colocalization of retrovirus and target cells.

Efficient gene transfer into hematopoietic stem cells offers a number of potential therapeutic applications. However, the relatively low titer of retroviral supernatants and the requirement for cell division to ensure integration have meant that transduction efficiency has been low. We have modified a flowthrough approach to cell transduction and have been able consistently to increase gene transfer efficiency into human hematopoietic progenitor cells. We transduced CD34 cells with retroviral vectors encoding a truncated nerve growth factor receptor (NGFR) or neo. Retroviral supernatant was pulled through 0.2-micron polycarbonated membranes, followed by placement of cells on the filter. In the absence of cytokines, the transduction efficiency of CD34 cells with a NGFR vector was increased 3-11-fold over that obtained at an identical MOI in liquid culture to produce 11%-44% transduction. Furthermore, both Thy1+ and Thy1- subsets in a total CD34 population were transduced with similar efficiency, and transduction with a neo vector, as measured by G418 resistance in clonogenic assays, increased 1.5-5-fold. The mechanism by which gene transfer is improved may reflect colocalization of cells and retrovirus. Costaining of cells transduced on the filter with an NGFR retrovirus with both an NGFR antibody and a gp70 antibody that recognizes viral coat protein revealed high-level coexpression. The levels of in vitro gene transfer we obtain are equivalent to those observed when CD34 cells are cocultured in liquid culture with cytokines. However, culture with cytokines may commit CD34 cells to differentiation and has produced disappointingly low levels of subsequent in vivo gene transfer. Gene marking studies using distinguishable retroviral vectors will provide a means of learning whether the effects of flowthrough transduction genuinely enhance the efficiency of gene transfer to human marrow-repopulating cells.

Antigens, CD↗

PubMed1999

Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170.

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

Animals↗