Using Visual Phenotypes to Dissect Sequence- Function Relationships and Complex Drug Responses Nicholas Hasle A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2020 Reading Committee: Douglas Fowler (Chair) Emily Hatch Hao Yuan Kueh Program Authorized to Offer Degree: Genome Sciences ©Copyright 2020 Nicholas Hasle University of Washington Abstract Using Visual Phenotypes to Dissect Sequence-Function Relationships and Complex Drug Responses Nicholas Hasle Chair of the Supervisory Committee: Douglas M. Fowler, PhD Genome Sciences Cellular morphology is a potent indicator of cellular function and dysfunction, but the relationships between morphology, genetic variants, and cellular state remain incompletely understood. In this thesis, I describe a method called Visual Cell Sorting, which can be used to systematically characterize cellular morphologies and other visual phenotypes of interest. In a Visual Cell Sorting experiment, automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. Visual Cell Sorting can be used to characterize hundreds of genetic variants according to a visual phenotype and to discover genes that are responsible for maintaining homeostasis in response to drug treatment. Visual Cell Sorting’s greatest strength is that a variety of downstream assays can be performed on the separated cells, which together can characterize a morphologic phenotype in a multimodal and systematic fashion. Table of Contents Abstract ................................................................................................................................ 3 Table of Contents ................................................................................................................. 4 Dedication ............................................................................................................................ 8 Introduction .........................................................................................................................10 Structure-Function Relationships: A Hallmark of Biology .................................................10 New Methods for Detecting Cell Morphology-Function Relationships ..............................11 Visual Cell Sorting: A Historical Perspective ....................................................................14 Chapter 1: Separation of Cells by Visual Phenotypes with Visual Cell Sorting ....................18 Introduction ......................................................................................................................18 Methods ...........................................................................................................................19 General reagents, DNA oligonucleotides and plasmids ................................................19 Cloning Visual Cell Sorting constructs ..........................................................................20 Cell lines ......................................................................................................................20 Lentivirus production ....................................................................................................20 Visual Cell Sorting: equipment and settings .................................................................21 Visual Cell Sorting: cell preparation, imaging, analysis and photoactivation .................21 Visual Cell Sorting: FACS on microscope-activated cells .............................................24 Selective photoactivation of cells expressing miRFP ....................................................25 Photoactivation of cells for 0, 50, 200, and 800 milliseconds ........................................25 Testing for photoactivation-induced toxicity with Annexin V and DAPI ..........................25 Testing for photoactivation-induced toxicity with RNA sequencing ...............................26 Results ............................................................................................................................26 Discussion .......................................................................................................................28 Chapter 2: Visual Cell Sorting to Examine Sequence-Function Relationships .....................31 Introduction ......................................................................................................................31 Methods ...........................................................................................................................32 Cloning constructs and individual variants ....................................................................32 Cloning the library of SV40 NLS variants......................................................................32 Recombination of single-variant SV40 NLS clones or the library into U-2 OS LLP- Blast/H3-Dendra2 Clone 4 cells .........................................................................................33 Visual Cell Sorting of cells expressing SV40 NLS library ..............................................33 Sorted SV40 NLS library genomic DNA preparation and sequencing ...........................34 Calculating NLS variant localization scores ..................................................................34 Results ............................................................................................................................35 Discussion .......................................................................................................................37 Chapter 3: Visual Cell Sorting to Dissect Complex Drug Responses ...................................41 Introduction ......................................................................................................................41 Methods ...........................................................................................................................42 Cloning .........................................................................................................................42 Cell line ........................................................................................................................43 Time-lapse imaging of cells treated with paclitaxel .......................................................43 Visual Cell Sorting of cells treated with paclitaxel .........................................................43 Extended description of the Visual Cell Sorting on cells treated with paclitaxel ............44 Single cell RNA sequencing of sorted, paclitaxel-treated populations ...........................44 Analysis of single cell RNA sequencing data ................................................................44 Differentially Expressed Genes Analysis ......................................................................45 Extended description of the differentially expressed genes analysis .............................45 Gene Set Enrichment Analysis .....................................................................................46 Results ............................................................................................................................46 Discussion .......................................................................................................................49 Chapter 4: Towards Comprehensive Dissection of the Function of Thousands of PTEN Variants.....................................................................................................................................52 Introduction ......................................................................................................................52 PTEN biology and its role in cancer and Mendelian disease ........................................52 Methods ...........................................................................................................................55 Cell culture ...................................................................................................................55 Cloning .........................................................................................................................55 Lentiviral production .....................................................................................................56 Preliminary growth experiment .....................................................................................57 Preliminary DNA damage experiment ..........................................................................57 Derivation of U-87MG landing pad cells .......................................................................57 U87MG MaxCyte recombination protocol .....................................................................58 Results ............................................................................................................................58 Future Directions .............................................................................................................59 Library cloning ..............................................................................................................59 Growth selections .........................................................................................................60 Single cell methods for assessing variant function .......................................................61