Multi-Scale Structure-Function Analysis of Mitochondrial Network Morphology and Respiratory State in Budding Yeast
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UNIVERSITY OF CALIFORNIA, IRVINE Multi-scale Structure-function Analysis of Mitochondrial Network Morphology and Respiratory State in Budding Yeast DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Engineering by Swee Siong Lim Dissertation Committee: Assistant Professor Susanne Rafelski, Chair Associate Professor Elliot Botvinick Professor Suzanne Sandmeyer Professor Vasan Venugopalan 2015 © 2015 Swee Siong Lim DEDICATION To my parents. ii TABLE OF CONTENTS Page LIST OF FIGURES vi LIST OF TABLES viii ACKNOWLEDGMENTS ix CURRICULUM VITAE x ABSTRACT OF THE DISSERTATION xii 1 Introduction 1 1.1 Mitochondrial structure .............................. 2 1.2 Mitochondrial function ............................... 3 1.3 The link between structure and function in mitochondrial remodeling ... 4 1.4 Pathological consequences of mitochondrial damage .............. 6 1.5 Motivation and goal of thesis ........................... 8 1.6 Budding yeast as a model organism for studying structure-function rela- tionship ........................................ 9 1.7 Overview of thesis ................................. 9 2 Structure-function mapping pipeline 12 2.1 Introduction ..................................... 13 2.1.1 Development of a structure function mapping pipeline ........ 15 2.2 Materials and Methods ............................... 16 2.2.1 Spinning disk microscopy platform ................... 16 2.2.2 Strain construction for visualization of matrix structure ........ 17 2.2.3 Cell preparation and loading of functional dye ............. 19 2.2.4 Image microscopy pipeline ........................ 22 2.2.5 Data preparation before input into pipeline ............... 25 2.2.6 Artifacts that may arise when mapping ΔΨ to mitochondrial network 26 2.2.7 Pipeline to map ΔΨ to mitochondrial network with normalization and scaling to control artifacts ...................... 27 2.2.8 Data wrangling – database structure ................... 28 iii 3 Modulating metabolic state 32 3.1 Introduction ..................................... 33 3.1.1 Parameters of the OXPHOS process ................... 35 3.1.2 Mitochondrial membrane potential (ΔΨ) as a bioenergetic indicator . 38 3.1.3 Oxygen consumption measurement of cellular respiration ...... 39 3.1.4 Variation of carbon source substrates and their expected bioener- getic measurements ............................ 40 3.2 Materials and Methods ............................... 41 3.2.1 O2 consumption rate measurement using a Clark electrode ...... 41 3.2.2 OCR measurement protocols ....................... 43 3.3 Results ........................................ 45 3.4 Discussion ...................................... 48 4 Membrane potential heterogeneity at the mitochondrial tubule level 53 4.1 Introduction ..................................... 54 4.2 Materials and Methods ............................... 57 4.2.1 Sampling of random distributions .................... 57 4.2.2 Autocorrelation curves ........................... 59 4.2.3 Power spectral density ........................... 61 4.2.4 Delta intensity ΔI(k) ............................ 64 4.2.5 Statistical testing with post-hoc multiple testing correction ...... 66 4.3 Results ........................................ 67 4.3.1 Mitochondrial tubules have nonrandom heterogeneity of ΔΨ .... 67 4.3.2 Mitochondrial tubules in respiratory conditions have less correla- tion of ΔΨ at large length scales compared to fermentative conditions 69 4.3.3 Mitochondrial tubules in respiratory conditions have thicker width and more uniform distribution of thickness compared to fermenta- tive conditions ............................... 70 4.4 Discussion ...................................... 74 5 Membrane potential heterogeneity at the mitochondrial network level 78 5.1 Introduction ..................................... 79 5.2 Materials and Methods ............................... 84 5.2.1 Data structure ................................ 84 5.2.2 Surface density as a measure of spatial density of mitochondria ... 85 5.2.3 Global and local measures of connectivity ................ 85 5.2.4 Statistical testing between conditions .................. 88 5.3 Results ........................................ 88 5.3.1 Mitochondrial surface density scales with network connectivity ... 88 5.3.2 Mitochondrial surface density does not correlate with ΔΨ or con- nectivity ................................... 91 5.3.3 Mitochondria with similar surface densities do not show a differ- ence in correlation with ΔΨ or connectivity measures ......... 92 5.3.4 Branchpoint regions have similar ΔΨ to non branchpoint regions .. 94 iv 5.3.5 ΔΨ of isolated mitochondrial fragments are no different from the rest of the network ............................. 96 5.4 Discussion ...................................... 99 6 Membrane potential heterogeneity at the cellular level 105 6.1 Introduction ..................................... 106 6.2 Materials and Methods ............................... 108 6.2.1 Picking of points to define the mother-bud axis ............ 108 6.2.2 Direction cosine based transformation matrix to realign the mother-bud cellular axis .......................... 109 6.2.3 Tracking functional heterogeneity during budding progression ... 114 6.3 Results ........................................ 115 6.3.1 Buds have mitochondria with higher ΔΨ compared to mother cells . 115 6.3.2 Mitochondria display different gradients of ΔΨ along the mother- bud axis ................................... 117 6.3.3 Mitochondrial ΔΨ asymmetry is maintained during the budding progression ................................. 118 6.4 Discussion ...................................... 122 7 Significance and future direction 125 7.1 Significance ..................................... 125 7.2 Improved spatial resolution ............................ 126 7.3 Genetically encoded functional sensors ..................... 128 7.4 Refinement in experimental setup ......................... 129 Bibliography 131 A Supplementary figures 144 B Statistical tables 147 C Database variables 155 D Source codes 158 D.1 Structure-function pipeline modules ....................... 158 D.2 Multi-scale database module ........................... 164 D.3 Mother-daughter transformation module .................... 170 v LIST OF FIGURES Page 2.1 Schematic diagram of the spinning disk microscope platform. ........ 17 2.2 Excitation-emission spectra of dsRed mitochondrial matrix marker ..... 18 2.3 Comparison of crosstalk levels in mRuby2 and dsRed. ............. 20 2.4 Plasmid construction details for pvt100-mRuby2 for cloning into SRY-124. 21 2.5 Glas18 surface treatment to improve signal to noise ratio ........... 22 2.6 Comparison of sequential channel vs sequential stack switching scheme. .. 24 2.7 ROI tracing for a budding yeast cell. ....................... 25 2.8 Structure-function mapping pipeline ....................... 29 2.9 Multi-scale structure-function database ..................... 31 3.1 Components of OXPHOS respiration ....................... 35 3.2 Respiration – PMF relationship of mitochondria ................ 37 3.3 Proportion of respiration from the input and outputs of ETC/OXPHOS ... 38 3.4 Definition of State 3, basal and State 4 respiration levels ............ 39 3.5 Diagram of a Clark electrode used for oxygen consumption rate (OCR) measurement .................................... 42 3.6 Raw oxygen consumption rate (OCR) data from one sampling run at a particular carbon source and OD600 reading. .................. 45 3.7 Respiration rate (OCR) as a function of optical density (OD600). ....... 46 3.8 Oxygen consumption rate (OCR) of yeast in different carbon sources. .... 47 3.9 Relationship between mitochondrial OCR, membrane potential (ΔΨ) and amount density (volume ratio) of yeast grown in different carbon sources. 49 4.1 Inner mitochondrial membrane structure contributes to ΔΨ heterogeneity . 55 4.2 Tubule level ΔΨ heterogeneity in actual and random distributions ...... 58 4.3 Autocorrelation curves of actual vs random distributions ........... 60 4.4 Autocorrelation curves of populations of cells growing in different carbon sources ........................................ 61 4.5 Power spectral density of actual vs random distributions ........... 62 4.6 Power spectral density of populations of cells growing in different carbon sources ........................................ 63 4.7 Delta intensity ΔI(k) curves ............................ 65 4.8 ΔI(k) actual for tubule populations growing in different carbon sources ... 66 4.9 Distribution for ΔI(k) for k=1 ........................... 69 4.10 Tubule thickness variation is not an artifact of matrix intensities ....... 71 vi 4.11 Distribution of mean tubule diameter per cell per population ......... 72 4.12 Autocorrelation and ΔI(k) curves of tubule diameter heterogeneity ..... 73 4.13 Mitochondria ultrastructure changes due to respiratory state ......... 76 5.1 Heterogeneity of structure-function in mitochondrial networks ........ 80 5.2 Global and local connectivity in mitochondrial networks ........... 83 5.3 Network connectivity is positively correlated with mitochondrial surface density ........................................ 89 5.4 Cells in fermentation have a higher regression coefficient for network connectivity–surface density ........................... 90 5.5 Mitochondrial membrane potential is not correlated with mitochondrial surface density .................................... 92