• X-epilectin: a novel epidermal fucolectin regulated by BMP signalling.

      Massé, Karine; Baldwin, Rebecca J; Barnett, Mark W; Jones, Elizabeth A; Molecular Physiology, Department of Biological Sciences, Warwick University, Coventry, UK. (2004-12)
      This paper reports the cloning and characterisation of a new posterior epidermal marker, X-epilectin, in Xenopus laevis. This gene encodes for a fucolectin, which belongs to the lectin superfamily of carbohydrate binding proteins and specifically binds fucose residues. RT-PCR and in situ hybridisation show that the expression of this gene is switched on during gastrulation and up-regulated during neurula stages and found expressed ubiquitously throughout the epidermis. From tailbud stages, the expression is limited to the dorsal posterior region of the embryo, suggesting that X-epilectin expression is regulated along anteroposterior and dorsoventral gradients during development. In the adult, X-epilectin is mainly expressed in intestinal components, kidney, spinal cord and skin. The effects of growth factors on the regulation of X-epilectin were studied. Change of the fate of animal caps into cement gland or dorsal mesoderm induces a down-regulation of X-epilectin expression in explants treated respectively with ammonium chloride and activin A. We also show that X-epilectin expression is down-regulated by Noggin and tBR and that this effect is inhibited by BMP4 over-expression, suggesting X-epilectin expression is mediated by the BMP signalling pathway.
    • X-linked inhibitor of apoptosis protein as a therapeutic target.

      Dean, Emma J; Ranson, Malcolm R; Blackhall, Fiona H; Dive, Caroline; Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. emma.dean@christie.nhs.uk (2007-11)
      Dysregulation of apoptosis has been shown to contribute to many diseases, including cancer formation, development and resistance, as well as neurodegenerative and autoimmune disorders. One mechanism through which tumour cells are believed to acquire resistance to apoptosis is by overexpression of X-linked inhibitor of apoptosis protein (XIAP), which belongs to a family of inhibitor of apoptosis proteins. When XIAP is overexpressed, cancer cells are rendered resistant to apoptosis, both intrinsically and in response to chemotherapy and radiotherapy. Significant progress has been made in targeting XIAP therapeutically, both directly and indirectly through the modulation of other molecules involved in the apoptotic pathway. This review introduces XIAP from its molecular origins, discusses its modulation and potential as a novel drug target, and considers future therapeutic perspectives.
    • X-ray diffraction from isolated metaphase chromosomes.

      Pardon, J; Richards, B; Skinner, L G; Ockey, Charles H; Searle Research Laboratories, Lane End Road, High Wycombe, Bucks, England (1973-05-15)
    • X:Map: annotation and visualization of genome structure for Affymetrix exon array analysis.

      Yates, Tim; Okoniewski, Michal J; Miller, Crispin J; Cancer Research UK, Bioinformatics Group, Paterson Institute for Cancer Research, The University of Manchester, Christie Hospital Site, Wilmslow Road, Withington, Manchester, M20 4BX, UK. (2008-01)
      Affymetrix exon arrays aim to target every known and predicted exon in the human, mouse or rat genomes, and have reporters that extend beyond protein coding regions to other areas of the transcribed genome. This combination of increased coverage and precision is important because a substantial proportion of protein coding genes are predicted to be alternatively spliced, and because many non-coding genes are known also to be of biological significance. In order to fully exploit these arrays, it is necessary to associate each reporter on the array with the features of the genome it is targeting, and to relate these to gene and genome structure. X:Map is a genome annotation database that provides this information. Data can be browsed using a novel Google-maps based interface, and analysed and further visualized through an associated BioConductor package. The database can be found at http://xmap.picr.man.ac.uk.
    • Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts.

      Goldberg, Martin W; Jenkins, H; Allen, Terence D; Whitfield, W G; Hutchison, C J; CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1995-11)
      Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of 'depleted' extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.
    • Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts.

      Nicolás, F J; Zhang, C; Hughes, M; Goldberg, Martin W; Watton, S J; Clarke, P R; Zeneca Laboratory of Molecular and Cellular Biology, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK. (1997-12)
      Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1). Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-beta/karyopherin-beta and importin-alpha/karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1.
    • Xeroderma pigmentosum group D haplotype predicts for response, survival, and toxicity after platinum-based chemotherapy in advanced nonsmall cell lung cancer.

      Booton, Richard; Ward, Timothy H; Heighway, Jim; Taylor, Pat; Power, Fiona; Ashcroft, Linda; Morris, Julie; Thatcher, Nick; Christie Hospital National Health Service Trust, Manchester, United Kingdom. r.booton@btopenworld.com (2006-06-01)
      BACKGROUND: The treatment of lung cancer has reached a therapeutic plateau. Several mechanisms of platinum resistance have been described, including the removal of platinum-DNA adduct by nucleotide excision repair (NER). Polymorphisms within the Xeroderma pigmentosum Group D protein (XPD), a member of the NER pathway, are associated with alterations in enzyme activity and may change sensitivity to platinum-based chemotherapy. The authors investigated the relation between XPD polymorphisms and treatment response, toxicity, and overall survival in patients who received platinum-based chemotherapy for advanced nonsmall cell lung cancer (NSCLC). METHODS: Between 2001 and 2002, 108 patients with chemotherapy-naive, advanced NSCLC were recruited. Associations between XPD312/751 polymorphisms and XPD haplotype and treatment response, toxicity. and survival were evaluated. RESULTS: Significant correlations were observed between XPD haplotype and Grade 4 neutropenia and overall survival together with a greater response to platinum-based chemotherapy for the XPD *A haplotype. CONCLUSIONS: The XPD haplotype may represent a useful pharmacogenomic marker of platinum-based chemotherapy in patients with advanced NSCLC and requires prospective validation.
    • The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage.

      Usui, Takeo; Maekawa, Hiromi; Pereira, Gislene; Schiebel, Elmar; The Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. (2003-09-15)
      The yeast protein Stu2 belongs to the XMAP215 family of conserved microtubule-binding proteins which regulate microtubule plus end dynamics. XMAP215-related proteins also bind to centrosomes and spindle pole bodies (SPBs) through proteins like the mammalian transforming acidic coiled coil protein TACC or the yeast Spc72. We show that yeast Spc72 has two distinct domains involved in microtubule organization. The essential 100 N-terminal amino acids of Spc72 interact directly with the gamma-tubulin complex, and an adjacent non-essential domain of Spc72 mediates binding to Stu2. Through these domains, Spc72 brings Stu2 and the gamma-tubulin complex together into a single complex. Manipulation of Spc72-Stu2 interaction at SPBs compromises the anchorage of astral microtubules at the SPB and surprisingly also influences the dynamics of microtubule plus ends. Permanently tethering Stu2 to SPBs by fusing it to a version of Spc72 that lacks the Stu2-binding site in part complements these defects in a manner which is dependent upon the microtubule-binding domain of Stu2. Thus, the SPB-associated Spc72-Stu2 complex plays a key role in regulating microtubule properties.
    • Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions.

      Maekawa, Hiromi; Usui, Takeo; Knop, Michael; Schiebel, Elmar; The Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. (2003-02-03)
      The budding yeast spindle aligns along the mother- bud axis through interactions between cytoplasmic microtubules (CMs) and the cell cortex. Kar9, in complex with the EB1-related protein Bim1, mediates contacts of CMs with the cortex of the daughter cell, the bud. Here we established a novel series of events that target Kar9 to the bud cortex. First, Kar9 binds to spindle pole bodies (SPBs) in G(1) of the cell cycle. Secondly, in G(1)/S the yeast Cdk1, Cdc28, associates with SPBs and phosphorylates Kar9. Thirdly, Kar9 and Cdc28 then move from the SPB to the plus end of CMs directed towards the bud. This movement is dependent upon the microtubule motor protein Kip2. Cdc28 activity is required to concentrate Kar9 at the plus end of CMs and hence to establish contacts with the bud cortex. The Cdc28-regulated localization of Kar9 is therefore an integral part of the program that aligns spindles.
    • Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery.

      Fischer, Tamás; Rodríguez-Navarro, Susana; Pereira, Gislene; Rácz, Attila; Schiebel, Elmar; Hurt, Ed; Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. (2004-09)
      Centrins are calmodulin-like proteins that function in the duplication of microtubule-organizing centres. Here we describe a new function of the yeast centrin Cdc31. We show that overproduction of a sequence, termed CID, in the carboxy-terminal domain of the nuclear export factor Sac3 titrates Cdc31, causing a dominant-lethal phenotype and a block in spindle pole body (SPB) duplication. Under normal conditions, the CID motif recruits Cdc31 and Sus1 (a subunit of the SAGA transcription complex) to the Sac3-Thp1 complex, which functions in mRNA export together with specific nucleoporins at the nuclear basket. A previously reported cdc31 temperature-sensitive allele, which is neither defective in SPB duplication nor Kic1 kinase activation, induces mRNA export defects. Thus, Cdc31 has an unexpected link to the mRNA export machinery.
    • The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal

      Maekawa, Hiromi; Priest, Claire; Lechner, Johannes; Pereira, Gislene; Schiebel, Elmar; Zentrum für Molekulare Biologie and 2Biochemie-Zentrum, Universität Heidelberg, 69120 Heidelberg, Germany. (2007-11-05)
      The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1-Bub2 guanosine triphosphatase-activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil-like molecule that interacts with the gamma-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.
    • Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments.

      Kiseleva, Elena; Allen, Terence D; Rutherford, Sandra A; Bucci, Mirella; Wente, Susan R; Goldberg, Martin W; Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK. m.w.goldberg@durham.ac.uk (2004-03)
      The nuclear pore complex (NPC) controls transport of macromolecules across the nuclear envelope. It is large and complex but appears to consist of only approximately 30 different proteins despite its mass of > 60MDa. Vertebrate NPC structure has been analyzed by several methods giving a comprehensive architectural model. Despite our knowledge of yeast nucleoporins, structural data is more limited and suggests the basic organization is similar to vertebrates, but may lack some peripheral and other components. Using field emission scanning electron microscopy to probe NPC structure we found that the yeast, like higher eukaryotic, NPCs contain similar peripheral components. We can detect cytoplasmic rings and evidence of nucleoplasmic rings in yeasts. A filamentous basket is present on the nucleoplasmic face and evidence for cytoplasmic filaments is shown. We observed a central structure, possibly the transporter, that which may be linked to the cytoplasmic ring by internal filaments. Immuno-gold labeling suggested that Nup159p may be attached to the cytoplasmic ring, whereas Nup116p may be associated, partly, with the cytoplasmic filaments. Analysis of a Nup57p mutant suggested a role in maintaining the stability of cytoplasmic components of the NPC. We conclude that peripheral NPC components appear similar in yeasts compared to higher organisms and present a revised model for yeast NPC structural composition.
    • ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response.

      Weston, Ria; Peeters, Hanneke; Ahel, Dragana; DNA Damage Response Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. (2012-07-15)
      To efficiently duplicate their genomic content, cells must overcome DNA lesions that interfere with processive DNA replication. These lesions may be removed and repaired, rather than just tolerated, to allow continuity of DNA replication on an undamaged DNA template. However, it is unclear how this is achieved at a molecular level. Here we identify a new replication-associated factor, ZRANB3 (zinc finger, RAN-binding domain containing 3), and propose its role in the repair of replication-blocking lesions. ZRANB3 has a unique structure-specific endonuclease activity, which is coupled to ATP hydrolysis. It cleaves branched DNA structures with unusual polarity, generating an accessible 3'-OH group in the template of the leading strand. Furthermore, ZRANB3 localizes to DNA replication sites and interacts with the components of the replication machinery. It is recruited to damaged replication forks via multiple mechanisms, which involve interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures. Collectively, our data support a role for ZRANB3 in the replication stress response and suggest new insights into how DNA repair is coordinated with DNA replication to maintain genome stability.
    • β2-syntrophin and Par-3 promote an apicobasal Rac activity gradient at cell-cell junctions by differentially regulating Tiam1 activity.

      Mack, Natalie A; Porter, Andrew P; Whalley, Helen J; Schwarz, J; Jones, R; Khaja, A; Bjartell, A; Anderson, K; Malliri, Angeliki; Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Manchester M20 4BX, UK. (2012-11)
      Although Rac and its activator Tiam1 are known to stimulate cell-cell adhesion, the mechanisms regulating their activity in cell-cell junction formation are poorly understood. Here, we identify β2-syntrophin as a Tiam1 interactor required for optimal cell-cell adhesion. We show that during tight-junction (TJ) assembly β2-syntrophin promotes Tiam1-Rac activity, in contrast to the function of the apical determinant Par-3 whose inhibition of Tiam1-Rac activity is necessary for TJ assembly. We further demonstrate that β2-syntrophin localizes more basally than Par-3 at cell-cell junctions, thus generating an apicobasal Rac activity gradient at developing cell-cell junctions. Targeting active Rac to TJs shows that this gradient is required for optimal TJ assembly and apical lumen formation. Consistently, β2-syntrophin depletion perturbs Tiam1 and Rac localization at cell-cell junctions and causes defects in apical lumen formation. We conclude that β2-syntrophin and Par-3 fine-tune Rac activity along cell-cell junctions controlling TJ assembly and the establishment of apicobasal polarity.