Abstract
Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.
Highlights
Macromolecular interactions between and amongst proteins and organelles mediate a considerable amount of biochemical signaling processes
With the goal of engineering such a pair, we first established a robust screen for reconstitution of Magnets dimerization using light-dependent accumulation of a protein at the outer mitochondrial membrane (Benedetti et al, 2018) (Fig. 1A), which is readily visible and quantifiable
The existing Magnets tools have two critical disadvantages, which preclude their wider adoption: 1) their weak dimerization efficiency necessitates the use of concatemers, which can perturb target proteins and slow kinetics, and 2) the low thermodynamic stability means that expression and maturation must occur at reduced temperatures, complicating cell culture experiments and ruling out mammalian in vivo work entirely
Summary
Macromolecular interactions between and amongst proteins and organelles mediate a considerable amount of biochemical signaling processes. Light offers much greater spatial and temporal resolution than drugs, and as such, optogenetic dimerizers are generally used to probe phenomena at cellular and subcellular scales. Photodimerizers have been successfully used to manipulate a variety of cellular processes, including signaling networks (Gasser et al, 2014; Grusch et al, 2014; Guglielmi et al, 2015; Idevall-Hagren et al, 2012; Toettcher et al, 2011, 2013; Wu et al, 2009) organelle trafficking (Bergeijk et al, 2015; Duan et al, 2015), nuclear import/export (Lerner et al, 2018; Niopek et al, 2014, 2016), cytoskeletal dynamics (Haren et al, 2018), and phase separation (Bracha et al, 2018; Dine et al, 2018; Shin et al, 2017), among others
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