• 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.