You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells}
Article:
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.
Introduction:
Macromolecular interactions between and amongst proteins and organelles mediate a considerable amount of biochemical signaling processes. A principal method of testing the physiological significance of such interactions is to drive their association with a user-supplied stimulus such as light or drugs. Typically, two different components, each fused to a specific protein, come together( ‘heterodimerize’) to reconstitute a given protein-protein interaction following addition of a small molecule( DeRose et al., 2013; Putyrski and Schultz, 2012; Spencer et al., 1993) or upon light illumination( Losi et al., 2018; Rost et al., 2017). 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. At the organism scale, light is much less invasive but suffers from penetration issues. 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., 2013; Toettcher et al., 2011; Wu et al., 2009) organelle trafficking( van Bergeijk et al., 2015; Duan et al., 2015), nuclear import/export( Lerner et al., 2018; Niopek et al., 2016; Niopek et al., 2014), cytoskeletal dynamics( van Haren et al., 2018), and phase separation( Bracha et al., 2018; Dine et al., 2018; Shin et al., 2017), among others. Both natural and synthetic photoswitches( Guntas et al., 2015; Losi et al., 2018; Lungu et al., 2012; Rost et al., 2017; Strickland et al., 2012) have been used for these studies, each with its own advantages and drawbacks. Limitations of existing systems include necessity of adding exogenous cofactors( Levskaya et al., 2009), large size adversely affecting function of targeted proteins( Kaberniuk et al., 2016; Kennedy et al., 2010; Levskaya et al., 2009; Yazawa et al., 2009), non-trivial levels of basal dimerization in the dark( Guntas et al., 2015; Hallett et al., 2016; Nijenhuis et al., 2020; Zimmerman et al., 2016), poor light-dependent dimerization efficiency( Kawano et al., 2015; Strickland et al., 2012), and improper homo-, instead of heterodimerization( Bugaj et al., 2013; Che et al., 2015; Duan et al., 2017; Taslimi et al., 2016). One popular photodimerizer pair is ‘Magnets’, engineered from the Neurospora crassa Vivid photoreceptor, which comprises an N-terminal Ncap domain responsible for homodimerization and a C-terminal light-oxygen-voltage-sensing( LOV) domain( Kawano et al., 2015). Magnets employ the ubiquitous cofactor flavin adenine dinucleotide( FAD) as the light-sensing moiety. The Magnets pair was engineered from the Vivid homodimer by introducing complementary charges, giving rise to nMag( negative Magnet) and pMag( positive Magnet). The two Magnets components are quite small( 150 aa) for photodimerizers, exhibit relatively fast association and dissociation kinetics, and function when fused to a broad range of proteins, including peripheral and intrinsic membrane proteins( Benedetti et al., 2018; Kawano et al., 2016; Kawano et al., 2015). Furthermore, heterodimerization of Magnets requires light-dependent activation of both components, rather than just one. This property results in low levels of background activity and allows induction of dimer formation with single-wavelength excitation in small cytoplasmic volumes( Benedetti et al., 2018). However, the Magnets system has two prominent shortcomings. First, the low thermodynamic stability of the Magnets components precludes their proper expression and folding at 37°C. Thus, they cannot be used in mammals. When used in cultured mammalian cells they require a preincubation at low temperature( 28°C) for 12 hr to allow expression and folding. Second, as the Magnets components heterodimerize with low efficiency, robust activation requires concatemerization( Furuya et al., 2017; Kawano et al., 2015), which may affect trafficking, motility and function of target proteins, create vector payload constraints, and give rise to recombination and/or silencing of the sequence repeats. Here, we overcome these limitations of the Magnets by structure-guided protein engineering and validation by cellular assays. The resulting reagents, ‘enhanced Magnets’( eMags), have greater thermal stability and dimerization efficiency, as well as faster association and dissociation kinetics. We confirmed their effectiveness in a variety of applications including protein recruitment to different organelles, the generation/expansion of organelle contact sites, and the rapid and reversible reconstitution of VAP-dependent inter-organelle tethers that have key regulatory functions in lipid transport.
Discussion:
In this work, we have both engineered a dramatically improved photodimerizer pair and used it in a set of experiments elucidating details of organellar interactions and cellular lipid metabolism and transport. In a previous study( Benedetti et al., 2018), we had compared multiple optogenetic dimerizer reagents and found that the Magnets system, based on orthogonalization of the Vivid LOV domain homodimer( Kawano et al., 2015), offers major advantages over other systems in several different assays. Magnets have rapid association and dissociation kinetics and require both monomers to undergo blue-light activation to permit dimerization. These properties make the background activation of Magnets low, so that they are well-suited to optogenetic modulation of small volumes and sub-cellular organelles. However, 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. To overcome these limitations, we established a robust cell-culture screen that captures dimerization efficiency, association and dissociation kinetics, and folding and maturation. This screen allowed us to identify variants encompassing mutations across the whole protein with particular focus on the dimer interface. Mutations were selected based on sequence alignments with thermophilic fungal Vivid domains and structure-guided design. After several rounds of mutagenesis and screening, we selected final ‘enhanced Magnets’( eMag) variants with nine mutations over the starting scaffolds. The eMag reagents showed greater dimerization efficiency – allowing use as monomers instead of concatemers, full function after their folding and maturation at 37°C, and faster association and dissociation kinetics than the original Magnets. We recently compared( Benedetti et al., 2018) the original Magnets system to Cry2/CIB1( Kennedy et al., 2010) and iLIDs( Guntas et al., 2015), using several of the assays also used in this study, for example protein recruitment to ER, mitochondria, or single lysosomes, and activity of OCRL phosphatase recruited to the plasma membrane. From this systematic comparison, several obvious themes emerged. Firstly, upon prolonged local( 3 x 3 μm) illumination, Magnets dimerization occurs and persists only in the irradiated region, while activated iLID dimers extend some distance from this region and activated Cry2/CIB1 dimers penetrate the entire cell. Secondly, recovery kinetics of Cry2/CIB1 after light offset are more than an order of magnitude slower than those of iLIDs and Magnets. Thirdly, the efficiency of light-driven recruitment of original Magnets is greater than that of iLIDs but somewhat lower than that of Cry2/CIB1 in whole cell illumination conditions. Importantly, use of Cry2/CIB1 involves a balance between the desired Cry2/CIB1 heterodimerization and counterproductive Cry2 oligomerization( Bugaj et al., 2013; Che et al., 2015; Duan et al., 2017; Taslimi et al., 2016). This balance is difficult to determine and could vary across cellular environments and target proteins. Finally, Cry2 is less effective when used in membrane-bound bait( Benedetti et al., 2018; Che et al., 2015; Hallett et al., 2016; Pathak et al., 2014), hampering its utility in applications such as those shown here( Cry2 is much larger than LOV domains and may perturb fused proteins and organelles). Relative to the original Magnets – which we systematically compared to Cry2/CIB1 and iLIDs in our previous study – eMags shows significant improvements in the dimerization efficiency, and association and dissociation kinetics. Taken together, eMags provides several advantages over Cry2/CIB1 and iLIDs for subcellular optogenetics and perhaps other settings as well. We have shown the benefits of the faster eMagsF clones for rapid manipulation. In the other direction, it is likely that grafting the photoactivation-extending mutations Val74Ile and/or Val85Ile( Zoltowski et al., 2009) could produce high-efficiency, thermostable versions of eMags with recovery kinetics on the order of minutes to hours, if experimenters desire bistable control of protein-protein interactions. We thoroughly validated the eMags constructs in a range of cellular assays both in whole cell and local irradiation conditions involving protein recruitment to different membranes, inter-organellar association, and bilayer lipid metabolism and trafficking. The success of the engineering effort validates the design strategy and shows that many mutations from thermophilic fungi grafted well to the scaffold of the Vivid photoreceptor of Neurospora crassa, a mesophilic fungus. These mutations improved packing, hydrogen bonding, and secondary-structure preference. These improved optogenetic dimerizers will be broadly applicable and useful for applications across diverse fields. Also, our protein design and cellular screening strategies will likely extend to other reagent optimization projects.
The cell relies on direct interactions among proteins and compartments called organelles to stay alive . Manipulating these interactions allows researchers to control a wide variety of cell behaviors . A system called ‘Magnets’ uses light to trigger interactions between proteins . Magnets uses a segment of a protein called Vivid from a common bread mold that responds to light . When light shines on two of these segments , it causes them to bind together , in a process known as dimerization . In the Magnets system , Vivid segments are attached to specific proteins or organelles . By using light , researchers can force their target molecules to come together and trigger signals that can change cell behavior . However , the Magnets system has limitations: its stability and low efficiency mean that the cells need to be kept at low temperatures and that several copies of Vivid are needed . These conditions can interfere with the activity of the target proteins . To expand the technique , Benedetti et al . added mutations to make the Vivid protein more similar to proteins found in fungi that thrive at temperatures around 50°C . These changes meant that the enhanced system could work at body temperature in mammals . Further mutations at the interface between the two Vivid segments improved the efficiency of dimerization . This enhanced version was put to the test in different applications , including delivering proteins to different organelles and bringing organelles together . The enhanced Magnets system should enable researchers to control a greater variety of signaling events in the cell . In addition , the methodology established for improving the efficiency of the Magnets system could be useful to researchers working on other proteins .