Copyright by Alice Zhao 2016
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Copyright by Alice Zhao 2016 The Dissertation Committee for Alice Zhao certifies that this is the approved version of the following dissertation: Cytoplasmic foci at the crossroads of artifactual science and biological function Committee: Edward M. Marcotte, Supervisor Andrew D. Ellington Yan (Jessie) Zhang Dean R. Appling Vishwanath R. Iyer Cytoplasmic foci at the crossroads of artifactual science and biological function by Alice Zhao, B.S.Bio DISSERTATION Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT AUSTIN MAY 2016 Dedication To my mother. The simplest lessons turn out to be the most important. And to those who (have) dedicate(d) their lives to open-access and transparency. Acknowledgements With great appreciation, I acknowledge the following individuals for their contributions to various aspects of my development as a scientist and a person. This body of research could not have been accomplished without their help, to each I owe my very deep gratitude. I would like to thank members of the Marcotte lab, former and current, for all have helped or influenced me in some way. Through the comradery of tackling science, I have learned valuable lessons from each and every one of us. In particular, I thank Traver Hart for his excellent mentorship which recruited me into graduate studies. I thank the expert adviser Zhihua Li, for his wealth of knowledge on experimental techniques. I thank Mark Tsechansky for his persistent encouragement and earnest fascination with science. I thank Jagannath Swaminathan, Dan Boutz, Fan Tu, and Anna Battenhouse for their skillful help and expertise at critical points of each project. I also thank Jeremy O’Connell, Kevin Drew, and Chris Yellman, for their kind and patient guidance. I thank the core proteomics facility staff and the sequencing facility staff, who have all consistently and reliably aided me with my questions on sample processing and analysis. I thank my committee members, Dr. Andy Ellington, Dr. Yan Jessie Zhang, Dr. Dean Appling, Dr. Vishy Iyer, and Dr. Edward Marcotte for their courageous support, motivation, and valuable inputs. I especially thank Edward Marcotte for being an exceptional role model for me and for generously providing me with an amazing environment to tinker and explore. I further extend my thanks to the students, post-docs, and faculty from various other labs and institutes who have given me their valuable advice, materials, and time. I am also thankful for the support and diligence from past and present administrative staff of the building and of the department, and for the cheery custodial team with whom I’ve spent many late evenings. Finally, I thank my friends and family for all of their love and support over the years, without which this work would not have been possible. v Cytoplasmic foci at the crossroads of artifactual science and biological function Publication No. ______ Alice Zhao, Ph.D. The University of Texas at Austin, 2016 Supervisor: Edward M. Marcotte Deciphering protein interaction and compartmentalization is crucial to understanding the molecular mechanisms that drive biological processes. Using various high throughput approaches, we have managed to score subcellular dynamic protein re-organization into supramolecular structures and map physical association networks to discover protein complexes on a proteome-wide level. However, the case by case studies of some of these novel structures and interactions reveal difficulties in interpreting their biological basis. This study offers insights into limits inherent in the molecular techniques used to investigate subcellular structures and protein interactions, describing a set of cautionary tales and critical analysis for deciphering cases of confounding data from orthogonal approaches. This study also offers a new experimental technique for high-throughput imaging assays with mammalian cell lines. vi Table of Contents Acknowledgements ........................................................................................ v List of Tables ............................................................................................. xiv List of Figures ............................................................................................. xv Introduction .................................................................................................. 1 Chapter 1: A human cell chip for high-throughput cell imaging .................. 6 1.1 Abstract ................................................................................................. 6 1.2 Introduction ........................................................................................... 7 1.2.1 Current platforms for compact high-content screening .............. 7 1.2.2 Inspirations from yeast cell microarrays ..................................... 9 1.2.3 Advantages of the new platform of human cell microarrays ............................................................................... 10 1.3 Development of the cell printing process ............................................. 11 1.3.1 Adapting microarray printing components ............................... 11 1.3.2 Proof of concept: detecting cellular states with high density cell chip-based immunofluorescence ............................. 14 1.3.3 Troubleshooting and optimization to increase robustness against assay-related mechanical insults ................. 16 1.4 Demonstration of cell chip multiplexing and utility ............................ 17 1.5 Discussion and Remarks ....................................................................... 22 1.5.1 Constraints of the human cell chip ........................................... 23 1.5.2 Applicability of the technology across fields of study .............. 24 1.6 Materials and Methods ......................................................................... 25 1.6.1 Cell culture ............................................................................... 25 vii 1.6.2 Printing cell arrays ................................................................... 25 1.6.3 Assaying printed slides ............................................................. 26 1.6.3.1 Immunofluorescence and imaging ........................... 26 1.6.3.2 Image signal quantitation ....................................... 27 Chapter 2: Reorganization of metabolic enzymes into megastructures and compartments ...................................................... 29 2.1 Abstract ............................................................................................... 29 2.2 Introduction ......................................................................................... 30 2.3 Types of intracellular bodies ................................................................ 31 2.4 Bacterial microcompartments .............................................................. 33 2.5 Aggregates with microbodies ................................................................ 35 2.6 Fibers and foci ...................................................................................... 38 2.6.1 Fibers of metabolic proteins: carbon utilization ....................... 39 2.6.1.1 Acetyl-CoA carboxylase .......................................... 39 2.6.1.2 β-glucosidase ........................................................... 40 2.6.2 Fibers of metabolic proteins: nitrogen utilization ..................... 40 2.6.2.1 Glutamine synthetase ............................................. 40 2.6.2.2 Glutamate dehydrogenase and glutamate synthetase ............................................................... 41 2.6.3 Fibers of metabolic proteins: nucleotide biosynthesis ............... 42 2.6.3.1 Cytidine triphosphate (CTP) synthase ................... 42 2.6.3.2 Inosine 5’-monophosphate dehydrogenase, types I and II .......................................................... 46 2.6.3.3 Purine biosynthesis and purinosomes ..................... 48 2.6.4 Large-scale screens in various systems reveal many additional intracellular foci and fibers ...................................... 51 viii 2.7 Three potential roles for intracellular bodies ....................................... 56 2.7.1 Catalytic efficiency and improved regulation ........................... 57 2.7.1.1 Substrate channeling ............................................... 57 2.7.1.2 Allostery and cooperativity ..................................... 58 2.7.2 Storage depots .......................................................................... 60 2.7.3 Aggregation of dysfunctional and/or misfolded proteins ..................................................................................... 62 2.8 Speculations and conclusions................................................................ 63 2.8.1 Are metabolic enzymes intrinsically more likely to self-assemble? ............................................................................ 64 2.8.2 Protein aggregation as an evolutionary compromise ................ 67 2.8.3 A case study in ambiguity: are CTP synthase fibers cytoskeletal elements or bacterial sickle-cell disease? ............... 69 2.8.4 High prevalence of GAT domains in assembled structures .................................................................................. 71 Chapter 3: Purine biosynthesis, purinosomes, and punctate foci formed by purine biosynthesis enzymes ............................................... 74 3.1