The role of PFK in the oscillatory yeast strain X2180

Muiyser, S. M. (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: The elucidation of mechanisms involved in glycolytic oscillations in yeast can lead to a deeper understanding of the metabolic organisation in living systems. These oscillations, detected in the concentrations of the glycolytic intermediates of yeast, are a well-studied topic in the context of cell populations and extracts. In a recent study sustained oscillations were detected in individually placed single cells of the X2180 yeast strain in a microfluidic flow-chamber. Literature states that the specific regulation of the phosphofructokinase (PFK) enzyme by the metabolites AMP and ATP are a necessary feature in the glycolytic pathway that enables these oscillations. This regulation was also observed in a previous study where a mathematical model for the oscillatory system was created by numerical optimisation of enzyme kinetic parameters in an experimentally validated detailed model of steady state glycolysis. One of the results of the optimisation was an increased inhibition of PFK by its substrate, ATP. The aim of the current study was to perform an experimental investigation into the enzyme kinetic characteristics of phosphofructokinase in the X2180 oscillatory strain, as well as in the VIN13 strain, which has not been tested for oscillations. To address this, we used wide-field microscopy to detect oscillations in the X2180 strain and to test for oscillations in VIN13, when they were both cultured under conditions that have been shown to elicit oscillations in X2180. We optimised a protocol for inducing oscillations in our local microscope setup, and subsequently detected oscillations in X2180, and not in VIN13. We were able to optically resolve oscillations on the single cell level which will allow for the investigation of synchronization between cells (a feature that is observed in oscillating populations) in future studies. We then performed a kinetic characterisation of the phosphofructokinase enzyme in both yeast strains to test whether differences in its regulatory characteristics could be the reason for oscillations in one strain and not the other. We found that there are differences in PFK kinetic parameters of these two strains. Finally, by using our experimentally determined PFK kinetic parameters in a non-oscillating mathematical model, and following the previously published optimisation steps for other enzymes, we were able to simulate oscillations for the X2180 strain but could not do so for VIN13. From this we conclude that VIN13 requires further kinetic characterisation to be able to determine if it exhibits oscillatory behaviour.

AFRIKAANSE OPSOMMING: Die toeligting van meganismes betrokke by glikolitiese ossillasies in gis kan aanleiding gee tot ’n dieper insig van die metaboliese organisasie van lewende sisteme. Hierdie ossillasies, wat in die konsentrasies van die tussenprodukte van glikolise waargeneem word, is ’n goed-bestudeerde onderwerp in die konteks van selpopulasies en ekstrakte. In ’n onlangse studie is volgehoue ossillasies waargeneem in individueel-geplaasde enkelselle van die X2180 gisstam, in ’n mikrofluïde vloeikamer. Bestaande literatuur wys dat die regulasie van die fosfofruktokinase (PFK) ensiem deur die metaboliete AMP en ATP ’n noodsaaklike kenmerk is in die glikolitiese padweg wat hierdie ossillasies moontlik maak. Hierdie regulasie is ook opgemerk in ’n vorige studie waar ’n wiskundige model van die ossillerende sisteem deur numeriese optimering van ensiemkinetiese parameters in ’n eksperimenteel-gevalideerde model van glikolise in ’n bestendige toestand uitgevoer is. Een van die resultate van die optimering was ’n verhoogde inhibisie van PFK deur sy substraat, ATP. Die doelstelling van die huidige studie was om ’n eksperimentele ondersoek na die ensiem-kinetiese eienskappe van PFK in die ossillerende X2180 stam, asook die VIN13 stam, wat nog nie vir ossillasies getoets is nie, in te stel. Ons het wye-veld mikroskopie gebruik om ossillasies in die X2180 stam te gewaar en te toets vir ossillasies in die VIN13 stam. Beide kulture is gekweek in toestande wat voorheen bewys is om ossillasies in X2180 te ontlok. Ons het ’n protokol om ossillasies te voorskyn te roep in ons mikroskoop opstelling geoptimeer, en ossillasies in X2180 waargeneem, maar nie in VIN13 nie. Dit was moontlik om ossillasies op enkel-selvlak opties te ontleed, wat voorsiening maak vir die verdere ondersoek van die sinkronisasie tussen selle (’n kenmerk wat waargeneem word in ossillerende populasies) in toekomstige studies. ’n Kinetiese karakterisering van PFK in beide gisstamme is uitgevoer om te toets of verskille in die regulatoriese eienskappe die oorsaak kan wees van ossillasies in die een stam en nie in die ander nie. Ons het gevind dat daar verskille tussen die kinetiese parameters van PFK van die twee stamme is. Ten slotte, deur gebruik te maak van ons eksperimenteel vasgestelde kinetiese parameters van PFK in ’n nie-ossillerende wiskundige model, en die vooraf gepubliseerde optimeringstappe vir ander ensieme te gebruik, was ons in staat om ossillasies vir die X2180 stam te simuleer, maar nie vir die VIN13 stam nie. Hieruit kan ons aflei dat VIN13 verdere kinetiese karakterisering vereis om te kan bepaal of dit ossillerende gedrag besit.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105956
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