mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging Benjamin C. Campbella,b,1, Elisa M. Nabelc,d,e, Mitchell H. Murdockb, Cristina Lao-Peregrinf, Pantelis Tsoulfasg, Murray G. Blackmoreh, Francis S. Leef,i,j, Conor Listona,b,f,i, Hirofumi Morishitac,d,e,k,l, and Gregory A. Petskoa,b,1,2 aHelen and Robert Appel Alzheimer’s Disease Research Institute, Weill Cornell Medicine, New York, NY 10021; bFeil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY 10021; cDepartment of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029; dMindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; eFriedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; fDepartment of Psychiatry, Weill Cornell Medicine, Cornell University, New York, NY 10021; gDepartment of Biomedical Sciences, Marquette University, Milwaukee, WI 53211; hDepartment of Neurological Surgery, Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL 33136; iSackler Institute for Developmental Psychobiology, Weill Cornell Medicine, Cornell University, New York, NY 10021; jDepartment of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY 10021; kDepartment of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and lDepartment of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY 10029 Contributed by Gregory A. Petsko, October 6, 2020 (sent for review January 24, 2020; reviewed by Matthew P. DeLisa and Michael Z. Lin) Although ubiquitous in biological studies, the enhanced green and The brightest GFPs recently developed are Clover (4), yellow fluorescent proteins (EGFP and EYFP) were not specifically mClover3 (5), and mNeonGreen (6), but they exhibit only minor optimized for neuroscience, and their underwhelming brightness differences in quantum yield (QY) and extinction coefficient and slow expression in brain tissue limits the fidelity of dendritic (EC) values, suggesting that these properties may have reached a spine analysis and other indispensable techniques for studying local maximum in FPs that contain a GYG-type chromophore neurodevelopment and plasticity. We hypothesized that EGFP’s (5). However, a biomolecule’s brightness in vitro hardly matters low solubility in mammalian systems must limit the total fluores- if it does not develop efficiently in a cell, and it has long been cence output of whole cells, and that improving folding efficiency known that a considerable fraction of FP molecules misfold and/ could therefore translate into greater brightness of expressing neu- or fail to form a functional chromophore at 37 °C (7), resulting in NEUROSCIENCE rons. By introducing rationally selected combinations of folding- aggregation-prone products incapable of fluorescence (8). enhancing mutations into GFP templates and screening for brightness We therefore reasoned that enhancing the folding efficiency and expression rate in human cells, we developed mGreenLan- and solubility of FPs in mammalian systems should yield a larger tern, a fluorescent protein having up to sixfold greater bright- fraction of functional emitting FP molecules per cell, increasing ness in cells than EGFP. mGreenLantern illuminates neurons in the mouse brain within 72 h, dramatically reducing lag time be- Significance tween viral transduction and imaging, while its high brightness improves detection of neuronal morphology using widefield, We have developed a fluorescent protein, mGreenLantern, that BIOPHYSICS AND COMPUTATIONAL BIOLOGY confocal, and two-photon microscopy. When virally expressed features exceptionally high brightness in mouse, bacterial, and to projection neurons in vivo, mGreenLantern fluorescence de- human cells (up to sixfold brighter than EGFP) and have dem- veloped four times faster than EYFP and highlighted long-range onstrated its superior ability to highlight neuronal morphology processes that were poorly detectable in EYFP-labeled cells. Ad- compared to EGFP and EYFP. Screening fluorescent protein ditionally, mGreenLantern retains strong fluorescence after tissue mutants based on whole-cell brightness while evaluating ex- clearing and expansion microscopy, thereby facilitating superreso- pression kinetics in lysate enabled us to identify variants lution and whole-brain imaging without immunohistochemistry. exhibiting striking divergences between their computed spec- mGreenLantern can directly replace EGFP/EYFP in diverse systems troscopic brightness and actual performance in cells. mGreen- due to its compatibility with GFP filter sets, recognition by EGFP Lantern additionally features unusually high chemical and antibodies, and excellent performance in mouse, human, and bac- thermodynamic stability and is compatible with existing GFP terial cells. Our screening and rational engineering approach is filter sets, excitation sources, commercial EGFP antibodies, ex- broadly applicable and suggests that greater potential of fluores- pansion microscopy, and whole-brain tissue clearing. Our cent proteins, including biosensors, could be unlocked using a hypothesis-driven engineering strategy represents a general- similar strategy. izable method with great potential to enhance the perfor- mance of constitutive reporters and GFP-based biosensors. GFP | fluorescent protein | neurobiology | imaging | protein engineering Author contributions: B.C.C., E.M.N., M.H.M., C.L.-P., P.T., M.G.B., F.S.L., C.L., H.M., and odern neuroscience research is placing increasing demands G.A.P. designed research; B.C.C., E.M.N., M.H.M., C.L.-P., P.T., M.G.B., F.S.L., C.L., and H.M. Mon fluorescent protein (FP) technology for robust perfor- performed research; B.C.C. contributed new reagents/analytic tools; B.C.C., E.M.N., M.H.M., C.L.-P., P.T., M.G.B., F.S.L., C.L., H.M., and G.A.P. analyzed data; B.C.C. wrote mance in new applications, including high-resolution interroga- the paper; and G.A.P. edited the paper. tion of intact biological systems through tissue clearing and Reviewers: M.P.D., Cornell University; and M.Z.L., Stanford University. expansion. With single-circuit studies and two-photon (2P) imaging The authors declare no competing interest. in the living brain becoming increasingly routine, there is a strong This open access article is distributed under Creative Commons Attribution-NonCommercial- need for brighter genetically encoded tags with efficient folding, NoDerivatives License 4.0 (CC BY-NC-ND). broad dispersal, and fast expression in brain tissue to facilitate un- 1To whom correspondence may be addressed. Email: [email protected] or ambiguous discrimination of individual neurons. Limitations in FP [email protected]. brightness have led researchers to use various enhancement strat- 2Present address: Ann Romney Institute for Neurologic Diseases, Department of Neurol- egies, including intracellular dye injection into GFP-expressing ogy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115. neurons (1) and antibody labeling even in transgenic animals (2), This article contains supporting information online at https://www.pnas.org/lookup/suppl/ which are strategies that consume additional time and resources doi:10.1073/pnas.2000942117/-/DCSupplemental. and present their own set of limitations (3). www.pnas.org/cgi/doi/10.1073/pnas.2000942117 PNAS Latest Articles | 1of12 Downloaded by guest on October 2, 2021 the cell’s total brightness and improving detection by fluorescence brighter than EGFP, respectively (Fig. 1A). Cellular brightness microscopy, independent of the FP’s molecular brightness (QY × trends were consistent across all three mammalian cell lines EC). Aided by the wealth of structure–activity information avail- tested (SI Appendix, Fig. S1). HeLa cells expressing FOLD1 were able for Aequorea victoria FPs (avFPs), we chose to forego random 3.3-fold brighter than EGFP and 2.2-fold brighter than Clover, a mutagenesis and instead rationally introduced sets of known avFP difference that cannot be explained by molecular brightness “folding mutations” into GFP templates, beginning with Clover, alone (SI Appendix, Table S1). and screened mutants from each round of selection for brightness Based on cellular brightness data and visual comparison of in Escherichia coli, brightness in mammalian cells, and then for E. coli plates, we suspected that the fluorescence intensity had rapid chromophore maturation. The GFPs were compared in vitro already peaked with mF1Y (FOLD1-A206K/N149Y) and was and in vivo against six widely used FPs. declining in subsequent mutants, as exemplified by our most The best-performing mutant, mGreenLantern, is a bright, mo- extensively modified variant, mF3CPK (SI Appendix, Table S2). nomeric FP tailored for accelerated expression and high bright- Initially, we had produced the double mutant, FOLD1-A206K/ ness in living cells, including neurons. The rational engineering of N149Y, before the single mutant, FOLD1-A206K, so we did not mGreenLantern, and its demonstrated superiority in neuronal know whether individual or concerted effects accounted for the highlighting experiments compared to enhanced green fluorescent improvement in cellular brightness. While producing the FOLD1- protein (EGFP) and enhanced yellow fluorescent protein (EYFP), V206K single mutant, we serendipitously introduced V206K into show that biological fluorophores can be optimized