Role of mto2 in temporal and spatial regulation of cytoplasmic microtubule nucleation in Schizosaccharomyces pombe

The microtubule [MT] cytoskeleton of S. pombe is a highly dynamic network of filaments that facilitates intracellular transport, determines cell polarity and plays an essential role in chromosome separation during mitosis. In fission yeast, MTs are nucleated in a temporally and spatially regulated manner from sites called Microtubule Organising Centres [MTOCs], through the activity of both the g-tubulin complex [g-TuC] and the Mto1/2 complex. The Mto1/2 complex determines the localisation of the g-TuC at MTOCs, which change throughout the cell cycle. As cells enter mitosis the cytoplasmic array of MT bundles depolymerise. They are replaced by the intranuclear mitotic spindle and cytoplasmic spindle pole bodyderived astral MTs that in turn give way to the formation of the post-anaphase array. Although much is known about the properties of each type of MT array, the mechanism by which the timing of MT nucleation at different MTOCs is regulated over the cell cycle remains unclear. In the Mto1/2 complex, Mto1 is thought to provide the primary interaction with the g-TuC, and Mto2 functions by reinforcing this interaction. Due to the lack of structural information for the Mto1/2 complex, the molecular mechanism of Mto1/2- mediated assembly of the g-TuC at MTOCs is unknown. The aim of my study is to investigate the possibility that the Mto1/2 complex is able to promote g-TuC assembly by forming a direct template. In addition, I will attempt to determine the molecular role of Mto2 within the Mto1/2 complex and examine ways in which regulation of Mto2 may influence the function the Mto1/2 complex at specific MTOCs. As part of the investigation into the mechanism of Mto2 function, an in vitro analysis of recombinant protein demonstrated that in the absence of Mto1, purified Mto2 is able to self-interact as a tetramer. I have confirmed this interaction in vivo and have also shown that Mto2 forms a dimer as cells enter mitosis. However, in the context of an Mto1/2 complex the significance of the change in Mto2 oligomeric state remains unknown. Hydrodynamic analysis of a truncated form of the Mto1/2 complex suggests that it may form a heterotetramer, a hypothesis which is consistent with the equimolar levels of Mto2 and Mto1 protein within the cell. This information provides some structural insight as to how the Mto1/2 complex may interact with the g-TuC at MTOCs. Further analysis of the Mto1/2 complex revealed that in vivo, the Mto1-Mto2 interaction is disrupted during mitosis. This was found to correlate with the hyperphosphorylation of Mto2, which occurs as cells enter mitosis. Subsequently, an in vitro kinase assay demonstrated that phosphorylation of the Mto1/2 complex reduces the stability of the complex. Mass spectrometry techniques and sequence conservation were used to identify several phosphorylated residues within Mto2 and the ability of these mutants to bind to Mto1 was analysed in vivo and in vitro. In summary, in this study I have uncovered a mechanism which allows fission yeast cells to regulate the nucleation of cytoplasmic MT nucleation in a cell-cycle dependent manner, through a phosphorylation-dependent remodelling of the Mto1/2 complex

Two distinct regions of Mto1 are required for normal microtubule nucleation and efficient association with the gamma-tubulin complex in vivo

Cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe involves the interacting proteins Mto1 and Mto2, which are thought to recruit the γ-tubulin complex (γ-TuC) to prospective microtubule organizing centers. Mto1 contains a short amino-terminal region (CM1) that is conserved in higher eukaryotic proteins implicated in microtubule organization, centrosome function and/or brain development. Here we show that mutations in the Mto1 CM1 region generate mutant proteins that are functionally null for cytoplasmic microtubule nucleation and interaction with the γ-TuC (phenocopying mto1Δ), even though the Mto1-mutant proteins localize normally in cells and can bind Mto2. Interestingly, the CM1 region is not sufficient for efficient interaction with the γ-TuC. Mutation within a different region of Mto1, outside CM1, abrogates Mto2 binding and also impairs cytoplasmic microtubule nucleation and Mto1 association with the γ-TuC. However, this mutation allows limited microtubule nucleation in vivo, phenocopying mto2Δ rather than mto1Δ. Further experiments suggest that Mto1 and Mto2 form a complex (Mto1/2 complex) independent of the γ-TuC and that Mto1 and Mto2 can each associate with the γ-TuC in the absence of the other, albeit extremely weakly compared to when both Mto1 and Mto2 are present. We propose that Mto2 acts cooperatively with Mto1 to promote association of Mto1/2 complex with the γ-TuC

Activation of the γ-Tubulin Complex by the Mto1/2 Complex

SummaryThe multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites [5–10]. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC. This was achieved through the construction of a “minimal” version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and γ-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing ∼13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of γ-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast γ-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes

Role of mto2 in temporal and spatial regulation of cytoplasmic microtubule nucleation in Schizosaccharomyces pombe

The microtubule [MT] cytoskeleton of S. pombe is a highly dynamic network of filaments that facilitates intracellular transport, determines cell polarity and plays an essential role in chromosome separation during mitosis. In fission yeast, MTs are nucleated in a temporally and spatially regulated manner from sites called Microtubule Organising Centres [MTOCs], through the activity of both the g-tubulin complex [g-TuC] and the Mto1/2 complex. The Mto1/2 complex determines the localisation of the g-TuC at MTOCs, which change throughout the cell cycle. As cells enter mitosis the cytoplasmic array of MT bundles depolymerise. They are replaced by the intranuclear mitotic spindle and cytoplasmic spindle pole bodyderived astral MTs that in turn give way to the formation of the post-anaphase array. Although much is known about the properties of each type of MT array, the mechanism by which the timing of MT nucleation at different MTOCs is regulated over the cell cycle remains unclear. In the Mto1/2 complex, Mto1 is thought to provide the primary interaction with the g-TuC, and Mto2 functions by reinforcing this interaction. Due to the lack of structural information for the Mto1/2 complex, the molecular mechanism of Mto1/2- mediated assembly of the g-TuC at MTOCs is unknown. The aim of my study is to investigate the possibility that the Mto1/2 complex is able to promote g-TuC assembly by forming a direct template. In addition, I will attempt to determine the molecular role of Mto2 within the Mto1/2 complex and examine ways in which regulation of Mto2 may influence the function the Mto1/2 complex at specific MTOCs. As part of the investigation into the mechanism of Mto2 function, an in vitro analysis of recombinant protein demonstrated that in the absence of Mto1, purified Mto2 is able to self-interact as a tetramer. I have confirmed this interaction in vivo and have also shown that Mto2 forms a dimer as cells enter mitosis. However, in the context of an Mto1/2 complex the significance of the change in Mto2 oligomeric state remains unknown. Hydrodynamic analysis of a truncated form of the Mto1/2 complex suggests that it may form a heterotetramer, a hypothesis which is consistent with the equimolar levels of Mto2 and Mto1 protein within the cell. This information provides some structural insight as to how the Mto1/2 complex may interact with the g-TuC at MTOCs. Further analysis of the Mto1/2 complex revealed that in vivo, the Mto1-Mto2 interaction is disrupted during mitosis. This was found to correlate with the hyperphosphorylation of Mto2, which occurs as cells enter mitosis. Subsequently, an in vitro kinase assay demonstrated that phosphorylation of the Mto1/2 complex reduces the stability of the complex. Mass spectrometry techniques and sequence conservation were used to identify several phosphorylated residues within Mto2 and the ability of these mutants to bind to Mto1 was analysed in vivo and in vitro. In summary, in this study I have uncovered a mechanism which allows fission yeast cells to regulate the nucleation of cytoplasmic MT nucleation in a cell-cycle dependent manner, through a phosphorylation-dependent remodelling of the Mto1/2 complex.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mre11 Dimers Coordinate DNA End Bridging and Nuclease Processing in Double-Strand-Break Repair

SummaryMre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD)

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining