Cell cycle dependent regulation of the Dam1 kinetochore complex by Cdk1

Kinetochore ankern Plus-Enden der Mikrotubuli an die Zentromere und übertragen deren Dynamik in die direkte Bewegung der Chromosomen. Es wurde gezeigt, daß der Hefe- spezifische Dam1-Komplex sowohl strukturelle als auch regulatorische Elemente beinhaltet, die zur Kinetochor-Mikrotubulikontaktfläche in der Bäckerhefe beitragen (Lampert and Westermann, 2011). Mutationen, die die Funktion dieses Komplexes in vivo beeinträchtigen, führen zu Chromosomen-Missegregation und zu gravierenden Spindeldefekten (Cheeseman et al., 2001a; Janke et al., 2002). Während die Rolle des Komplexes in Bezug auf das Etablieren von Kinetochore-Mikrotubuliverbindungen ausgiebig erforscht ist, ist es nach wie vor nicht klar wie der Komplex zur Erhaltung der Spindelintegrität beiträgt. Ein Hinweis für den Mechanismus, der diesem Phänotypen unterliegt, kommt von einer neuesten Studie, in der gezeigt wurde, daß Cdc28 (Cdk1)-abhängige Phosphorylierung des Dam1-Komplexes die Dynamik der Mikrotubuli in der Metaphase und Anaphase A beeinflußt (Higuchi and Uhlmann, 2005). Die Aufreiningung des Dam1-Komplexes aus Hefeextrakten und dessen weiterführende massenspektrometrische Analyse enthüllte weitere Cdk1-Phosphorylierungsstellen. Diese Entdeckung machte es möglich eine übergreifende Studie in Bezug auf die Regulierung des Dam1-Komplexes durch Phosphorylierung sowohl in vivo als auch in vitro durchzuführen. Zu diesem Zweck wurde eine Serie an rekombinanten Dam1 Phosphomutanten darauf getestet, in wie fern die Haupteigenschaften des Komplexes wie dessen Stabilität, dessen Fähigkeit Ringe auszubilden und dessen Mikrotubulibindungsaffinität durch Phosphorylierung beeinflußt werden. Weiters wurden Hefestämme, in denen die spezifischen Phosphorylierungsstellen mutiert waren, weitgehend untersucht, um deren in vivo Effekt zu charakterisieren. Dabei wurde spezielles Augenmerk auf die Veränderungen der Dam1-Komplexlokalisierung als auch der Spindeldynamik gelegt. Diese Untersuchungen weisen darauf hin, daß die Phosphoregulierung des Komplexes Verbindungen, die sich zwischen zwei Dam1- Komplexen befinden, und dadurch die Ringbildung beeinflußt, was sich in einer veränderten Spindeldynamik äußert. Dieser regulatorische Mechanismus, der der Funktion des Dam1-Komplexes unterliegt, bekräftigt die Vorstellung der Existenz des Dam1-Ringmodels in vivo.Kinetochores anchor microtubule (MT) plus-ends at the centromere and translate their dynamics into directed movement of chromosomes during mitosis. The fungal-specific Dam1 complex was previously reported to constitute a major structural and regulatory element of the kinetochore-microtubule interface in budding yeast (Lampert and Westermann, 2011). Mutations that cripple Dam1 function in vivo phenotypically result in chromosome mis- segregation and severe spindle defects (Cheeseman et al., 2001a; Janke et al., 2002). While the role of the complex in establishing kinetochore-microtubule attachments has been intensively studied it is less clear how Dam1 directly contributes to maintenance of spindle integrity. A mechanistic hint comes from a previous study that demonstrated that Cdc28 (Cdk1)-dependent phospho-regulation of Dam1 influences microtubule dynamics in metaphase and anaphase A after artificially induced sister-chromatid separation (Higuchi and Uhlmann, 2005). Purification of the Dam1 complex from yeast extracts and further mass-spec analysis revealed additional Cdk1-sites allowing to conduct a comprehensive study on Dam1 phospho- regulation in vitro and in vivo. To this end, a series of recombinant Dam1 phospho-mutants have been probed for changes in major Dam1 complex properties such as complex stability, ring formation and microtubule binding affinity. The generation of several Dam1 phospho- mutant yeast strains allowed to characterize the effects in vivo by monitoring changes in Dam1 complex localization patterns and measuring altered spindle microtubule dynamics. The data suggest that Cdk1 phospho-regulation influences inter-complex connections and thereby ring formation that influences the dynamics of the mitotic spindle. This regulatory mechanism of Dam1 complex function strengthens the argument for the existence of the Dam1 ring model in vivo

Validation of a novel analytical method for metabolomics and exposomics – an essay

Im Zentrum dieser Arbeit steht eine neuartige instrumentelle HPLC-HRMS Methode, welche für die simultane Analyse von (hydrophilem) Metabolom und (internem) Exposom entwickelt wurde. Die gleichzeitige Erfassung endogener Metabolite und weniger polarer Xenobiotika wird durch Erweiterung des Polaritätsspektrums mittels paralleler HILIC- und RP-Chromatographie ermöglicht, ohne dadurch die Laufzeit der Analyse zu erhöhen. Der Auftrag der Arbeit besteht darin, die Methode so weit als möglich nach behördlichen Empfehlungen zu validieren, wie sie im Rahmen klinischer Studien verlangt werden. Die Leitlinie Bioanalytical Method Validation – Guidance for Industry der U.S.-amerikaischen Lebensmittelüberwachungs- und Arzneimittelbehörde U.S. Food and Drug Administration (U.S. FDA) dient dabei als Orientierungshilfe zur Einschätzung der behördlichen Erwartungen. Durch Auswertung bereits vorhandenen Datenmaterials gelingt eine grundlegende Charakterisierung der Methode, welche die Eignung für die kombinierte metabolomics-exposomics-Forschung untermauert. Das besonderes Augenmerk der Untersuchung gilt dabei den Vorteilen, aber auch möglichen Komplikationen durch den 13C-markierten Pichia pastoris-Zellextrakt, der hier als interner Multikomponentenstandard zur Anwedung kommt. Eine vollständige Validierung nach U.S. FDA kann letzten Endes nicht erzielt werden. Die Gründe dafür – validierungsstrategische wie metabolomics-spezifische – werden in der Arbeit ausführlich diskutiert. Möglicherweise können künftige Validierungsvorhaben davon profitieren.The central motif of this work is to face the challenge of validating an absolute quantification proce-dure based on a newly developed LC-MS method for simultaneous metabolomics and exposomics analysis in a way that approximates the validation needs for a clinical study. The presented method is a novel HPLC-HRMS approach employing simultaneous chromatography under complementing elu-tion conditions: Hydrophilic interaction liquid chromatography (HILIC) provides good separation con-ditions for the more hydrophilic metabolites, whereas reversed phase (RP) chromatography poses a better environment for analyzing compounds of higher lipophilicity, a property quite common for small molecule pharmaceuticals. To get a sense of a medicines authority’s regulatory expectations of analytical method validation, the U.S. FDA’s guideline Bioanalytical Method Validation – Guidance for Industry is sought for advice. The guideline conveniently suggests strategies how to evaluate the ca-pabilities of a chromatographic method. It serves as a starting point for approaching the specific is-sues of an HPLC-HRMS metabolomics method. An already acquired data set is exploited to gain deeper insight into the capabilities and weak spots of the novel dual-chromatography HPLC-HRMS method and in deed suggests its versatility for com-bined metabolomics and exposomics research. With the exception of a few targeted metabolites with overlapping HILIC and RP peaks, the 2D chromatography concept saves time without considera-ble drawbacks for the quality of the analysis. This work especially maps out the benefits of internal standardization with a fully 13C-labelled metabolite extract from Pichia pastoris while, on the other hand, also discussing its possible pitfalls. Analyzing the data arises questions that will have to be ad-dressed by further research. Linking basic university research with a highly regulated application environment is an ambitious endeavor, which, in the course of this work, turned out to be hardly feasible for the current state of method development. Nonetheless, the thesis collates general issues specific for a full validation of a metabolomics method according to medicines authorities’ provisions. Among them are the complexi-ty of matrix effect assessment and the problem that an approach via blank matrix, as suggested by the guideline, is not viable for metabolomics. Internal standardization with a fully 13C-labeled yeast cell extract is presented as a possible solution. Dedicated evaluation is needed but lies beyond the scope of this thesis

Integrated exposomics/metabolomics for rapid exposure and effect analyses

The totality of environmental exposures and lifestyle factors, commonly referred to as the exposome, are poorly understood. Measuring the myriad of chemicals that humans are exposed to is immensely challenging and identifying disrupted metabolic pathways is even more complex. Here, we present a novel technological approach for the comprehensive, rapid and integrated analysis of the endogenous human metabolome and the chemical exposome. By combining reverse-phase and hydrophilic interaction liquid chromatography and fast polarity switching, molecules with highly diverse chemical structures can be analyzed in 15 minutes with a single analytical run as both column’s effluents are combined before analysis. Standard reference materials and authentic standards were evaluated to critically benchmark performance. Highly sensitive median limits of detection (LOD) with 0.04 µM for >140 quantitatively assessed endogenous metabolites and 0.08 ng/mL for the >100 model xenobiotics and human estrogens in solvent were obtained. In matrix, the median LOD values were higher with 0.7 ng/mL (urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the dual-column approach’s applicability, real-life urine samples from sub-Saharan Africa (high exposure scenario) and Europe (low exposure scenario) were assessed in a targeted and non-targeted manner. Our LC-HRMS approach demonstrates the feasibility of quantitatively and simultaneously assessing the endogenous metabolome and the chemical exposome for the high-throughput measurement of environmental drivers of disease

Integrated Exposomics/Metabolomics for Rapid Exposure and Effect Analyses

The totality of environmental exposures and lifestyle factors, commonly referred to as the exposome, is poorly understood. Measuring the myriad of chemicals that humans are exposed to is immensely challenging, and identifying disrupted metabolic pathways is even more complex. Here, we present a novel technological approach for the comprehensive, rapid, and integrated analysis of the endogenous human metabolome and the chemical exposome. By combining reverse-phase and hydrophilic interaction liquid chromatography (HILIC) and fast polarity-switching, molecules with highly diverse chemical structures can be analyzed in 15 min with a single analytical run as both column’s effluents are combined before analysis. Standard reference materials and authentic standards were evaluated to critically benchmark performance. Highly sensitive median limits of detection (LODs) with 0.04 μM for >140 quantitatively assessed endogenous metabolites and 0.08 ng/mL for the >100 model xenobiotics and human estrogens in solvent were obtained. In matrix, the median LOD values were higher with 0.7 ng/mL (urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the dual-column approach’s applicability, real-life urine samples from sub-Saharan Africa (high-exposure scenario) and Europe (low-exposure scenario) were assessed in a targeted and nontargeted manner. Our liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach demonstrates the feasibility of quantitatively and simultaneously assessing the endogenous metabolome and the chemical exposome for the high-throughput measurement of environmental drivers of diseases

Integrated Exposomics/Metabolomics for Rapid Exposure and Effect Analyses

The totality of environmental exposures and lifestyle factors, commonly referred to as the exposome, is poorly understood. Measuring the myriad of chemicals that humans are exposed to is immensely challenging, and identifying disrupted metabolic pathways is even more complex. Here, we present a novel technological approach for the comprehensive, rapid, and integrated analysis of the endogenous human metabolome and the chemical exposome. By combining reverse-phase and hydrophilic interaction liquid chromatography (HILIC) and fast polarity-switching, molecules with highly diverse chemical structures can be analyzed in 15 min with a single analytical run as both column’s effluents are combined before analysis. Standard reference materials and authentic standards were evaluated to critically benchmark performance. Highly sensitive median limits of detection (LODs) with 0.04 μM for >140 quantitatively assessed endogenous metabolites and 0.08 ng/mL for the >100 model xenobiotics and human estrogens in solvent were obtained. In matrix, the median LOD values were higher with 0.7 ng/mL (urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the dual-column approach’s applicability, real-life urine samples from sub-Saharan Africa (high-exposure scenario) and Europe (low-exposure scenario) were assessed in a targeted and nontargeted manner. Our liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach demonstrates the feasibility of quantitatively and simultaneously assessing the endogenous metabolome and the chemical exposome for the high-throughput measurement of environmental drivers of diseases