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A Brüning-Richardson,
J Bond,
R Alsiary,
J Richardson,
D A Cairns,
L McCormack,
R Hutson,
P Burns,
N Wilkinson,
G D Hall, E E Morrison,
S M Bell
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ABSTRACT: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers.
Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data.
A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.
Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.
British Journal of Cancer 05/2011; 104(10):1602-10. · 5.04 Impact Factor
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A Br|[uuml]|ning-Richardson,
J Bond,
R Alsiary,
J Richardson,
D A Cairns,
L McCormack,
R Hutson,
P Burns,
N Wilkinson,
G D Hall, E E Morrison,
S M Bell
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ABSTRACT: Background: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers.
British Journal of Cancer 04/2011; 104(10):1602-1610. · 5.04 Impact Factor
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ABSTRACT: The microtubule (MT)-associated protein EB1 localizes to and promotes growth at MT plus ends. The MT depolymerizing kinesin MCAK has also been reported to track growing MT plus ends. Here, we confirm that human MCAK colocalizes with EB1 at growing MT ends when expressed as a GFP fusion protein in transfected cells. We show that MCAK associates with the C-terminus of EB1 and EB3 but much less efficiently with RP1. EB1 associates with the N-terminal localization and regulatory domain in MCAK but not with the motor domain of the protein. The interaction is competitive with the binding of other EB1 ligands and does not require MTs. Knockdown of EB1 expression using siRNA impaired the ability of GFP-MCAK to localize to MT tips in transfected cells. We propose that MCAK is targeted to growing MT ends by EB1, that MCAK is held in an inactive conformation when associated with EB1 and that this could provide the basis for a mechanism that facilitates rapid switching between phases of MT growth and depolymerization.
Oncogene 05/2008; 27(17):2494-500. · 6.37 Impact Factor
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E E Morrison
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ABSTRACT: Microtubule dynamic instability is fundamentally important to the way cells respond to their environment and segregate their genetic material. A disparate class of proteins defined by their localisation to growing microtubule plus ends ('+TIPS') play a key role in controlling microtubule dynamics and organisation. They directly impact upon the behaviour of the microtubule tip and link this structure to interfaces that include kinetochores and the cortex of the cell. Surprisingly, some +TIPs also have important functions at the microtubule minus end. These properties contribute to the important roles played by +TIPs in processes such as mitosis and cell migration. This review examines how recent advances have impacted our understanding of +TIP function in mammalian cells, with emphasis on the emergence of the EB1 family as a core component of +TIP activities. An overview of the use of +TIP imaging as a tool for the cell biologist is also presented.
Cellular and Molecular Life Sciences CMLS 03/2007; 64(3):307-17. · 6.57 Impact Factor
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ABSTRACT: The adenomatous polyposis coli (APC) tumor suppressor protein is mutated in most colorectal carcinomas. In addition to its role in WNT signaling it is proposed to be involved in both cell migration and mitosis. Although a variety of studies have shown an APC localization along lateral membranes of adjacent epithelial cells the existence of a cortical APC localization in mammalian cells remains controversial. To address this we have used matched rat epithelial (NRK-52E) and fibroblast (NRK-49F) cell lines to investigate the localization of APC. Subconfluent cultures of NRK-52E and -49F cells displayed microtubule-associated APC populations by immunostaining. However, confluent NRK-52E, but not -49F monolayers, exhibited a cortical APC distribution. Cortical APC localized in close proximity to a number of cell junction proteins in a microtubule-independent manner while calcium switch experiments suggested that APC was recruited to the cortex only when junction assembly was complete. Confluent NRK-49F and -52E cells also showed contrasting APC localizations in response to monolayer wounding. Our data suggests APC cortical localization is a feature of confluent epithelioid cells and that the subcellular distribution of APC is therefore dependent upon both cell type and context.
Cell Motility and the Cytoskeleton 09/2006; 63(8):483-92. · 4.19 Impact Factor
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ABSTRACT: Cajal bodies are intra-nuclear structures enriched in proteins involved in transcription and mRNA processing. In this study, immunofluorescence microscopy experiments using a highly specific antibody to actin revealed nuclear actin spots that colocalized in part with p80 coilin-positive Cajal bodies. Actin remained associated with Cajal bodies in cells extracted to reveal the nuclear matrix. Adenovirus infection, which is known to disassemble Cajal bodies, resulted in loss of actin from these structures late in infection. In infected cells, nuclear actin was observed to relocate to structures at the periphery of the nucleus, inside the nuclear envelope. Based on these findings, it is suggested that actin may play an important role in the organization or function of the Cajal body.
Experimental Cell Research 03/2005; 303(2):229-39. · 3.58 Impact Factor
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ABSTRACT: Gastrointestinal carcinomas frequently disseminate within the abdominal cavity to form secondary peritoneal metastases. Invasion of the peritoneal mesothelium is fundamental to this process, yet the underlying invasive mechanisms remain unclear. Preliminary in vitro work suggested that tumour cells can induce mesothelial apoptosis, representing a novel mechanism of peritoneal invasion. We examined the role of tumour cell-induced mesothelial apoptosis and explored the role of the death ligand/receptor system, Fas Ligand/Fas, as mediators of the apoptotic process. Cultured human mesothelial cells were used to establish in vitro co-culture models with the SW480 colonic cancer cell line. Tumour-induced mesothelial apoptosis was confirmed by phase-contrast microscopy and apoptotic detection assays. Human mesothelial cells and SW480 tumour cells constitutively expressed Fas and Fas Ligand mRNA and protein as determined by RT-PCR and confocal fluorescent microscopy. Stimulation of human mesothelial cells with anti-Fas monoclonal antibody or crosslinked soluble Fas Ligand-induced apoptosis, confirming the functional status of the Fas receptor. Pretreatment of SW480 cells with a blocking recombinant anti-Fas Ligand monoclonal antibody significantly reduced mesothelial apoptosis, indicating that tumour-induced mesothelial apoptosis may, in part, be mediated via a Fas-dependent mechanism. This represents a novel mechanism of mesothelial invasion and offers several new targets for therapeutic intervention.
British Journal of Cancer 05/2004; 90(7):1437-42. · 5.04 Impact Factor
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ABSTRACT: When myoblasts fuse into myotubes, the organisation of the cytoskeleton changes dramatically. For example, microtubules emanate in a radial array form the centrosome in myoblasts, but form linear arrays not linked to a centrosome in myotubes. It is not clear how these linear arrays are formed and nucleated. They could arise in a number of ways: by nucleation and release from centrosomal like structures, cytoplasmic assembly, breakage/severing or nucleation from non-centrosomal sites. To test which of the above mechanisms or combination of mechanisms are responsible we investigated the re-formation of microtubules after depolymerisation by nocodazole, using antibodies against pericentrin, gamma-tubulin, EB1, and tyrosinated alpha-tubulin. In myoblasts, we found that when microtubules were allowed to recover after complete depolymerisation with nocodazole, microtubule recovery began within 1 min and was complete after 5 min. Microtubules grew out from the centrosome, which was positively stained for gamma-tubulin or pericentrin. In untreated myotubes, microtubules were arranged in linear arrays, with EB1 at their ends. The pericentriolar protein, pericentrin was arranged in a band around the nucleus as well as discrete spots in the cytoplasm. In contrast, the microtubule nucleating protein gamma-tubulin was not found in a band around the nucleus, but was found in several punctuate spots throughout the cytoplasm. Further, when microtubules were allowed to recover, after complete depolymerisation with nocodazole, recovery was not as rapid as that seen in myoblasts, and we found that regrowth began with the formation of short microtubule fragments throughout the cytoplasm. Gamma-tubulin was associated with these fragments. These results suggest that in myotubes, nucleation of microtubules can be non-centrosomal.
Journal of Muscle Research and Cell Motility 02/2003; 24(4-6):301-8. · 1.98 Impact Factor
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ABSTRACT: When myoblasts fuse into myotubes, the organisation of the cytoskeleton changes dramatically. For example, microtubules emanate
in a radial array form the centrosome in myoblasts, but form linear arrays not linked to a centrosome in myotubes. It is not
clear how these linear arrays are formed and nucleated. They could arise in a number of ways: by nucleation and release from
centrosomal like structures, cytoplasmic assembly, breakage/severing or nucleation from non-centrosomal sites. To test which
of the above mechanisms or combination of mechanisms are responsible we investigated the re-formation of microtubules after
depolymerisation by nocodazole, using antibodies against pericentrin, γ-tubulin, EB1, and tyrosinated α-tubulin. In myoblasts,
we found that when microtubules were allowed to recover after complete depolymerisation with nocodazole, microtubule recovery
began within 1 min and was complete after 5 min. Microtubules grew out from the centrosome, which was positively stained for
γ-tubulin or pericentrin. In untreated myotubes, microtubules were arranged in linear arrays, with EB1 at their ends. The
pericentriolar protein, pericentrin was arranged in a band around the nucleus as well as discrete spots in the cytoplasm.
In contrast, the microtubule nucleating protein γ-tubulin was not found in a band around the nucleus, but was found in several
punctuate spots throughout the cytoplasm. Further, when microtubules were allowed to recover, after complete depolymerisation
with nocodazole, recovery was not as rapid as that seen in myoblasts, and we found that regrowth began with the formation
of short microtubule fragments throughout the cytoplasm. γ-tubulin was associated with these fragments. These results suggest
that in myotubes, nucleation of microtubules can be non-centrosomal.
Journal of Muscle Research and Cell Motility 01/2003; 24(4):303-310. · 1.98 Impact Factor
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ABSTRACT: EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.
Molecular Biology of the Cell 11/2002; 13(10):3627-45. · 4.94 Impact Factor
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ABSTRACT: EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending neurites in these cells. In N2A axons, analysis of the ratio of EB1/beta-tubulin fluorescence demonstrated that the distal tip region contained the highest proportion of polymerising microtubules. Time-lapse confocal imaging of an EB1-GFP fusion protein in transfected N2A cells directly revealed the dynamics of microtubule extension in neurites, and demonstrated the existence of unusual, discrete knots of microtubule polymerisation at the periphery of non-process bearing cells which may represent an early event in neurite outgrowth. We conclude that EB1 localisation can be used to identify and analyse sites of microtubule polymerisation at a high resolution during neurite development, a process to which it may contribute.
Molecular Brain Research 02/2002; 98(1-2):145-52. · 2.00 Impact Factor
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ABSTRACT: Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.
European Journal of Cell Biology 01/2002; 80(12):749-53. · 2.81 Impact Factor
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ABSTRACT: The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.
Oncogene 05/2000; 19(15):1950-8. · 6.37 Impact Factor
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ABSTRACT: Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.
European Journal of Cancer 04/2000; 36(4):525-32. · 5.54 Impact Factor
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ABSTRACT: The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.
Oncogene 01/1999; 17(26):3471-7. · 6.37 Impact Factor
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ABSTRACT: The fate of herpes simplex virus 1 (HSV-1) tegument proteins during infection in Vero cells was investigated immunochemically. Input virion-associated VP13/14 and VP16 localized to the nucleus early in infection, while VP1/2 localized to the nuclear envelope of the cell and VP22 could not be detected using monoclonal antibody P43. Western blotting suggested that virion-associated VP13/14, VP16 and VP22 were stable in infected cells whereas VP1/2 appeared to be processed or modified. Further studies showed that P43 recognized a phosphorylation-sensitive epitope in VP22 and suggested that virion-associated VP22 was phosphorylated upon entry to the cell. VP13/14 and VP16 were easily extracted from cells early in infection whereas VP22 was largely insoluble. Phosphatase treatment of soluble extracts caused a shift in the molecular mass of VP16 showing it was phosphorylated. As infection progressed VP16 was observed in discrete nuclear compartments where it co-localized with ICP8 and the capsid-associated protein VP22a. VP13/14 was also observed in the nucleus. P43 immunostaining appeared around 6 h post-infection as punctate nuclear foci which often localized to the edge of VP16-immunoreactive areas. Punctate P43 cytoplasmic staining appeared around 12 h post-infection. By 18 h the nuclear pattern had disappeared and an extensive cytoplasmic stain was observed which closely overlapped that of other tegument proteins. On the basis of these data we suggest that virion-associated VP22 is phosphorylated upon entry of the virus into the cell and that unphosphorylated VP22, which is preferentially recognized by P43, becomes available later in infection, initially in the nucleus, for packaging into virions.
Journal of General Virology 11/1998; 79 ( Pt 10):2517-28. · 3.36 Impact Factor
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ABSTRACT: The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.
Journal of Virology 10/1998; 72(9):7108-14. · 5.40 Impact Factor
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ABSTRACT: The adenomatous polyposis coli tumour suppressor protein is highly expressed in developing rodent brain, but its function is unclear. Recent studies have suggested a role for this protein in regulating microtubule dynamics. Neuro 2A mouse neuroblastoma cells were previously thought not to express this protein. Using immunochemical techniques, this report corrects this observation. Immunoreactive bands of a size consistent with that of the full-length protein were observed by western blotting. Using immunocytochemistry, punctate immunoreactivity localized to areas of the cell containing microtubules, particularly neurite growth cones, in a distribution suggesting a role in neuritogenesis and growth cone extension. The protein did not localize to actin-rich cellular structures, and perturbation of the actin cytoskeleton had no effect upon this distribution. Treatment of cells with taxol to stabilize microtubules caused the concentration of the immunoreactive puncta to the tips of microtubules and areas along the axis of potential microtubule assembly. Treatment of cells with the microtubule disrupting reagent nocodazole showed that over shorter times the punctate distribution was not dependent upon polymerized microtubules. However, at longer incubation times a decrease in punctate immunostaining was observed. These results indicate that the intracellular distribution of the adenomatous polyposis coli protein is dependent upon microtubule but not actin dynamics. A role for this protein in the regulation of directed microtubule assembly is suggested.
Neuroscience 12/1997; 81(2):553-63. · 3.38 Impact Factor
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ABSTRACT: Beta-catenin is known to associate with the tumour suppressor protein adenomatous polyposis coli (APC), which is highly expressed in developing brain. We have therefore investigated the distribution of beta-catenin and APC in primary cultures of mouse neocortex. Western blotting demonstrated the presence of a single beta-catenin species in our cultures. Immunocytochemistry showed that beta-catenin was plasma membrane associated and concentrated in growth cones in cultured neurons. The APC tumour suppressor protein was also concentrated in growth cones. In glial cells, beta-catenin was localised at cell-cell contacts in a manner similar to that previously described in other cell types. This data suggests a role for both APC and beta-catenin in neuronal growth cones, and for beta-catenin in the formation of cell to cell contacts between glia.
Neuroscience Letters 11/1997; 235(3):129-32. · 2.11 Impact Factor
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ABSTRACT: The herpes simplex virus type 1 (HSV-1) capsid protein VP24 (encoded by UL26) was expressed as a GST-fusion protein and used to prepare a group of monoclonal antibodies. These were used to characterize the protein in capsids and virus infected cells and demonstrated that it exists as two polypeptide species. The nature of the relationship between these two species was investigated and found to be associated with disulphide bonding. Under non-reducing conditions a species corresponding to dimers of VP24 was identified in preparations of B capsids, the site of action of the proteinase. Biochemical subcellular fractionation studies suggested that only cleaved forms of UL26 and UL26.5 gene products could be detected in the nucleus of the infected cell at early times post-infection.
Journal of General Virology 04/1997; 78 ( Pt 3):671-5. · 3.36 Impact Factor
