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Engineered Illumination Devices for Optogenetic Control of Cellular Signaling Dynamics bioRxiv preprint doi: https://doi.org/10.1101/675892; this version posted June 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 TITLE 2 Engineered illumination devices for optogenetic control of cellular signaling dynamics 3 AUTHORS 4 Nicole A. Repina1,2, Thomas McClave3, Xiaoping Bao4,5, Ravi S. Kane6, David V. Schaffer1,7,8,9* 5 1Department of Bioengineering, University of California, Berkeley, California 94720 6 2Graduate Program in Bioengineering, University of California, San Francisco and University of 7 California, Berkeley, California 94720 8 3Department of Physics, University of California, Berkeley, California 94720 9 4Department of Chemical and Biomolecular Engineering, University of California, Berkeley, 10 California 94720 11 5Present address: Davidson School of Chemical Engineering, Purdue University, West Lafayette, 12 IN 47907 13 6School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, 14 Georgia 30332 15 7Department of Chemical and Biomolecular Engineering, University of California, Berkeley, 16 California 94720 17 8Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720 18 9Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 19 *Corresponding author. Email: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/675892; this version posted June 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 20 ABSTRACT 21 Spatially and temporally varying patterns of morphogen signals during development drive cell fate 22 specification at the proper location and time. However, current in vitro methods typically do not 23 allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus 24 insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that 25 optogenetic Wnt/b-catenin pathway activation can be controlled at user-defined intensities, 26 temporal sequences, and spatial patterns using novel engineered illumination devices for 27 optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical 28 design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to 29 enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein- 30 protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent 31 response in optoWnt activation and downstream Brachyury expression in human embryonic stem 32 cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue 33 patterning that models embryonic presentation of Wnt signals in vitro. The engineered LAVA 34 devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal 35 optogenetic control of cell signaling for applications in developmental and cell biology. bioRxiv preprint doi: https://doi.org/10.1101/675892; this version posted June 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 36 INTRODUCTION 37 Cell fate decisions are governed by dynamic, spatially and temporally varying signals from the 38 cellular environment. In particular, during development, morphogen gradients orchestrate the 39 dynamic, coordinated movement and differentiation of embryonic cell populations (Arnold & 40 Robertson 2009). Genetic perturbation and biomolecular treatment with pathway agonists or 41 inhibitors have provided insight into the critical regulators of embryogenesis, yet spatially-varying 42 interactions between cell subpopulations and time-varying signal dynamics and duration 43 thresholds remain largely unstudied due to a lack of tools to perturb signaling dynamics in 44 biological systems (Arnold & Robertson 2009; Oates et al. 2009). 45 Optogenetic methods aim to address this need for techniques that control such cell signal 46 dynamics. With optogenetics, light-responsive proteins from plants, algae, and bacteria have 47 been adapted to control signaling and protein-protein interactions in mammalian cells (Boyden et 48 al. 2005; Nagel et al. 2003; Hegemann & Nagel 2013). In particular, a variety of photosensory 49 domains have been discovered, optimized, and repurposed to place intracellular signaling 50 pathways under light control, capabilities that offer the unique advantage of using light patterns 51 to stimulate signaling in a specific location and at a specific time (Repina et al. 2017). Within 52 mammalian cells, optogenetic tools that control protein-protein interactions have for example 53 been developed for diverse signaling pathways such as Wnt/b-catenin (Bugaj et al. 2013; Repina 54 et al. 2019), Ras/Raf/Mek/Erk (Toettcher et al. 2011; Johnson & Toettcher 2019; Toettcher et al. 55 2013; Johnson et al. 2017), fibroblast growth factor (FGF) (Kainrath et al. 2017), and Rho-family 56 GTPase signaling (Levskaya et al. 2009; Yazawa et al. 2009; Strickland et al. 2012; Guntas et al. 57 2015; Bugaj et al. 2015; Wang et al. 2010). Optogenetic methods have also been recently applied 58 to studies in developmental biology in key model organisms (Johnson & Toettcher 2018). For 59 example, in Drosophila and zebrafish embryos, dynamic control of signaling elucidated the bioRxiv preprint doi: https://doi.org/10.1101/675892; this version posted June 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 60 spatiotemporal limits of ERK (Johnson et al. 2017; Johnson & Toettcher 2019), Nodal (Sako et 61 al. 2016), and Bicoid (Huang et al. 2017) patterning and how local modulation of cell migration 62 and contractility drives tissue morphogenesis (Izquierdo et al. 2018; Guglielmi et al. 2015; Čapek 63 et al. 2019). Furthermore, in embryonic stem cell models for mammalian development, we have 64 recently achieved optogenetic control of canonical Wnt signaling and elucidated mechanisms of 65 tissue self-organization during mesendoderm differentiation (Bugaj et al. 2013; Repina et al. 66 2019). 67 To activate the variety of photosensory domains developed for cellular signaling pathway control, 68 there must be complementary development of optical tools for cell culture illumination. However, 69 current optical methods lack practical applicability to routine cell culture stimulation and/or lack 70 critical characterization and functionality. In particular, microscope-based systems are widely 71 used for optogenetic photostimulation that implement one- or two-photon excitation to scan a 72 single diffraction-limited spot (Packer et al. 2013; Prakash et al. 2012; Packer et al. 2015; Carrillo- 73 Reid et al. 2016; Nikolenko et al. 2007) or project multi-spot light patterns (Papagiakoumou et al. 74 2010; Pégard et al. 2017; Papagiakoumou et al. 2008; Hernandez et al. 2016) onto the biological 75 sample. While such systems are essential for high-resolution in vivo experiments and precise 76 manipulation of neural circuits, their application to intracellular signal pathway activation in cell 77 cultures is limited by the system complexity, low throughput, high cost, and need for continuous 78 environmental control (Yizhar et al. 2011). For manipulating signaling pathways where speed and 79 single-cell spatial resolution are often less critical, the paramount photostimulation criteria are 80 high-throughput control of multiple parallel biological conditions, defined illumination parameters, 81 and compatibility with established cell culture formats and assays. Several methods have been 82 developed for such photostimulation of signal pathways in cell cultures, though they can lack 83 critical characterization and functionality. For example, a simple panel of light emitting diodes bioRxiv preprint doi: https://doi.org/10.1101/675892; this version posted June 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 84 (LEDs) enables optogenetic activation of a tissue culture plate, but lacks multi-well, high- 85 throughput control (Müller et al. 2014; Shao et al. 2018) and spatiotemporal patterning capability 86 (Tucker et al. 2014). Multi-well control can be achieved by incorporating a microcontroller and 87 LED drivers that set user-defined intensities for separate wells (Olson et al. 2014; Gerhardt et al. 88 2016). Though such multi-well devices have advanced the throughput of optogenetic studies, 89 current implementations have not characterized key performance parameters such as uniformity 90 of illumination, spatial or temporal light patterning resolution, and quantification of device 91 overheating and cell toxicity (Gerhardt et al. 2016; E. A. Davidson et al. 2013; Lee et al. 2013; 92 Hannanta-Anan & Chow 2016; Bugaj et al. 2018; Hennemann et al. 2018; Richter et al. 2015; 93 Olson et al. 2014). Several such designs also rely on modification of expensive instrumentation 94 (Richter et al. 2015) or focus on bacterial culture tubes (Olson et al. 2014). 95 We have designed a programmable illumination system for photostimulation of multi-well plates 96
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