Next generation single-molecule techniques: Imaging, labeling, and manipulation in vitro and in cellulo.

Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.

A Guide to Fluorescence Lifetime Microscopy and Förster's Resonance Energy Transfer in Neuroscience.

Fluorescence lifetime microscopy (FLIM) and Förster's resonance energy transfer (FRET) are advanced optical tools that neuroscientists can employ to interrogate the structure and function of complex biological systems in vitro and in vivo using light. In neurobiology they are primarily used to study protein-protein interactions, to study conformational changes in protein complexes, and to monitor genetically encoded FRET-based biosensors. These methods are ideally suited to optically monitor changes in neurons that are triggered optogenetically. Utilization of this technique by neuroscientists has been limited, since a broad understanding of FLIM and FRET requires familiarity with the interactions of light and matter on a quantum mechanical level, and because the ultra-fast instrumentation used to measure fluorescent lifetimes and resonance energy transfer are more at home in a physics lab than in a biology lab. In this overview, we aim to help neuroscientists overcome these obstacles and thus feel more comfortable with the FLIM-FRET method. Our goal is to aid researchers in the neuroscience community to achieve a better understanding of the fundamentals of FLIM-FRET and encourage them to fully leverage its powerful ability as a research tool. Published 2020. U.S. Government.

Development of a Cysteine-Conjugatable Disulfide FRET Probe: Influence of Charge on Linker Cleavage and Payload Trafficking for an Anti-HER2 Antibody Conjugate.

Disulfide-linked bioconjugates allow the delivery of pharmacologically active or other cargo to specific tissues in a redox-sensitive fashion. However, an understanding of the kinetics, subcellular distribution, and mechanism of disulfide cleavage in such bioconjugates is generally lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based FRET probe that can be readily synthesized, modified, and conjugated to a cysteine-containing biomolecule to enable real-time monitoring of disulfide cleavage during receptor-mediated endocytosis in cells. We demonstrate the utility of this probe to study disulfide reduction during HER2 receptor-mediated uptake of a Cys-engineered anti-HER2 THIOMAB antibody. We found that introduction of positive, but not negative, charges in the probe improved retention of the BODIPY catabolite. This permitted the observation of significant disulfide cleavage in endosomes or lysosomes on par with proteolytic cleavage of a similarly charged valine-citrulline peptide-based probe. In general, the FRET probe we describe should enable real-time cellular monitoring of disulfide cleavage in other targeted delivery systems for mechanistic or diagnostic applications. Furthermore, modifications to the released BODIPY moiety permit evaluation of physicochemical properties that govern lysosomal egress or retention, which may have implications for the development of next-generation antibody-drug conjugates.

Optical voltage imaging in neurons: moving from technology development to practical tool.

A central goal in neuroscience is to determine how the brain's neuronal circuits generate perception, cognition and emotions and how these lead to appropriate behavioural actions. A methodological platform based on genetically encoded voltage indicators (GEVIs) that enables the monitoring of large-scale circuit dynamics has brought us closer to this ambitious goal. This Review provides an update on the current state of the art and the prospects of emerging optical GEVI imaging technologies.

Strategic labelling approaches for RNA single-molecule spectroscopy.

Most single-molecule techniques observing RNA in vitro or in vivo require fluorescent labels that have to be connected to the RNA of interest. In recent years, a plethora of methods has been developed to achieve site-specific labelling, in many cases under near-native conditions. Here, we review chemical as well as enzymatic labelling methods that are compatible with single-molecule fluorescence spectroscopy or microscopy and show how these can be combined to offer a large variety of options to site-specifically place one or more labels in an RNA of interest. By either chemically forming a covalent bond or non-covalent hybridization, these techniques are prerequisites to perform state-of-the-art single-molecule experiments.

Role of green fluorescent proteins and their variants in development of FRET-based sensors.

Since the last decade, a lot of advancement has been made to understand biological processes involving complex intracellular pathways. The major challenge faced was monitoring and trafficking of metabolites in real time. Although a range of quantitative and imaging techniques have been developed so far, the discovery of green fluorescent proteins (GFPs) has revolutionized the advancement in the field of metabolomics. GFPs and their variants have enabled researchers to 'paint' a wide range of biological molecules. Fluorescence resonance energy transfer (FRET)-based genetically encoded sensors is a promising technology to decipher the real-time monitoring of the cellular events inside living cells. GFPs and their variants, due to their intrinsic fluorescence properties, are extensively being used nowadays in cell-based assays. This review focuses on structure and function of GFP and its derivatives, mechanism emission and their use in the development of FRET-based sensors for metabolites.

Genetically encoded fluorescent voltage indicators: are we there yet?

In order to understand how brain activity produces adaptive behavior we need large-scale, high-resolution recordings of neuronal activity. Fluorescent genetically encoded voltage indicators (GEVIs) offer the potential for these recordings to be performed chronically from targeted cells in a minimally invasive manner. As the number of GEVIs successfully tested for in vivo use grows, so has the number of open questions regarding the improvements that would facilitate broad adoption of this technology that surpasses mere 'proof of principle' studies. Our aim in this review is not to provide a status check of the current state of the field, as excellent publications covering this topic already exist. Here, we discuss specific questions regarding GEVI development and application that we think are crucial in achieving this goal.

Genetically Encoded Voltage Indicators: Opportunities and Challenges.

UNLABELLED: A longstanding goal in neuroscience is to understand how spatiotemporal patterns of neuronal electrical activity underlie brain function, from sensory representations to decision making. An emerging technology for monitoring electrical dynamics, voltage imaging using genetically encoded voltage indicators (GEVIs), couples the power of genetics with the advantages of light. Here, we review the properties that determine indicator performance and applicability, discussing both recent progress and technical limitations. We then consider GEVI applications, highlighting studies that have already deployed GEVIs for biological discovery. We also examine which classes of biological questions GEVIs are primed to address and which ones are beyond their current capabilities. As GEVIs are further developed, we anticipate that they will become more broadly used by the neuroscience community to eavesdrop on brain activity with unprecedented spatiotemporal resolution. SIGNIFICANCE STATEMENT: Genetically encoded voltage indicators are engineered light-emitting protein sensors that typically report neuronal voltage dynamics as changes in brightness. In this review, we systematically discuss the current state of this emerging method, considering both its advantages and limitations for imaging neural activity. We also present recent applications of this technology and discuss what is feasible now and what we anticipate will become possible with future indicator development. This review will inform neuroscientists of recent progress in the field and help potential users critically evaluate the suitability of genetically encoded voltage indicator imaging to answer their specific biological questions.

Fluorescence resonance energy transfer (FRET)-based biosensors: visualizing cellular dynamics and bioenergetics.

Förster (or fluorescence) resonance energy transfer (FRET) is a process involving the radiation-less transfer of energy from a "donor" fluorophore to an "acceptor" fluorophore. FRET technology enables the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as the monitoring of protein-protein interactions, protein-DNA interactions, and protein conformational changes. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time. Lately, applications of FRET-based biosensors range from basic biological to biomedical disciplines. Despite the diverse applications of FRET, FRET-based sensors still face many challenges. There is an increasing need for higher fluorescence resolution and improved specificity of FRET biosensors. Additionally, as more FRET-based technologies extend to medical diagnostics, the affordability of FRET reagents becomes a significant concern. Here, we will review current advances and limitations of FRET-based biosensor technology and discuss future FRET applications.

FRET microscopy in the living cell: different approaches, strengths and weaknesses.

New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter-molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra-molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio-temporal protein-protein interaction studies open new avenues for FRET in biological research.

The new era of bioluminescence resonance energy transfer technology.

Bioluminescence resonance energy transfer (BRET) assay is a comparatively new cell-based assay technology that is assuming more prominent roles in the field of studying protein-protein interactions, protein dimerization and signal transduction. In the last few years BRET related research has gained significant momentum in terms of adding versatility in the assay format as well as a variety of new applications where it has been suitably used. Beyond the scope of quantitative measurement of protein-protein interactions and protein dimerization, molecular imaging applications based on BRET assays have broaden its scope as a great tool for high-throughput screening (HTS) of pharmacologically important compounds. This article will highlight the landmarks in BRET research, with those which have significant contributions towards making it an attractive single format assay that shuttles between in vitro and in vivo measurements.

FRET for lab-on-a-chip devices - current trends and future prospects.

This review focuses on the use of Förster Resonance Energy Transfer (FRET) to monitor intra- and intermolecular reactions occurring in microfluidic reactors. Microfluidic devices have recently been used for performing highly efficient and miniaturised biological assays for the analysis of biological entities such as cells, proteins and nucleic acids. Microfluidic assays are characterised by nanolitre to femtolitre reaction volumes, which necessitates the adoption of a sensitive optical detection scheme. FRET serves as a strong 'spectroscopic ruler' for elucidating the tertiary structure of biomolecules, as the efficiency of the non-radiative energy transfer is extremely sensitive to nanoscale changes in the separation between donor and acceptor markers attached to the biomolecule of interest. In this review, we will review the implementation of various microfluidic assays which employ FRET for diverse applications in the biomedical field, along with the advantages and disadvantages of the various approaches. The future prospects for development of microfluidic devices incorporating FRET detection will be discussed.

Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors.

The spatiotemporal patterns of ion and metabolite levels in living cells are important in understanding signal transduction and metabolite flux. Imaging approaches using genetically encoded sensors are ideal for detecting such molecule dynamics, which are hard to capture otherwise. Recent years have seen iterative improvements and evaluations of sensors, which in turn are starting to make applications in more challenging experimental settings possible. In this review, we will introduce recent progress made in the variety and properties of biosensors, and how biosensors are used for the measurement of metabolite and ion in live cells. The emerging field of applications, such as parallel imaging of two separate molecules, high-resolution transport studies and high-throughput screening using biosensors, will be discussed.

FRET and mechanobiology.

Since the development of green fluorescent protein (GFP) and other fluorescent proteins (FPs) with distinct colors, genetically-encoded probes and biosensors have been widely applied to visualize the molecular localization and activities in live cells. In particular, biosensors based on fluorescence resonance energy transfer (FRET) have significantly advanced our understanding of the dynamic molecular hierarchy at subcellular levels. These biosensors have also been extensively applied in recent years to study how cells perceive the mechanical environment and transmit it into intracellular molecular signals (i.e. mechanotransduction). In this review, we will first provide a brief introduction of the recent development of FPs. Different FRET biosensors based on FPs will then be described. The last part of the review will be dedicated to the introduction of examples applying FRET biosensors to visualize mechanotransduction in live cells. In summary, the integration of FRET technology and the different cutting-edge mechanical stimulation systems can provide powerful tools to allow the elucidation of the mechanisms regulating mechanobiology at cellular and molecular levels in normal and pathophysiological conditions.

Elucidation of the gating of the GIRK channel using a spectroscopic approach.

The traditional view of G protein-coupled receptor (GPCR)-mediated signalling puts the players in this signalling cascade, namely the GPCR, the G protein and its effector, as individual components in space, where the signalling specificity is obtained mainly by the interaction of the GPCR and the Galpha subunits of the G protein. A question is then raised as to how fidelity in receptor signalling is achieved, given that many systems use the same components of the G protein signalling machinery. One possible mechanism for obtaining the specific flow of the downstream signals, from the activated G protein to its specific effector target, in a timely manner, is compartmentalization, a spatial arrangement of the complex in a rather restricted space. Here we review our recent findings related to these issues, using the G protein-coupled potassium channel (GIRK) as a model effector and fluorescence-based approaches to reveal how the signalling complex is arranged and how the G protein exerts its action to activate the GIRK channel in intact cells.

Potential clinical applications of quantum dots.

The use of luminescent colloidal quantum dots in biological investigations has increased dramatically over the past several years due to their unique size-dependent optical properties and recent advances in biofunctionalization. In this review, we describe the methods for generating high-quality nanocrystals and report on current and potential uses of these versatile materials. Numerous examples are provided in several key areas including cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. We also explore toxicity issues surrounding these materials and speculate about the future uses of quantum dots in a clinical setting.

Fluorescent protein tools for studying protein dynamics in living cells: a review.

We have witnessed remarkable advances over the past decade in the application of optical techniques to visualize the genetically encoded fluorescent proteins (FPs) in living systems. The imaging of the FPs inside living cells has become an essential tool for studies of cell biology and physiology. FPs are now available that span the visible spectrum from deep blue to deep red, providing a wide choice of genetically encoded fluorescent markers. Furthermore, some FPs have been identified that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells. These additions to the FP toolbox are now being used for some very innovative live-cell imaging applications. Here, we will highlight the characteristics and uses of a few of these exceptional probes. Many different optical methods can be combined with the FPs from marine organisms to provide quantitative measurements in living systems.

The BRET technology and its application to screening assays.

The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor. Since its first description in 1999, several versions of BRET have been described using different substrates and energy donor/acceptor couples. Today, BRET is considered as one of the most versatile techniques for studying the dynamics of protein-protein interactions in living cells. Various studies have applied BRET-based assays to screen new receptor ligands and inhibitors of disease-related-proteases. Inhibitors of protein-protein interactions are likely to become a new major class of therapeutic drugs, and BRET technology is expected to play an important role in the identification of such compounds. This review describes the original BRET-based methodology, more recent variants, and potential applications to drug screening.