Measuring and modelling autophagic flux

Du Toit, Andre (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Introduction. Autophagy is a dynamic process that is responsible for cellular protein degradation, which involves sequestering of bulk cytoplasm and its delivery to lysosomes where degradation and recycling occurs. Autophagy is vital for cellular function and can be induced during periods of nutrient deprivation for the recycling of proteins and for removing potentially harmful proteins and organelles. A reduction in the autophagic degradative capacity has been linked to several diseases such as those associated with neurodegeneration. These attributes make autophagy an attractive therapeutic target; clinical trials using autophagy inducers have already shown promising results. In order to successfully exploit autophagy, it is crucial to determine whether the autophagic flux is too high or too low, and adjust it accordingly. However, the accurate measurement of autophagic flux still remains a challenge. Aims. The aim of this project was therefore, rst, to develop a novel method to accurately measure autophagic ux. Second, to assess autophagy using conventional techniques and compare it with the new approach. Our third aim was to construct a kinetic model of the autophagic system that could simulate our experimentally generated data and thereby help us understand the contribution of the different processes involved in autophagy and its dynamic behaviour. Methods. We made use of uorescent-based imaging to acquire z-stack images of mouse embryonic broblasts that stably express GFP-LC3. Images were processed and the total autophagic vesicles pool size was measured using ImageJ with the WatershedCounting3D plugin. Cells were cultured in the presence of an acidotrophic uorescent dye that allows (in-combination with GFP-LC3) the visualisation of autophagosomes, autophagolysosomes and lysosomes. Cells were encased in a humidi ed atmosphere in the presence of 5% CO2 at 37 C in a microscope slide of the IX81 Olympus microscope. First we determined the concentration of ba lomycin A1 required for the complete inhibition of the autophagosome and lysosome fusion process. We calculated the autophagic ux as the initial rate of increase in the number of autophagosomes after inhibition of fusion. Second, we increased autophagosomal synthesis through induction with 25 nM rapamycin and again calculated the autophagic ux from the initial rate of increase in autophagosomes after fusion inhibition. In parallel, we assessed changes in the autophagic markers LC3-II and p62 with Western blot analysis and in the morphology of autophagic vesicles with electron microscopy at time points suggested by the uorescent experimental data. A kinetic model of the autophagic system was constructed and parameterised so as to t the experimental data. Computational modelling was done with the Python Simulator for Cellular Systems (PySCeS). Results. Although we found that 100 nM ba lomycin A1 was su cient to inhibit the fusion of autophagosomes and lysosomes, we chose to use 400 nM ba lomycin A1 in order to be absolutely sure the inhibition was complete. Induction of autophagosomal synthesis with 25 nM rapamycin increased the autophagic ux in MEF cells from its basal value of 25.4 autophagosomes/cell/hr to 105.4 autophagosomes/cell/hr. The transition time, i.e., the time required to clear the autophagosomal pool, decreased from its basal value of 0.53 hr to 0.16 hr after induction. Similarly the transition times for the basal and induced autophagolysosomal pools were 6.7 hr and 2.4 hr. Whereas with our uorescence microscopy method we measured a four-fold increase in autophagic ux from the basal to the induced state, traditional approaches such as Western blot analysis measure only a two-fold increase; electron microscopy proved to be inadequate for assessing autophagic vesicles. Autophagosomes constituted a small percentage of the total GFP-LC3-positive vacuoles. Upon induction with rapamycin the number of autophagosomes/cell increased slightly from 13 to 17, whereas the number of autophagolysosomes/cell increased considerably from 165 to 251. Autophagosomal size was about four times smaller than autophagolysosomal size. Simulating the autophagic system with our kinetic model provided an excellent t to the experimental data. Conclusion. Our novel approach quanti es autophagic variables such as the ux and the number of the di erent types of autophagic vesices accurately at single cell level, and, used in combination with kinetic modelling of the dynamics of autophagy, hold promise for future therapeutic application.

AFRIKAANSE OPSOMMING: Inleiding. Autofagie is 'n dinamiese proses wat verantwoordelik is vir sellul^ ere prote en degradasie, 'n proses waarin sitoplasma in vesikels gesekwestreer word en na lisosome afgelewer word waar degradasie en herwinning plaasvind. Autofagie is noodsaaklik vir sellul^ere funksie en kan ge nduseer word tydens periodes van voedingstoftekorte vir die herwinning van prote ene vir energiedoeleindes en as 'n meganisme vir die verwydering van potensieel skadelike prote ene en organelle. 'n Afname in die autofagiese degradasiekapasiteit is al geassosieer met verskeie siektes soos neurodegenerasie. Hierdie eienskappe maak autofagie 'n aantreklike terapeutiese teiken. Studies met induseerders van autofagie het reeds belowende resultate in kliniese proewe getoon. Om autofagie suksesvol te benut is dit noodsaaklik om te bepaal of die autofagiese uksie te hoog of te laag is, en om dit dan dienooreenkomstig aan te pas. Die akkurate meting van autofagiese fluksie was egter tot nou toe 'n uitdaging. Doel. Die doel van hierdie projek was om, eerstens,'n nuwe metode te ontwikkel om autofagiese uksie akkuraat te meet. 'n Tweede doel was om autofagie met konvensionele tegnieke te assesseer en dan met die nuwe benadering te vergelyk. Die derde doel was om 'n kinetiese model van die autofagie sisteem te bou wat die eksperimentele data kan simuleer en ons sodoende help om die bydrae van die verskillende prosesse betrokke by autofagie tot die dinamiese gedrag van autofagie te verstaan. Metodes. Fluoressensie-gebaseerde afbeelding is gebruik om z-stapel beelde van muis embrioniese broblaste wat stabiele GFP-LC3 uitdruk te verkry. Hierdie beelde is verwerk en die totale autofagiese vesikelpoelgrootte is bepaal met ImageJ en dieWatershedCounting 3D sisteem. Selle is gekweek in die teenwoordigheid van 'n asidotro ese uoresserende kleurstof wat, in kombinasie met GFP-LC3, die visualisering van autofagosome, autofagolisosome en lisosome moontlik maak. Selle is omhul in 'n gehumidi seerde atmosfeer in die teenwoordigheid van 5% CO2 by 37 C in 'n mikroskoopsky e van die Olympus IX81 mikroskoop. Ons het eers die konsentrasie van ba lomisien A1 nodig om die fusie van autofagosome en lisosome volkome te inhibeer bepaal. Die autofagiese uksie is toe bereken as die aanvanklike snelheid waarmee autofagosome toeneem na inhibisie van fusie. Daarna het ons autofagosomale sintese verhoog deur induksie met 25 nM rapamisien en weer die uksie gemeet as die aanvanklike snelheid waarmee autofagosome toeneem na inhibisie van fusie. Parallel aan hierdie eksperimente het ons die veranderings in die autofagiese merkers LC3-II en p62 met Westernklad analise en die veranderings in die morfologie van autofagiese vesikels met elektronmikroskopie bepaal by tydspunte afgelei uit die uoressensie eksperimentele data. Rekenaarmatige modellering is gedoen met die Python Simulator for Cellular Systems (PySCeS). Resultate. Alhoewel ons gevind het dat 100 nM ba lomisien A1 voldoende was om die fusie van autofagosome en lisosome te inhibeer, het ons 400 nM ba lomisien gebruik om absoluut seker te maak dat die inhibisie volledig was. Induksie van autofagosomale sintese met 25 nM rapamisien het die autofagiese uksie in MEF selle van die basale waarde van 25.4 autofagosome/sel/uur na 105.4 autofagosome/sel/uur verhoog. Die transisietyd, nl. die tyd nodig om die autofagosomale poel te vervang, het verminder van die basale waarde van 0.53 uur na die ge nduseerde waarde van 0.16 uur. Soortgelyk het die transisietyd vir die autofagolisosomale poel afgeneem van 6.7 uur voor induksie and 2.4 uur na induksie. Waar ons met die uoressensie mikroskopie metode 'n viervoudige toename in autofagiese uksie gemeet het van basale na ge nduseerde vlakke, het tradisionele metodes soosWesternklad analise slegs 'n tweevoudige verhoging gemeet; elektronmikroskopie was nie bevoeg om autofagiese vesikels te assesseer nie. Autofagosome het maar 'n klein persentasie van die totale GFP-LC3-positiewe vakuole uitgemaak. Na induksie met rapamisien het die aantal autofagosome/sel e ens verhoog van 13 na 17, terwyl die aantal autofagolisosome/sel aansienlik verhoog het van 165 na 251. Die grootte van autofagosome was ongeveer vier maal kleiner as die van autofagolisosome. Simulering van die autofagiese sisteem met ons kinetiese model het uitstekend op die eksperimentele data gepas. Gevolgtrekking. Ons nuwe benadering kwanti seer autofagiese veranderlikes soos uksie en die aantal van die verskillende tipes autofagiese vesikels akkuraat op enkelselvlak, en hou, in kombinasie met kinetiese modellering van die dinamika van autofagie, belofte in vir toekomstige terapeutiese toepassings.

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