Bio


Our lab applies biochemical and engineering principles to the development of protein-based tools for imaging and control of biochemical processes. Topics of investigation include fluorescent proteins structure and biophysics, fluorescent protein-based biosensors, neuronal activity sensors, spatiotemporal analysis of protein translation pathways, chemical control of protein translation, and light-responsive proteins.

Academic Appointments


Honors & Awards


  • Burroughs Wellcome Career Award for Medical Scientists, Burroughs Wellcome Foundation (2007-2013)
  • Damon Runyon-Rachleff Cancer Innovation Award, Damon Runyon Foundation (2012-2014)
  • Pioneer Award, NIH (2013-2018)
  • Roger Tsien Award for Excellence in Chemical Biology, World Molecular Imaging Society (2019)

Professional Education


  • BA, Harvard University, Biochemical Sciences (1994)
  • PhD, Harvard Medical School, Biological & Biomedical Sciences, Lab of Michael E. Greenberg (2002)
  • MD, UCLA, Medicine (2004)
  • Postdoctoral Fellowship, UCSD, Lab of Roger Y. Tsien (2009)

Current Research and Scholarly Interests


Our lab applies biochemical and engineering principles to the development of protein-based tools for investigating biology in living animals. Topics of investigation include fluorescent protein-based voltage indicators, synthetic light-controllable proteins, bioluminescent reporters, and applications to studying animal models of disease.

2023-24 Courses


Stanford Advisees


Graduate and Fellowship Programs


All Publications


  • A positively tuned voltage indicator for extended electrical recordings in the brain. Nature methods Evans, S. W., Shi, D., Chavarha, M., Plitt, M. H., Taxidis, J., Madruga, B., Fan, J. L., Hwang, F., van Keulen, S. C., Suomivuori, C., Pang, M. M., Su, S., Lee, S., Hao, Y. A., Zhang, G., Jiang, D., Pradhan, L., Roth, R. H., Liu, Y., Dorian, C. C., Reese, A. L., Negrean, A., Losonczy, A., Makinson, C. D., Wang, S., Clandinin, T. R., Dror, R. O., Ding, J. B., Ji, N., Golshani, P., Giocomo, L. M., Bi, G., Lin, M. Z. 2023; 20 (7): 1104-1113

    Abstract

    Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

    View details for DOI 10.1038/s41592-023-01913-z

    View details for PubMedID 37429962

  • Kinase-Modulated Bioluminescent Indicators Enable Noninvasive Imaging of Drug Activity in the Brain. ACS central science Wu, Y., Walker, J. R., Westberg, M., Ning, L., Monje, M., Kirkland, T. A., Lin, M. Z., Su, Y. 2023; 9 (4): 719-732

    Abstract

    Aberrant kinase activity contributes to the pathogenesis of brain cancers, neurodegeneration, and neuropsychiatric diseases, but identifying kinase inhibitors that function in the brain is challenging. Drug levels in blood do not predict efficacy in the brain because the blood-brain barrier prevents entry of most compounds. Rather, assessing kinase inhibition in the brain requires tissue dissection and biochemical analysis, a time-consuming and resource-intensive process. Here, we report kinase-modulated bioluminescent indicators (KiMBIs) for noninvasive longitudinal imaging of drug activity in the brain based on a recently optimized luciferase-luciferin system. We develop an ERK KiMBI to report inhibitors of the Ras-Raf-MEK-ERK pathway, for which no bioluminescent indicators previously existed. ERK KiMBI discriminates between brain-penetrant and nonpenetrant MEK inhibitors, reveals blood-tumor barrier leakiness in xenograft models, and reports MEK inhibitor pharmacodynamics in native brain tissues and intracranial xenografts. Finally, we use ERK KiMBI to screen ERK inhibitors for brain efficacy, identifying temuterkib as a promising brain-active ERK inhibitor, a result not predicted from chemical characteristics alone. Thus, KiMBIs enable the rapid identification and pharmacodynamic characterization of kinase inhibitors suitable for treating brain diseases.

    View details for DOI 10.1021/acscentsci.3c00074

    View details for PubMedID 37122464

    View details for PubMedCentralID PMC10141594

  • Optobiochemistry: Genetically Encoded Control of Protein Activity by Light. Annual review of biochemistry Seong, J., Lin, M. Z. 2021

    Abstract

    Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light.Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

    View details for DOI 10.1146/annurev-biochem-072420-112431

    View details for PubMedID 33781076

  • A compact synthetic pathway rewires cancer signaling to therapeutic effector release. Science (New York, N.Y.) Chung, H. K., Zou, X. n., Bajar, B. T., Brand, V. R., Huo, Y. n., Alcudia, J. F., Ferrell, J. E., Lin, M. Z. 2019; 364 (6439)

    Abstract

    An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9-mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.

    View details for PubMedID 31048459

  • StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins. Nature methods Jacobs, C. L., Badiee, R. K., Lin, M. Z. 2018; 15 (7): 523–26

    Abstract

    Robust approaches for chemogenetic control of protein function would have many biological applications. We developed stabilizable polypeptide linkages (StaPLs) based on hepatitis C virus protease. StaPLs undergo autoproteolysis to cleave proteins by default, whereas protease inhibitors prevent cleavage and preserve protein function. We created StaPLs responsive to different clinically approved drugs to bidirectionally control transcription with zinc-finger-based effectors, and used StaPLs to create single-chain, drug-stabilizable variants of CRISPR-Cas9 and caspase-9.

    View details for PubMedID 29967496

  • Optical control of cell signaling by single-chain photoswitchable kinases. Science Zhou, X. X., Fan, L. Z., Li, P., Shen, K., Lin, M. Z. 2017; 355 (6327): 836-842

    Abstract

    Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.

    View details for DOI 10.1126/science.aah3605

    View details for PubMedID 28232577

  • Fluorescent indicators for simultaneous reporting of all four cell cycle phases. Nature methods Bajar, B. T., Lam, A. J., Badiee, R. K., Oh, Y., Chu, J., Zhou, X. X., Kim, N., Kim, B. B., Chung, M., Yablonovitch, A. L., Cruz, B. F., Kulalert, K., Tao, J. J., Meyer, T., Su, X., Lin, M. Z. 2016

    Abstract

    A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

    View details for DOI 10.1038/nmeth.4045

    View details for PubMedID 27798610

  • High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nature neuroscience St-Pierre, F., Marshall, J. D., Yang, Y., Gong, Y., Schnitzer, M. J., Lin, M. Z. 2014; 17 (6): 884-889

    Abstract

    Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience. We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential. ASAP1 demonstrated on and off kinetics of ∼2 ms, reliably detected single action potentials and subthreshold potential changes, and tracked trains of action potential waveforms up to 200 Hz in single trials. With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

    View details for DOI 10.1038/nn.3709

    View details for PubMedID 24755780

  • Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein NATURE METHODS Chu, J., Haynes, R. D., Corbel, S. Y., Li, P., Gonzalez-Gonzalez, E., Burg, J. S., Ataie, N. J., Lam, A. J., Cranfill, P. J., Baird, M. A., Davidson, M. W., Ng, H., Garcia, K. C., Contag, C. H., Shen, K., Blau, H. M., Lin, M. Z. 2014; 11 (5): 572-578

    Abstract

    A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

    View details for DOI 10.1038/NMETH.2888

    View details for Web of Science ID 000335873400026

    View details for PubMedID 24633408

    View details for PubMedCentralID PMC4008650

  • Imaging of Evoked Cortical Depolarizations Using Either ASAP2s, or chi-VSFP, or Di-4-Anepps, or Autofluorescence Optical Signals. Journal of integrative neuroscience Milicevic, K. D., Zhu, M. H., Barbeau, B. L., Baser, O., Erol, Z. Y., Liu, L. X., Lin, M. Z., Antic, S. D. 2023; 22 (6): 160

    Abstract

    Population voltage imaging is used for studying brain physiology and brain circuits. Using a genetically encoded voltage indicator (GEVI), "VSFP" or "ASAP2s", or a voltage-sensitive dye, Di-4-Anepps, we conducted population voltage imaging in brain slices. The resulting optical signals, optical local field potentials (LFPs), were used to evaluate the performances of the 3 voltage indicators.In brain slices prepared from VSFP-transgenic or ASAP2s-transgenic mice, we performed multi-site optical imaging of evoked cortical depolarizations - compound excitatory postsynaptic potentials (cEPSPs). Optical signal amplitudes (ΔF/F) and cEPSP decay rates (OFF rates) were compared using analysis of variance (ANOVA) followed by unpaired Student's t test (31-104 data points per voltage indicator).The ASAP2s signal amplitude (ΔF/F) was on average 3 times greater than Di-4-Anepps, and 7 times greater than VSFP. The optical cEPSP decay (OFF rate) was the slowest in Di-4-Anepps and fastest in ASAP2s. When ASAP2s expression was weak, we observed slow, label-free (autofluorescence, metabolic) optical signals mixed into the ASAP2s traces. Fast hyperpolarizations, that typically follow depolarizing cortical transients (afterhyperpolarizations), were prominent in ASAP2s but not present in the VSFP and Di-4-Anepps experiments.Experimental applications for ASAP2s may potentially include systems neuroscience studies that require voltage indicators with large signal amplitude (ΔF/F), fast decay times (fast response time is needed for monitoring high frequency brain oscillations), and/or detection of brain patches in transiently hyperpolarized states (afterhyperpolarization).

    View details for DOI 10.31083/j.jin2206160

    View details for PubMedID 38176939

  • Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits. Cancer cell Sanchez-Aguilera, A., Masmudi-Martín, M., Navas-Olive, A., Baena, P., Hernández-Oliver, C., Priego, N., Cordón-Barris, L., Alvaro-Espinosa, L., García, S., Martínez, S., Lafarga, M., Lin, M. Z., Al-Shahrour, F., Menendez de la Prida, L., Valiente, M. 2023

    Abstract

    A high percentage of patients with brain metastases frequently develop neurocognitive symptoms; however, understanding how brain metastasis co-opts the function of neuronal circuits beyond a tumor mass effect remains unknown. We report a comprehensive multidimensional modeling of brain functional analyses in the context of brain metastasis. By testing different preclinical models of brain metastasis from various primary sources and oncogenic profiles, we dissociated the heterogeneous impact on local field potential oscillatory activity from cortical and hippocampal areas that we detected from the homogeneous inter-model tumor size or glial response. In contrast, we report a potential underlying molecular program responsible for impairing neuronal crosstalk by scoring the transcriptomic and mutational profiles in a model-specific manner. Additionally, measurement of various brain activity readouts matched with machine learning strategies confirmed model-specific alterations that could help predict the presence and subtype of metastasis.

    View details for DOI 10.1016/j.ccell.2023.07.010

    View details for PubMedID 37652007

  • Kinase-Modulated Bioluminescent Indicators Enable Noninvasive Imaging of Drug Activity in the Brain ACS CENTRAL SCIENCE Wu, Y., Walker, J. R., Westberg, M., Ning, L., Monje, M., Kirkland, T. A., Lin, M. Z., Su, Y. 2023
  • An optimized bioluminescent substrate for non-invasive imaging in the brain. Nature chemical biology Su, Y., Walker, J. R., Hall, M. P., Klein, M. A., Wu, X., Encell, L. P., Casey, K. M., Liu, L. X., Hong, G., Lin, M. Z., Kirkland, T. A. 2023

    Abstract

    Bioluminescence imaging (BLI) allows non-invasive visualization of cells and biochemical events in vivo and thus has become an indispensable technique in biomedical research. However, BLI in the central nervous system remains challenging because luciferases show relatively poor performance in the brain with existing substrates. Here, we report the discovery of a NanoLuc substrate with improved brain performance, cephalofurimazine (CFz). CFz paired with Antares luciferase produces greater than 20-fold more signal from the brain than the standard combination of D-luciferin with firefly luciferase. At standard doses, Antares-CFz matches AkaLuc-AkaLumine/TokeOni in brightness, while occasional higher dosing of CFz can be performed to obtain threefold more signal. CFz should allow the growing number of NanoLuc-based indicators to be applied to the brain with high sensitivity. Using CFz, we achieve video-rate non-invasive imaging of Antares in brains of freely moving mice and demonstrate non-invasive calcium imaging of sensory-evoked activity in genetically defined neurons.

    View details for DOI 10.1038/s41589-023-01265-x

    View details for PubMedID 36759751

  • Track: Protein Phase Separation in Biomolecular Condensates Using Phase Separation as Mechanism to Enhance Inducibility of a Synthetic Therapeutic Pathway Chau, J., Westberg, M., Song, D., Lin, M. WILEY. 2023
  • Non-invasive bioluminescent imaging of kinase inhibition in mouse brain Su, Y., Wu, Y., Lin, M. WILEY. 2023
  • Rational Design of Improved and Novel Photodissociable GFPs and RFPs Westberg, M., Trigo, M. L., Devenish, S., Huang, P., Lin, M. Z. WILEY. 2023
  • Combinatorial effects of RhoA and Cdc42 on the actin cytoskeleton revealed by photoswitchable GEFs SENSORS AND ACTUATORS B-CHEMICAL Ryu, H., Lee, H., Ju, J., Park, J., Oh, E., Lin, M. Z., Seong, J. 2022; 369
  • Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude. Cell reports Ju, J., Lee, H. N., Ning, L., Ryu, H., Zhou, X. X., Chun, H., Lee, Y. W., Lee-Richerson, A. I., Jeong, C., Lin, M. Z., Seong, J. 2022; 40 (2): 111080

    Abstract

    How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

    View details for DOI 10.1016/j.celrep.2022.111080

    View details for PubMedID 35830815

  • Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell Labanieh, L., Majzner, R. G., Klysz, D., Sotillo, E., Fisher, C. J., Vilches-Moure, J. G., Pacheco, K. Z., Malipatlolla, M., Xu, P., Hui, J. H., Murty, T., Theruvath, J., Mehta, N., Yamada-Hunter, S. A., Weber, E. W., Heitzeneder, S., Parker, K. R., Satpathy, A. T., Chang, H. Y., Lin, M. Z., Cochran, J. R., Mackall, C. L. 2022

    Abstract

    Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

    View details for DOI 10.1016/j.cell.2022.03.041

    View details for PubMedID 35483375

  • A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3. Scientific reports Kim, B. B., Wu, H., Hao, Y. A., Pan, M., Chavarha, M., Zhao, Y., Westberg, M., St-Pierre, F., Wu, J. C., Lin, M. Z. 2022; 12 (1): 3678

    Abstract

    A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in the presence of sample motion. We performed combinatorial multi-site mutagenesis on a cyan-excitable red fluorescent protein to create the bright and monomeric mCyRFP3, which proved to be uniquely non-perturbing when fused to the GEVI ASAP3. The green/red ratio from ASAP3-mCyRFP3 (ASAP3-R3) reported voltage while correcting for motion artifacts, allowing the visualization of membrane voltage changes in contracting cardiomyocytes and throughout the cell cycle of motile cells.

    View details for DOI 10.1038/s41598-022-07313-1

    View details for PubMedID 35256624

  • A Bright, Nontoxic, and Non-aggregating red Fluorescent Protein for Long-Term Labeling of Fine Structures in Neurons. Frontiers in cell and developmental biology Ning, L., Geng, Y., Lovett-Barron, M., Niu, X., Deng, M., Wang, L., Ataie, N., Sens, A., Ng, H., Chen, S., Deisseroth, K., Lin, M. Z., Chu, J. 2022; 10: 893468

    Abstract

    Red fluorescent proteins are useful as morphological markers in neurons, often complementing green fluorescent protein-based probes of neuronal activity. However, commonly used red fluorescent proteins show aggregation and toxicity in neurons or are dim. We report the engineering of a bright red fluorescent protein, Crimson, that enables long-term morphological labeling of neurons without aggregation or toxicity. Crimson is similar to mCherry and mKate2 in fluorescence spectra but is 100 and 28% greater in molecular brightness, respectively. We used a membrane-localized Crimson-CAAX to label thin neurites, dendritic spines and filopodia, enhancing detection of these small structures compared to cytosolic markers.

    View details for DOI 10.3389/fcell.2022.893468

    View details for PubMedID 35846353

  • FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs. Methods in molecular biology (Clifton, N.J.) Bajar, B. T., Guan, X., Lam, A., Lin, M. Z., Yasuda, R., Laviv, T., Chu, J. 2022; 2438: 31-43

    Abstract

    With the development of fluorescent proteins (FPs) and advanced optical microscopy techniques, Förster or fluorescence resonance energy transfer (FRET) has become a powerful tool for real-time noninvasive visualization of a variety of biological processes, including kinase activities, with high spatiotemporal resolution in living cells and organisms. FRET can be detected in appropriately configured microscopes as changes in fluorescence intensity, lifetime, and anisotropy. Here, we describe the preparation of samples expressing FP-based FRET sensors for RhoA kinase, intensity- and lifetime-based FRET imaging, and postimaging data analysis.

    View details for DOI 10.1007/978-1-0716-2035-9_2

    View details for PubMedID 35147933

  • Optical control of fast and processive engineered myosins in vitro and in living cells. Nature chemical biology Ruijgrok, P. V., Ghosh, R. P., Zemsky, S. n., Nakamura, M. n., Gong, R. n., Ning, L. n., Chen, R. n., Vachharajani, V. T., Chu, A. E., Anand, N. n., Eguchi, R. R., Huang, P. S., Lin, M. Z., Alushin, G. M., Liphardt, J. T., Bryant, Z. n. 2021

    Abstract

    Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.

    View details for DOI 10.1038/s41589-021-00740-7

    View details for PubMedID 33603247

  • Simultaneous Detection of Four Cell Cycle Phases with Live Fluorescence Imaging. Methods in molecular biology (Clifton, N.J.) Bajar, B. T., Lin, M. Z. 2021; 2274: 25-35

    Abstract

    Visualizing progression through the cell cycle provides valuable information for the study of development, tissue maintenance, and dysregulated growth in proliferative diseases, such as cancer. Developments in fluorescent biosensors have facilitated dynamic tracking of molecular processes, including the cell cycle. The genetically encoded set of fluorescent indicators, Fucci4, enables the visualization of transitions between each cell cycle phase. Here, we describe a method to track progression through each cell cycle phase using Fucci4 in live epifluorescence imaging. In principle, this approach can be adapted to in vitro time-lapse imaging of any four spectrally resolvable fluorescent indicators.

    View details for DOI 10.1007/978-1-0716-1258-3_3

    View details for PubMedID 34050459

  • Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging. ACS chemical biology Liu, S., Su, Y., Lin, M. Z., Ronald, J. A. 2021

    Abstract

    Bioluminescence imaging (BLI) using luciferase reporters is an indispensable method for the noninvasive visualization of cell populations and biochemical events in living animals. BLI is widely performed with preclinical rodent models to understand disease processes and evaluate potential cell- or gene-based therapies. However, in vivo BLI remains constrained by low photon production and tissue attenuation, limiting the sensitivity of reporting from small numbers of cells in deep locations and hindering its application to larger animal models. This Review highlights recent advances in the development of luciferase systems that improve the sensitivity of in vivo BLI and discusses the expanding array of biological applications.

    View details for DOI 10.1021/acschembio.1c00549

    View details for PubMedID 34780699

  • Integrated Neurophotonics: Toward Dense Volumetric Interrogation of Brain Circuit Activity-at Depth and in Real Time. Neuron Moreaux, L. C., Yatsenko, D., Sacher, W. D., Choi, J., Lee, C., Kubat, N. J., Cotton, R. J., Boyden, E. S., Lin, M. Z., Tian, L., Tolias, A. S., Poon, J. K., Shepard, K. L., Roukes, M. L. 2020; 108 (1): 66–92

    Abstract

    We propose a new paradigm for dense functional imaging of brain activity to surmount the limitations of present methodologies. We term this approach "integrated neurophotonics"; it combines recent advances in microchip-based integrated photonic and electronic circuitry with those from optogenetics. This approach has the potential to enable lens-less functional imaging from within the brain itself to achieve dense, large-scale stimulation and recording of brain activity with cellular resolution at arbitrary depths. We perform a computational study of several prototype 3D architectures for implantable probe-array modules that are designed to provide fast and dense single-cell resolution (e.g., within a 1-mm3 volume of mouse cortex comprising 100,000 neurons). We describe progress toward realizing integrated neurophotonic imaging modules, which can be produced en masse with current semiconductor foundry protocols for chip manufacturing. Implantation of multiple modules can cover extended brain regions.

    View details for DOI 10.1016/j.neuron.2020.09.043

    View details for PubMedID 33058767

  • Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nature methods Su, Y., Walker, J. R., Park, Y., Smith, T. P., Liu, L. X., Hall, M. P., Labanieh, L., Hurst, R., Wang, D. C., Encell, L. P., Kim, N., Zhang, F., Kay, M. A., Casey, K. M., Majzner, R. G., Cochran, J. R., Mackall, C. L., Kirkland, T. A., Lin, M. Z. 2020

    Abstract

    Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

    View details for DOI 10.1038/s41592-020-0889-6

    View details for PubMedID 32661427

  • On the cutting edge: protease-based methods for sensing and controlling cell biology. Nature methods Chung, H. K., Lin, M. Z. 2020

    Abstract

    Sequence-specific proteases have proven to be versatile building blocks for tools that report or control cellular function. Reporting methods link protease activity to biochemical signals, whereas control methods rely on engineering proteases to respond to exogenous inputs such as light or chemicals. In turn, proteases have inherent control abilities, as their native functions are to release, activate or destroy proteins by cleavage, with the irreversibility of proteolysis allowing sustained downstream effects. As a result, protease-based synthetic circuits have been created for diverse uses such as reporting cellular signaling, tuning protein expression, controlling viral replication and detecting cancer states. Here, we comprehensively review the development and application of protease-based methods for reporting and controlling cellular function in eukaryotes.

    View details for DOI 10.1038/s41592-020-0891-z

    View details for PubMedID 32661424

  • Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo. Nature methods Wu, J., Liang, Y., Chen, S., Hsu, C., Chavarha, M., Evans, S. W., Shi, D., Lin, M. Z., Tsia, K. K., Ji, N. 2020

    Abstract

    Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345mum below the brain surface in head-fixed awake mice.

    View details for DOI 10.1038/s41592-020-0762-7

    View details for PubMedID 32123392

  • Two-Photon Voltage Imaging of Spontaneous Activity from Multiple Neurons Reveals Network Activity in Brain Tissue. iScience Li, B. n., Chavarha, M. n., Kobayashi, Y. n., Yoshinaga, S. n., Nakajima, K. n., Lin, M. Z., Inoue, T. n. 2020; 23 (8): 101363

    Abstract

    Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the function of neuronal circuits. The combination of the fast scanning by random-access multiphoton microscopy (RAMP) and the latest two-photon-compatible high-performance fluorescent genetically encoded voltage indicators (GEVIs) has enabled action potential detection in deep layers in in vivo brain. However, neuron connectivity analysis on optically recorded action potentials from multiple neurons in brain tissue has yet to be achieved. With high expression of a two-photon-compatible GEVI, ASAP3, via in utero electroporation and RAMP, we achieved voltage recording of spontaneous activities from multiple neurons in brain slice. We provide evidence for the developmental changes in intralaminar horizontal connections in somatosensory cortex layer 2/3 with a greater sensitivity than calcium imaging. This method thus enables investigation of neuronal network connectivity at the cellular resolution in brain tissue.

    View details for DOI 10.1016/j.isci.2020.101363

    View details for PubMedID 32717641

  • An Axonal Blueprint: Generating Neuronal Polarity with Light-Inducible Proteins CELL CHEMICAL BIOLOGY Lin, M. Z. 2019; 26 (12): 1634–36
  • An Axonal Blueprint: Generating Neuronal Polarity with Light-Inducible Proteins. Cell chemical biology Lin, M. Z. 2019; 26 (12): 1634-1636

    Abstract

    In this issue of Cell Chemical Biology, Woo et al. (2019) show that activation of a photoinducible form of the TrkB protein in a single neurite induces multiple aspects of axonal differentiation. This study exemplifies the ability of optical methods to relate protein functions to complex phenotypes in living cells.

    View details for DOI 10.1016/j.chembiol.2019.12.003

    View details for PubMedID 31951578

  • Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice. Cell Villette, V., Chavarha, M., Dimov, I. K., Bradley, J., Pradhan, L., Mathieu, B., Evans, S. W., Chamberland, S., Shi, D., Yang, R., Kim, B. B., Ayon, A., Jalil, A., St-Pierre, F., Schnitzer, M. J., Bi, G., Toth, K., Ding, J., Dieudonne, S., Lin, M. Z. 2019; 179 (7): 1590

    Abstract

    Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling invivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking ofspikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

    View details for DOI 10.1016/j.cell.2019.11.004

    View details for PubMedID 31835034

  • Novel NanoLuc substrates enable bright and sustained bioluminescence imaging in animals Walker, J., Park, Y., Smith, T., Wang, D., Hall, M., Liu, L., Hurst, R., Su, Y., Encell, L., Kim, N., Casey, K., Kirkland, T., Lin, M. AMER CHEMICAL SOC. 2019
  • Novel substrates for NanoLuc luciferase with improved brightness and signal duration for bioluminescence imaging in vivo Walker, J. R., Park, Y., Lin, M., Kirkland, T. A., Hall, M. P., Encell, L. P., Oh, Y., Liu, L. AMER ASSOC CANCER RESEARCH. 2019
  • SYNTHETIC BIOLOGY A compact synthetic pathway rewires cancer signaling to therapeutic effector release SCIENCE Chung, H. K., Zou, X., Bajar, B. T., Brand, V. R., Huo, Y., Alcudia, J. F., Ferrell, J. E., Lin, M. Z. 2019; 364 (6439): 451-+
  • An orange calcium-modulated bioluminescent indicator for non-invasive activity imaging NATURE CHEMICAL BIOLOGY Oh, Y., Park, Y., Cho, J. H., Wu, H., Paulk, N. K., Liu, L., Kim, N., Kay, M. A., Wu, J. C., Lin, M. Z. 2019; 15 (5): 433-+
  • An orange calcium-modulated bioluminescent indicator for non-invasive activity imaging. Nature chemical biology Oh, Y., Park, Y., Cho, J. H., Wu, H., Paulk, N. K., Liu, L. X., Kim, N., Kay, M. A., Wu, J. C., Lin, M. Z. 2019

    Abstract

    Fluorescent indicators are used widely to visualize calcium dynamics downstream of membrane depolarization or G-protein-coupled receptor activation, but are poorly suited for non-invasive imaging in mammals. Here, we report a bright calcium-modulated bioluminescent indicator named Orange CaMBI (Orange Calcium-modulated Bioluminescent Indicator). Orange CaMBI reports calcium dynamics in single cells and, in the context of a transgenic mouse, reveals calcium oscillations in whole organs in an entirely non-invasive manner.

    View details for PubMedID 30936501

  • Kinase pathway inhibition restores PSD95 induction in neurons lacking fragile X mental retardation protein. Proceedings of the National Academy of Sciences of the United States of America Yang, Y. n., Geng, Y. n., Jiang, D. n., Ning, L. n., Kim, H. J., Jeon, N. L., Lau, A. n., Chen, L. n., Lin, M. Z. 2019

    Abstract

    Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates translation of numerous mRNA targets, some of which are present at synapses. While protein synthesis deficits have long been postulated as an etiology of FXS, how FMRP loss affects distributions of newly synthesized proteins is unknown. Here we investigated the role of FMRP in regulating expression of new copies of the synaptic protein PSD95 in an in vitro model of synaptic plasticity. We find that local BDNF application promotes persistent accumulation of new PSD95 at stimulated synapses and dendrites of cultured neurons, and that this accumulation is absent in FMRP-deficient mouse neurons. New PSD95 accumulation at sites of BDNF stimulation does not require known mechanisms regulating FMRP-mRNA interactions but instead requires the PI3K-mTORC1-S6K1 pathway. Surprisingly, in FMRP-deficient neurons, BDNF induction of new PSD95 accumulation can be restored by mTORC1-S6K1 blockade, suggesting that constitutively high mTORC1-S6K1 activity occludes PSD95 regulation by BDNF and that alternative pathways exist to mediate induction when mTORC1-S6K1 is inhibited. This study provides direct evidence for deficits in local protein synthesis and accumulation of newly synthesized protein in response to local stimulation in FXS, and supports mTORC1-S6K1 pathway inhibition as a potential therapeutic approach for FXS.

    View details for DOI 10.1073/pnas.1812056116

    View details for PubMedID 31118285

  • A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality CELL Daigle, T. L., Madisen, L., Hage, T. A., Valley, M. T., Knoblich, U., Larsen, R. S., Takeno, M. M., Huang, L., Gu, H., Larsen, R., Mills, M., Bosma-Moody, A., Siverts, L., Walker, M., Graybuck, L. T., Yao, Z., Fong, O., Thuc Nghi Nguyen, Garren, E., Lenz, G. H., Chavarha, M., Pendergraft, J., Harrington, J., Hirokawa, K. E., Harris, J. A., Nicovich, P. R., McGraw, M. J., Ollerenshaw, D. R., Smith, K. A., Baker, C. A., Ting, J. T., Sunkin, S. M., Lecoq, J., Lin, M. Z., Boyden, E. S., Murphy, G. J., da Costa, N. M., Waters, J., Li, L., Tasic, B., Zeng, H. 2018; 174 (2): 465-+

    Abstract

    Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

    View details for PubMedID 30007418

  • Excitation wavelength optimization improves photostability of ASAP-family GEVIs MOLECULAR BRAIN Xu, F., Shi, D., Lau, P., Lin, M. Z., Bi, G. 2018; 11: 32

    Abstract

    Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low-intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements.

    View details for PubMedID 29866136

  • A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription. ACS chemical biology Zhou, X. X., Zou, X., Chung, H. K., Gao, Y., Liu, Y., Qi, L. S., Lin, M. Z. 2018; 13 (2): 443-448

    Abstract

    Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

    View details for DOI 10.1021/acschembio.7b00603

    View details for PubMedID 28938067

    View details for PubMedCentralID PMC5820652

  • Understanding CRY2 interactions for optical control of intracellular signaling NATURE COMMUNICATIONS Duan, L., Hope, J., Ong, Q., Lou, H., Kim, N., McCarthy, C., Acero, V., Lin, M. Z., Cui, B. 2017; 8: 547

    Abstract

    Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2-CRY2 homo-oligomerization and CRY2-CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2-CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2-CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2-CIB1 and CRY2-CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2-CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems.Cryptochrome 2 (CRY2) can form light-regulated CRY2-CRY2 homo-oligomers or CRY2-CIB1 hetero-dimers, but modulating these interactions is difficult owing to the lack of interaction mechanism. Here the authors identify the interactions facilitating homo-oligomers and introduce mutations to create low and high oligomerization versions.

    View details for PubMedID 28916751

  • The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins TRENDS IN BIOCHEMICAL SCIENCES Rodriguez, E. A., Campbell, R. E., Lin, J. Y., Lin, M. Z., Miyawaki, A., Palmer, A. E., Shu, X., Zhang, J., Tsien, R. Y. 2017; 42 (2): 111-129

    Abstract

    Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

    View details for DOI 10.1016/j.tibs.2016.09.010

    View details for Web of Science ID 000393635200005

  • Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. eLife Chamberland, S. n., Yang, H. H., Pan, M. M., Evans, S. W., Guan, S. n., Chavarha, M. n., Yang, Y. n., Salesse, C. n., Wu, H. n., Wu, J. C., Clandinin, T. R., Toth, K. n., Lin, M. Z., St-Pierre, F. n. 2017; 6

    Abstract

    Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and inDrosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

    View details for PubMedID 28749338

    View details for PubMedCentralID PMC5584994

  • A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription Zhou, X. X., Zou, X., Chung, H. K., Gao, Y., Liu, Y., QI, L. S., Lin, M. Z. 2017: 443–48

    Abstract

    Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

    View details for DOI 10.1021/acschembio.7b00603

    View details for PubMedCentralID PMC5820652

  • Cell-Type-Specific Optical Recording of Membrane Voltage Dynamics in Freely Moving Mice CELL Marshall, J. D., Li, J. Z., Zhang, Y., Gong, Y., St-Pierre, F., Lin, M. Z., Schnitzer, M. J. 2016; 167 (6): 1650-?

    Abstract

    Electrophysiological field potential dynamics are of fundamental interest in basic and clinical neuroscience, but how specific cell types shape these dynamics in the live brain is poorly understood. To empower mechanistic studies, we created an optical technique, TEMPO, that records the aggregate trans-membrane voltage dynamics of genetically specified neurons in freely behaving mice. TEMPO has >10-fold greater sensitivity than prior fiber-optic techniques and attains the noise minimum set by quantum mechanical photon shot noise. After validating TEMPO's capacity to track established oscillations in the delta, theta, and gamma frequency bands, we compared the D1- and D2-dopamine-receptor-expressing striatal medium spiny neurons (MSNs), which are interspersed and electrically indistinguishable. Unexpectedly, MSN population dynamics exhibited two distinct coherent states that were commonly indiscernible in electrical recordings and involved synchronized hyperpolarizations across both MSN subtypes. Overall, TEMPO allows the deconstruction of normal and pathologic neurophysiological states into trans-membrane voltage activity patterns of specific cell types.

    View details for DOI 10.1016/j.cell.2016.11.021

    View details for Web of Science ID 000389470500024

    View details for PubMedID 27912066

    View details for PubMedCentralID PMC5382987

  • The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends in biochemical sciences Rodriguez, E. A., Campbell, R. E., Lin, J. Y., Lin, M. Z., Miyawaki, A., Palmer, A. E., Shu, X., Zhang, J., Tsien, R. Y. 2016

    Abstract

    Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

    View details for DOI 10.1016/j.tibs.2016.09.010

    View details for PubMedID 27814948

  • Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins. Nature methods Laviv, T., Kim, B. B., Chu, J., Lam, A. J., Lin, M. Z., Yasuda, R. 2016

    Abstract

    We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity.

    View details for DOI 10.1038/nmeth.4046

    View details for PubMedID 27798609

  • A Guide to Fluorescent Protein FRET Pairs SENSORS Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., Chu, J. 2016; 16 (9)

    Abstract

    Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.

    View details for DOI 10.3390/s16091488

    View details for Web of Science ID 000385527700145

    View details for PubMedID 27649177

    View details for PubMedCentralID PMC5038762

  • Genetically encoded indicators of neuronal activity. Nature neuroscience Lin, M. Z., Schnitzer, M. J. 2016; 19 (9): 1142-1153

    Abstract

    Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.

    View details for DOI 10.1038/nn.4359

    View details for PubMedID 27571193

  • Structure-guided wavelength tuning in far-red fluorescent proteins. Current opinion in structural biology Ng, H., Lin, M. Z. 2016; 39: 124-133

    Abstract

    In recent years, protein engineers have succeeded in tuning the excitation spectra of natural fluorescent proteins from green wavelengths into orange and red wavelengths, resulting in the creation of a series of fluorescent proteins with emission in the far-red portions of the optical spectrum. These results have arisen from the synergistic combination of structural knowledge of fluorescent proteins, chemical intuition, and high-throughput screening methods. Here we review structural features found in autocatalytic far-red fluorescent proteins, and discuss how they add to our understanding of the biophysical mechanisms of wavelength tuning in biological chromophores.

    View details for DOI 10.1016/j.sbi.2016.07.010

    View details for PubMedID 27468111

  • A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nature biotechnology Chu, J., Oh, Y., Sens, A., Ataie, N., Dana, H., Macklin, J. J., Laviv, T., Welf, E. S., Dean, K. M., Zhang, F., Kim, B. B., Tang, C. T., Hu, M., Baird, M. A., Davidson, M. W., Kay, M. A., Fiolka, R., Yasuda, R., Kim, D. S., Ng, H., Lin, M. Z. 2016; 34 (7): 760-767

    Abstract

    Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

    View details for DOI 10.1038/nbt.3550

    View details for PubMedID 27240196

  • Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo CELL Yang, H. H., St-Pierre, F., Sun, X., Ding, X., Lin, M. Z., Clandinin, T. R. 2016; 166 (1): 245-257

    Abstract

    A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

    View details for DOI 10.1016/j.cell.2016.05.031

    View details for Web of Science ID 000380254400025

    View details for PubMedID 27264607

  • Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments. Developmental cell Welf, E. S., Driscoll, M. K., Dean, K. M., Schäfer, C., Chu, J., Davidson, M. W., Lin, M. Z., Danuser, G., Fiolka, R. 2016; 36 (4): 462-475

    Abstract

    The microenvironment determines cell behavior, but the underlying molecular mechanisms are poorly understood because quantitative studies of cell signaling and behavior have been challenging due to insufficient spatial and/or temporal resolution and limitations on microenvironmental control. Here we introduce microenvironmental selective plane illumination microscopy (meSPIM) for imaging and quantification of intracellular signaling and submicrometer cellular structures as well as large-scale cell morphological and environmental features. We demonstrate the utility of this approach by showing that the mechanical properties of the microenvironment regulate the transition of melanoma cells from actin-driven protrusion to blebbing, and we present tools to quantify how cells manipulate individual collagen fibers. We leverage the nearly isotropic resolution of meSPIM to quantify the local concentration of actin and phosphatidylinositol 3-kinase signaling on the surfaces of cells deep within 3D collagen matrices and track the many small membrane protrusions that appear in these more physiologically relevant environments.

    View details for DOI 10.1016/j.devcel.2016.01.022

    View details for PubMedID 26906741

    View details for PubMedCentralID PMC4784259

  • Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting SCIENTIFIC REPORTS Bajar, B. T., Wang, E. S., Lam, A. J., Kim, B. B., Jacobs, C. L., Howe, E. S., Davidson, M. W., Lin, M. Z., Chu, J. 2016; 6

    Abstract

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.

    View details for DOI 10.1038/srep20889

    View details for Web of Science ID 000370196500001

    View details for PubMedID 26879144

    View details for PubMedCentralID PMC4754705

  • Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens. Biochemistry Yan, D., Weisshaar, M., Lamb, K., Chung, H. K., Lin, M. Z., Plemper, R. K. 2015; 54 (36): 5589-5604

    Abstract

    Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either pathogen or both, we have attempted co-infection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted the assay to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppressed the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nanoluciferase, the assay performed well on a human respiratory cell line and supports multicycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the National Institutes of Health Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed antimyxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose-response counterscreens using a standard recRSV reporter strain expressing Renilla luciferase.

    View details for DOI 10.1021/acs.biochem.5b00623

    View details for PubMedID 26307636

    View details for PubMedCentralID PMC4719150

  • Optical control of biological processes by light-switchable proteins WILEY INTERDISCIPLINARY REVIEWS-DEVELOPMENTAL BIOLOGY Fan, L. Z., Lin, M. Z. 2015; 4 (5): 545-554

    Abstract

    Cellular processes such as proliferation, differentiation, or migration depend on precise spatiotemporal coordination of protein activities. Correspondingly, reaching a quantitative understanding of cellular behavior requires experimental approaches that enable spatial and temporal modulation of protein activity. Recently, a variety of light-sensitive protein domains have been engineered as optogenetic actuators to spatiotemporally control protein activity. In the present review, we discuss the principle of these optical control methods and examples of their applications in modulating signaling pathways. By controlling protein activity with spatiotemporal specificity, tunable dynamics, and quantitative control, light-controllable proteins promise to accelerate our understanding of cellular and organismal biology. WIREs Dev Biol 2015, 4:545-554. doi: 10.1002/wdev.188 For further resources related to this article, please visit the WIREs website.

    View details for DOI 10.1002/wdev.188

    View details for Web of Science ID 000359429900006

    View details for PubMedID 25858669

    View details for PubMedCentralID PMC4529752

  • Tunable and reversible drug control of protein production via a self-excising degron. Nature chemical biology Chung, H. K., Jacobs, C. L., Huo, Y., Yang, J., Krumm, S. A., Plemper, R. K., Tsien, R. Y., Lin, M. Z. 2015; 11 (9): 713-720

    Abstract

    An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe small molecule-assisted shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, resulting in the production of an untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized copies of the protein. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto an RNA virus for which no licensed inhibitors exist. As SMASh does not require the permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh involves only a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.

    View details for DOI 10.1038/nchembio.1869

    View details for PubMedID 26214256

    View details for PubMedCentralID PMC4543534

  • Tunable and reversible drug control of protein production via a self-excising degron NATURE CHEMICAL BIOLOGY Chung, H. K., Jacobs, C. L., Huo, Y., Yang, J., Krumm, S. A., Plemper, R. K., Tsien, R. Y., Lin, M. Z. 2015; 11 (9): 713-?

    Abstract

    An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe small molecule-assisted shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, resulting in the production of an untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized copies of the protein. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto an RNA virus for which no licensed inhibitors exist. As SMASh does not require the permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh involves only a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.

    View details for DOI 10.1038/NCHEMBIO.1869

    View details for Web of Science ID 000359954700016

    View details for PubMedCentralID PMC4543534

  • Optical control of biological processes by light-switchable proteins. Wiley interdisciplinary reviews. Developmental biology Fan, L. Z., Lin, M. Z. 2015; 4 (5): 545-554

    Abstract

    Cellular processes such as proliferation, differentiation, or migration depend on precise spatiotemporal coordination of protein activities. Correspondingly, reaching a quantitative understanding of cellular behavior requires experimental approaches that enable spatial and temporal modulation of protein activity. Recently, a variety of light-sensitive protein domains have been engineered as optogenetic actuators to spatiotemporally control protein activity. In the present review, we discuss the principle of these optical control methods and examples of their applications in modulating signaling pathways. By controlling protein activity with spatiotemporal specificity, tunable dynamics, and quantitative control, light-controllable proteins promise to accelerate our understanding of cellular and organismal biology. WIREs Dev Biol 2015, 4:545-554. doi: 10.1002/wdev.188 For further resources related to this article, please visit the WIREs website.

    View details for DOI 10.1002/wdev.188

    View details for PubMedID 25858669

    View details for PubMedCentralID PMC4529752

  • Designs and sensing mechanisms of genetically encoded fluorescent voltage indicators. Current opinion in chemical biology St-Pierre, F., Chavarha, M., Lin, M. Z. 2015; 27: 31-38

    Abstract

    Neurons tightly regulate the electrical potential difference across the plasma membrane with millivolt accuracy and millisecond resolution. Membrane voltage dynamics underlie the generation of an impulse, the transduction of impulses from one end of the neuron to the other, and the release of neurotransmitters. Imaging these voltage dynamics in multiple neurons simultaneously is therefore crucial for understanding how neurons function together within circuits in intact brains. Genetically encoded fluorescent voltage sensors have long been desired to report voltage in defined subsets of neurons with optical readout. In this review, we discuss the diverse strategies used to design and optimize protein-based voltage sensors, and highlight the chemical mechanisms by which different classes of reporters sense voltage. To guide neuroscientists in choosing an appropriate sensor for their applications, we also describe operating trade-offs of each class of voltage indicators.

    View details for DOI 10.1016/j.cbpa.2015.05.003

    View details for PubMedID 26079047

    View details for PubMedCentralID PMC4553077

  • Experimental systems for optogenetic control of protein activity with photodissociable fluorescent proteins Conference on Optical Techniques in Neurosurgery, Neurophotonics, and Optogenetics II Zhou, X. X., Lin, M. Z. SPIE-INT SOC OPTICAL ENGINEERING. 2015

    View details for DOI 10.1117/12.2081242

    View details for Web of Science ID 000353410600040

  • Investigating neuronal function with optically controllable proteins. Frontiers in molecular neuroscience Zhou, X. X., Pan, M., Lin, M. Z. 2015; 8: 37-?

    Abstract

    In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.

    View details for DOI 10.3389/fnmol.2015.00037

    View details for PubMedID 26257603

    View details for PubMedCentralID PMC4508517

  • Optobiology: optical control of biological processes via protein engineering BIOCHEMICAL SOCIETY TRANSACTIONS Kim, B., Lin, M. Z. 2013; 41: 1183-1188

    Abstract

    Enabling optical control over biological processes is a defining goal of the new field of optogenetics. Control of membrane voltage by natural rhodopsin family ion channels has found widespread acceptance in neuroscience, due to the fact that these natural proteins control membrane voltage without further engineering. In contrast, optical control of intracellular biological processes has been a fragmented effort, with various laboratories engineering light-responsive properties into proteins in different manners. In the present article, we review the various systems that have been developed for controlling protein functions with light based on vertebrate rhodopsins, plant photoregulatory proteins and, most recently, the photoswitchable fluorescent protein Dronpa. By allowing biology to be controlled with spatiotemporal specificity and tunable dynamics, light-controllable proteins will find applications in the understanding of cellular and organismal biology and in synthetic biology.

    View details for DOI 10.1042/BST20130150

    View details for Web of Science ID 000324910200011

    View details for PubMedID 24059506

  • Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applications. Current opinion in chemical biology Zhou, X. X., Lin, M. Z. 2013; 17 (4): 682-690

    Abstract

    Reversibly photoswitchable fluorescent proteins (RSFPs) are fluorescent proteins whose fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner. In the last 10 years, many new RSFPs have been developed and novel applications in cell imaging discovered that rely on their photoswitching properties. This review will describe research on the mechanisms of reversible photoswitching and recent applications using RSFPs. While cis-trans isomerization of the chromophore is believed to be the general mechanism for most RSFPs, structural studies reveal diversity in the details of photoswitching mechanisms, including different effects of protonation, chromophore planarity, and pocket flexibility. Applications of RSFPs include new types of live-cell superresolution imaging, tracking of protein movements and interactions, information storage, and optical control of protein activity.

    View details for DOI 10.1016/j.cbpa.2013.05.031

    View details for PubMedID 23876529

  • Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy NATURE NEUROSCIENCE Butko, M. T., Yang, J., Geng, Y., Kim, H. J., Jeon, N. L., Shu, X., Mackey, M. R., Ellisman, M. H., Tsien, R. Y., Lin, M. Z. 2012; 15 (12): 1742-?

    Abstract

    Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.

    View details for DOI 10.1038/nn.3246

    View details for Web of Science ID 000311706700022

    View details for PubMedID 23103964

    View details for PubMedCentralID PMC3509268

  • New Alternately Colored FRET Sensors for Simultaneous Monitoring of Zn2+ in Multiple Cellular Locations PLOS ONE Miranda, J. G., Weaver, A. L., Qin, Y., Park, J. G., Stoddard, C. I., Lin, M. Z., Palmer, A. E. 2012; 7 (11)

    Abstract

    Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn²⁺based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²⁺ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²⁺ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²⁺. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²⁺ uptake simultaneously in two compartments, revealing that nuclear Zn²⁺ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²⁺ more slowly and with a delay of 600-700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²⁺ and reveal that nuclear Zn²⁺ is buffered at a higher level than cytosolic Zn²⁺.

    View details for DOI 10.1371/journal.pone.0049371

    View details for Web of Science ID 000311885300033

    View details for PubMedID 23173058

    View details for PubMedCentralID PMC3500285

  • Optical Control of Protein Activity by Fluorescent Protein Domains SCIENCE Zhou, X. X., Chung, H. K., Lam, A. J., Lin, M. Z. 2012; 338 (6108): 810-814

    Abstract

    Fluorescent proteins (FPs) are widely used as optical sensors, whereas other light-absorbing domains have been used for optical control of protein localization or activity. Here, we describe light-dependent dissociation and association in a mutant of the photochromic FP Dronpa, and we used it to control protein activities with light. We created a fluorescent light-inducible protein design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains associate and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control.

    View details for DOI 10.1126/science.1226854

    View details for Web of Science ID 000310839500048

    View details for PubMedID 23139335

    View details for PubMedCentralID PMC3702057

  • Improving FRET dynamic range with bright green and red fluorescent proteins NATURE METHODS Lam, A. J., St-Pierre, F., Gong, Y., Marshall, J. D., Cranfill, P. J., Baird, M. A., McKeown, M. R., Wiedenmann, J., Davidson, M. W., Schnitzer, M. J., Tsien, R. Y., Lin, M. Z. 2012; 9 (10): 1005-?

    Abstract

    A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

    View details for DOI 10.1038/NMETH.2171

    View details for Web of Science ID 000309519300023

    View details for PubMedID 22961245

    View details for PubMedCentralID PMC3461113

  • Beyond the rainbow: new fluorescent proteins brighten the infrared scene NATURE METHODS Lin, M. Z. 2011; 8 (9): 726-728

    Abstract

    Two fluorescent proteins that emit in the far-red and infrared range for imaging applications in cells and in vivo are described.

    View details for Web of Science ID 000294439100007

    View details for PubMedID 21878918

  • Toward the Second Generation of Optogenetic Tools JOURNAL OF NEUROSCIENCE Knoepfel, T., Lin, M. Z., Levskaya, A., Tian, L., Lin, J. Y., Boyden, E. S. 2010; 30 (45): 14998-15004

    Abstract

    This mini-symposium aims to provide an integrated perspective on recent developments in optogenetics. Research in this emerging field combines optical methods with targeted expression of genetically encoded, protein-based probes to achieve experimental manipulation and measurement of neural systems with superior temporal and spatial resolution. The essential components of the optogenetic toolbox consist of two kinds of molecular devices: actuators and reporters, which respectively enable light-mediated control or monitoring of molecular processes. The first generation of genetically encoded calcium reporters, fluorescent proteins, and neural activators has already had a great impact on neuroscience. Now, a second generation of voltage reporters, neural silencers, and functionally extended fluorescent proteins hold great promise for continuing this revolution. In this review, we will evaluate and highlight the limitations of presently available optogenic tools and discuss where these technologies and their applications are headed in the future.

    View details for DOI 10.1523/JNEUROSCI.4190-10.2010

    View details for Web of Science ID 000284096300014

    View details for PubMedID 21068304

    View details for PubMedCentralID PMC2997431

  • TimeSTAMP tagging of newly synthesized proteins. Current protocols in protein science / editorial board, John E. Coligan ... [et al.] Lin, M. Z., Tsien, R. Y. 2010; Chapter 26: Unit 26 5-?

    Abstract

    The ability to quantify or visualize newly synthesized proteins has important uses in cell biology. For example, a researcher may wish to quantify basal or inducible rates of translation of a specific gene of interest, or detect subcellular locations of newly synthesized copies of a protein in order to study the role of new protein synthesis in the growth of specialized cellular structures. In this unit, the TimeSTAMP method for labeling of newly synthesized copies of a protein of interest is described. In the TimeSTAMP method, the experimenter expresses a protein of interest as a fusion with a cis-acting protease and an epitope tag, both of which are removed by default protease activity. Addition of a specific protease inhibitor then allows preservation of the tag on subsequently synthesized proteins. Finally, the tag is detected by immunological methods.

    View details for DOI 10.1002/0471140864.ps2605s59

    View details for PubMedID 20155731

    View details for PubMedCentralID PMC2853805

  • Autofluorescent Proteins with Excitation in the Optical Window for Intravital Imaging in Mammals CHEMISTRY & BIOLOGY Lin, M. Z., McKeown, M. R., Ng, H., Aguilera, T. A., Shaner, N. C., Campbell, R. E., Adams, S. R., Gross, L. A., Ma, W., Alber, T., Tsien, R. Y. 2009; 16 (11): 1169-1179

    Abstract

    Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, because of absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the "optical window" above 600 nm are therefore highly desirable. We report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.

    View details for DOI 10.1016/j.chembiol.2009.10.009

    View details for Web of Science ID 000273207900010

    View details for PubMedID 19942140

  • Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome SCIENCE Shu, X., Royant, A., Lin, M. Z., Aguilera, T. A., Lev-Ram, V., Steinbach, P. A., Tsien, R. Y. 2009; 324 (5928): 804-807

    Abstract

    Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M(-1) cm(-1); and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.

    View details for DOI 10.1126/science.1168683

    View details for Web of Science ID 000265832400052

    View details for PubMedID 19423828

  • Characterization of Engineered Channel rhodopsin Variants with Improved Properties and Kinetics BIOPHYSICAL JOURNAL Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. 2009; 96 (5): 1803-1814

    Abstract

    Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile(170) of ChEF to Val (yielding "ChIEF") accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.

    View details for DOI 10.1016/j.bpj.2008.11.034

    View details for Web of Science ID 000266376500012

    View details for PubMedID 19254539

  • A drug-controllable tag for visualizing newly synthesized proteins in cells and whole animals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lin, M. Z., Glenn, J. S., Tsien, R. Y. 2008; 105 (22): 7744-7749

    Abstract

    Research on basic cellular processes involving local production or delivery of proteins, such as activity-dependent synaptic modification in neurons, would benefit greatly from a robust, nontoxic method to visualize selectively newly synthesized copies of proteins of interest within cells, tissues, or animals. We report a technique for covalent labeling of newly synthesized proteins of interest based on drug-dependent preservation of epitope tags. Epitope tags are removed from proteins of interest immediately after translation by the activity of a sequence-specific protease until the time a protease inhibitor is added, after which newly synthesized protein copies retain their tags. This method, which we call TimeSTAMP for time-specific tagging for the age measurement of proteins, allows sensitive and nonperturbative visualization and quantification of newly synthesized proteins of interest with exceptionally tight temporal control. We demonstrate applications of TimeSTAMP in retrospectively identifying growing synapses in cultured neurons and in visualizing the distribution of recently synthesized proteins in intact fly brains.

    View details for DOI 10.1073/pnas.0803060105

    View details for Web of Science ID 000256648600023

    View details for PubMedID 18511556

    View details for PubMedCentralID PMC2402386

  • Selective labeling of proteins with chemical probes in living cells PHYSIOLOGY Lin, M. Z., Wang, L. 2008; 23 (3): 131-141

    Abstract

    Selective labeling of proteins with small molecules introduces novel chemical and physical properties into proteins, enabling the target protein to be investigated or manipulated with various techniques. Different methods for labeling proteins in living cells have been developed by using protein domains, small peptides, or single amino acids. Their application in cells and in vivo has yielded novel insights into diverse biological processes.

    View details for Web of Science ID 000256685400002

    View details for PubMedID 18556466

  • Improving the photostability of bright monomeric orange and red fluorescent proteins NATURE METHODS Shaner, N. C., Lin, M. Z., McKeown, M. R., Steinbach, P. A., Hazelwood, K. L., Davidson, M. W., Tsien, R. Y. 2008; 5 (6): 545-551

    Abstract

    All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

    View details for Web of Science ID 000256308200019

    View details for PubMedID 18454154

  • Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse NEURON Sahin, M., Greer, P. L., Lin, M. Z., Poucher, H., Eberhart, J., Schmidt, S., Wright, T. M., Shamah, S. M., O'Connel, S., Cowan, C. W., Hu, L., Goldberg, J. L., Debant, A., Corfas, G., Krull, C. E., Greenberg, M. E. 2005; 46 (2): 191-204

    Abstract

    Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.

    View details for DOI 10.1016/j.neuron.2005.01.030

    View details for Web of Science ID 000228674800007

    View details for PubMedID 15848799

  • Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis DEVELOPMENTAL CELL Datta, S. R., Ranger, A. M., Lin, M. Z., Sturgill, J. F., Ma, Y. C., Cowan, C. W., Dikkes, P., Korsmeyer, S. J., Greenberg, M. E. 2002; 3 (5): 631-643

    Abstract

    Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.

    View details for Web of Science ID 000179230900007

    View details for PubMedID 12431371

  • Neurotrophins use the Erk5 pathway to mediate a retrograde survival response NATURE NEUROSCIENCE Watson, F. L., Heerssen, H. M., BHATTACHARYYA, A., Klesse, L., Lin, M. Z., Segal, R. A. 2001; 4 (10): 981-988

    Abstract

    Growth factors synthesized and released by target tissues promote survival and differentiation of innervating neurons. This retrograde signal begins when growth factors bind receptors at nerve terminals. Activated receptors are then endocytosed and transported through the axon to the cell body. Here we show that the mitogen-activated protein kinase (MAPK) signaling pathways used by neurotrophins during retrograde signaling differ from those used following direct stimulation of the cell soma. During retrograde signaling, endocytosed neurotrophin receptors (Trks) activate the extracellular signal-related protein kinase 5 (Erk5) pathway, leading to nuclear translocation of Erk5, phosphorylation of CREB, and enhanced neuronal survival. In contrast, Erk1/2, which mediates nuclear responses following direct cell body stimulation, does not transmit a retrograde signal. Thus, the Erk5 pathway has a unique function in retrograde signaling. Differential activation of distinct MAPK pathways may enable an individual growth factor to relay information that specifies the location and the nature of stimulation.

    View details for Web of Science ID 000171325800011

    View details for PubMedID 11544482

  • EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin CELL Shamah, S. M., Lin, M. Z., Goldberg, J. L., Estrach, S., Sahin, M., Hu, L., Bazalakova, M., NEVE, R. L., Corfas, G., Debant, A., Greenberg, M. E. 2001; 105 (2): 233-244

    Abstract

    Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.

    View details for Web of Science ID 000168384300010

    View details for PubMedID 11336673

  • Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms CELL Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z., Zubiaga, A., Hua, X. X., Fan, G. P., Greenberg, M. E. 2001; 104 (3): 365-376

    Abstract

    The mechanisms by which neural stem cells give rise to neurons, astrocytes, or oligodendrocytes are beginning to be elucidated. However, it is not known how the specification of one cell lineage results in the suppression of alternative fates. We find that in addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis. Thus, two distinct mechanisms are involved in the activation and suppression of gene expression during cell-fate specification by neurogenin.

    View details for Web of Science ID 000167003100007

    View details for PubMedID 11239394

  • EphB receptors interact with NMDA receptors and regulate excitatory synapse formation CELL Dalva, M. B., Takasu, M. A., Lin, M. Z., Shamah, S. M., Hu, L., Gale, N. W., Greenberg, M. E. 2000; 103 (6): 945-956

    Abstract

    EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, as perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function.

    View details for Web of Science ID 000165801300013

    View details for PubMedID 11136979

  • Rapid nuclear responses to target-derived neurotrophins require retrograde transport of ligand-receptor complex JOURNAL OF NEUROSCIENCE Watson, F. L., Heerssen, H. M., Moheban, D. B., Lin, M. Z., Sauvageot, C. M., BHATTACHARYYA, A., Pomeroy, S. L., Segal, R. A. 1999; 19 (18): 7889-7900

    Abstract

    Target-derived neurotrophins initiate signals that begin at nerve terminals and cross long distances to reach the cell bodies and regulate gene expression. Neurotrophin receptors, Trks, themselves serve as retrograde signal carriers. However, it is not yet known whether the retrograde propagation of Trk activation reflects movement of Trk receptors from neurites to cell bodies or reflects serial activation of stationary Trk molecules. Here, we show that neurotrophins selectively applied to distal neurites of sensory neurons rapidly induce phosphorylation of the transcription factor cAMP response element-binding protein (CREB) and also cause a slower increase in Fos protein expression. Both nuclear responses require activation of neurotrophin receptors (Trks) at distal nerve endings and retrograde propagation of Trk activation to the nerve cell bodies. Using photobleach and recovery techniques to follow biologically active, green fluorescent protein (GFP)-tagged BDNF receptors (TrkB-GFP) in live cells during retrograde signaling, we show that TrkB-GFP moves rapidly from neurites to the cell bodies. This rapid movement requires ligand binding, Trk kinase activity, and intact axonal microtubules. When they reach the cell bodies, the activated TrkB receptors are in a complex with ligand. Thus, the retrograde propagation of activated TrkB from neurites to cell bodies, although rapid, reflects microtubule-dependent transport of phosphorylated Trk-ligand complexes. Moreover, the relocation of activated Trk receptors from nerve endings to cell bodies is required for nuclear signaling responses. Together, these data support a model of retrograde signaling whereby rapid vesicular transport of ligand-receptor complex from the neurites to the cell bodies mediates the nuclear responses.

    View details for Web of Science ID 000082539900023

    View details for PubMedID 10479691

  • Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor CELL Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., ANDERSON, M. J., Arden, K. C., Blenis, J., Greenberg, M. E. 1999; 96 (6): 857-868

    Abstract

    Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

    View details for Web of Science ID 000079300100012

    View details for PubMedID 10102273