Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

Non fitting based FRET–FLIM analysis approaches applied to quantify protein–protein interactions in live cells

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET–FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET–FLIM to still be quantitative.     

Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.

BACKGROUND: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in F?rster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. METHODS: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. RESULTS: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET. CONCLUSIONS: We report a well-designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal-based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET.     

Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces

The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions—the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton’s tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions.     

Techniques for detecting protein-protein interactions in living cells: principles,limitations, and recent progress

Detecting protein-protein interactions(PPIs) provides fundamental information for understanding biochemical processes such as the transduction of signals from one cellular location to another; however, traditional biochemical techniques cannot provide sufficient spatio-temporal information to elucidate these molecular interactions in living cells. Over the past decade, several new techniques have enabled the identification and characterization of PPIs. In this review, we summarize three main techniques for detecting PPIs in vivo, focusing on their basic principles and applications in biological studies. We place a special emphasis on their advantages and limitations, and, in particular, we introduced some uncommon new techniques, such as single-molecule FRET(smFRET), FRET-fluorescence lifetime imaging microscopy(FRET-FLIM), cytoskeleton-based assay for protein-protein interaction(CAPPI) and single-molecule protein proximity index(smPPI), highlighting recent improvements to the established techniques. We hope that this review will provide a valuable reference to enable researchers to select the most appropriate technique for detecting PPIs.     

Combining mechanical and optical approaches to dissect cellular mechanobiology

Mechanical force modulates a wide array of cell physiological processes. Cells sense and respond to mechanical stimuli using a hierarchy of structural complexes spanning multiple length scales, including force-sensitive molecules and cytoskeletal networks. Understanding mechanotransduction, i.e., the process by which cells convert mechanical inputs into biochemical signals, has required the development of novel biophysical tools that allow for probing of cellular and subcellular components at requisite time, length, and force scales and technologies that track the spatio-temporal dynamics of relevant biomolecules. In this review, we begin by discussing the underlying principles and recent applications of atomic force microscopy, magnetic twisting cytometry, and traction force microscopy, three tools that have been widely used for measuring the mechanical properties of cells and for probing the molecular basis of cellular mechanotransduction. We then discuss how such tools can be combined with advanced fluorescence methods for imaging biochemical processes in living cells in the context of three specific problem spaces. We first focus on fluorescence resonance energy transfer, which has enabled imaging of intra- and inter-molecular interactions and enzymatic activity in real time based on conformational changes in sensor molecules. Next, we examine the use of fluorescence methods to probe force-dependent dynamics of focal adhesion proteins. Finally, we discuss the use of calcium ratiometric signaling to track fast mechanotransductive signaling dynamics. Together, these studies demonstrate how single-cell biomechanical tools can be effectively combined with molecular imaging technologies for elucidating mechanotransduction processes and identifying mechanosensitive proteins.     

Quantitative imaging of protein interactions in the cell nucleus

Over the past decade, genetically encoded fluorescent proteins have become widely used as noninvasive markers in living cells. The development of fluorescent proteins, coupled with advances in digital imaging, has led to the rapid evolution of live-cell imaging methods. These approaches are being applied to address biological questions of the recruitment, co-localization, and interactions of specific proteins within particular subcellular compartments. In the wake of this rapid progress, however, come important issues associated with the acquisition and analysis of ever larger and more complex digital imaging data sets. Using protein localization in the mammalian cell nucleus as an example, we will review some recent developments in the application of quantitative imaging to analyze subcellular distribution and co-localization of proteins in populations of living cells. In this report, we review the principles of acquiring fluorescence resonance energy transfer (FRET) microscopy measurements to define the spatial relationships between proteins. We then discuss how fluorescence lifetime imaging microscopy (FLIM) provides a method that is independent of intensity-based measurements to detect localized protein interactions with spatial resolution. Finally, we consider potential problems associated with the expression of proteins fused to fluorescent proteins for FRET-based measurements from living cells.     

A comparative analysis of resonance energy transfer methods for Alzheimer related protein-protein interactions in living cells

Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are extensively used to analyze protein interactions occurring in living cells. Although these two techniques are broadly applied in cellular biology, comparative analysis of their strengths and limitations is lacking. To this end, we analyzed a small network of proteins involved in the amyloidogenic processing of the Alzheimer β-amyloid precursor using FRET based cytometry, BRET, and fluorescence lifetime imaging microscopy (FLIM). Using all three methods, we were able to detect the interactions of the amyloid precursor protein with APBB1, APBB2, and APP itself. And we found an unreported interacting pair, APP-APH1A. In addition, we show that these four interacting pairs exhibit a strong FRET correlation with the acceptor/donor expression ratios. Overall the FRET based cytometry was the most sensitive and reliable approach to screen for new interacting proteins. Therefore, we applied FRET based cytometry to study competitive binding of two proteins, APBB1 and APBB2, with the same APP target.     

In vivo FLIM-FRET measurements of recombinant proteins expressed in filamentous fungi

Fluorescence imaging techniques are extremely powerful tools in cell biology, providing valuable insights into the structure and function of biomolecules in their native environments. In particular, the use of Förster resonance energy transfer (FRET) has become increasingly important to obtain information on interactions on the nanoscale, in turn providing insights into molecular behaviour inside living cells. This review describes the basic principles of FRET and fluorescence lifetime imaging microscopy (FLIM) and their application in analyses of protein interactions inside living fungal cells.     

Artificial induction of autophagy around polystyrene beads in nonphagocytic cells

Autophagy is an intracellular event that acts as an innate cellular defense mechanism to kill invading bacteria such as group A Streptococcus in nonphagocytic epithelial-like cells. The cellular events underlying autophagosome formation upon bacterial invasion remain unclear due to the biochemical complexity associated with uncharacterized bacterial components, and the difficulty of predicting the location as well as the timing of where/when autophagosome formation will take place. To overcome these problems, we monitored autophagosome formation in living nonphagocytic cells by inducing autophagy around artificial micrometer-sized beads instead of bacteria. Beads conjugated with bio-reactive molecules provide a powerful tool for examining biochemical properties in vitro. However, this technique has not been applied to living cells, except for phagocytes, because the beads cannot be easily incorporated into nonphagocytic cells. Here we report that micrometer-sized polystyrene beads coated with transfection reagents containing cationic lipids can be incorporated into nonphagocytic cells, and that autophagy can be efficiently induced around the beads in these cells. Monitoring the process of autophagosome formation for pH-sensitive fluorescent dye (pHrodo)-conjugated beads by fluorescence live cell imaging combined with correlative light and electron microscopy, we found that autophagosomes are formed around the beads after partial breakdown of the endosomal membrane. In addition, the beads were subsequently transferred to lysosomes within a couple of hours. Our findings demonstrate the cellular responses that lead to autophagy in response to pathogen invasion.     

Imaging [Ca2+]i dynamics during signal transduction

The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.     

A method for the direct identification of differentiating muscle cells by a fluorescent mitochondrial dye

Identification of differentiating muscle cells generally requires fixation, antibodies directed against muscle specific proteins, and lengthy staining processes or, alternatively, transfection of muscle specific reporter genes driving GFP expression. In this study, we examined the possibility of using the robust mitochondrial network seen in maturing muscle cells as a marker of cellular differentiation. The mitochondrial fluorescent tracking dye, MitoTracker, which is a cell-permeable, low toxicity, fluorescent dye, allowed us to distinguish and track living differentiating muscle cells visually by epi-fluorescence microscopy. MitoTracker staining provides a robust and simple detection strategy for living differentiating cells in culture without the need for fixation or biochemical processing.     

Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer

Much of the mechanism by which Wnt signaling drives proliferation during oncogenesis is attributed to its regulation of the cell cycle. Here, we show how Wnt/β‐catenin signaling directs another hallmark of tumorigenesis, namely Warburg metabolism. Using biochemical assays and fluorescence lifetime imaging microscopy (FLIM) to probe metabolism in vitro and in living tumors, we observe that interference with Wnt signaling in colon cancer cells reduces glycolytic metabolism and results in small, poorly perfused tumors. We identify pyruvate dehydrogenase kinase 1 (PDK1) as an important direct target within a larger gene program for metabolism. PDK1 inhibits pyruvate flux to mitochondrial respiration and a rescue of its expression in Wnt‐inhibited cancer cells rescues glycolysis as well as vessel growth in the tumor microenvironment. Thus, we identify an important mechanism by which Wnt‐driven Warburg metabolism directs the use of glucose for cancer cell proliferation and links it to vessel delivery of oxygen and nutrients.     

PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells

We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.     

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.     

Backstep scanning ion conductance microscopy as a tool for long term investigation of single living cells

Scanning ion conductance microscopy (SICM) is a suitable tool for imaging surfaces of living cells in a contact-free manner. We have shown previously that SICM in backstep mode allows one to trace the outlines of entire cell somata and to detect changes in cellular shape and volume. Here we report that SICM can be employed to quantitatively observe cellular structures such as cell processes of living cells as well as cell somata of motile cells in the range of hours.     

Real-time monitoring of cell elasticity reveals oscillating myosin activity

The cytoskeleton is the physical and biochemical interface for a large variety of cellular processes. Its complex regulation machinery is involved upstream and downstream in various signaling pathways. The cytoskeleton determines the mechanical properties of a cell. Thus, cell elasticity could serve as a parameter reflecting the behavior of the system rather than reflecting the specific properties of isolated components. In this study, we used atomic force microscopy to perform real-time monitoring of cell elasticity unveiling cytoskeletal dynamics of living bronchial epithelial cells. In resting cells, we found a periodic activity of the cytoskeleton. Amplitude and frequency of this spontaneous oscillation were strongly affected by intracellular calcium. Experiments reveal that basal cell elasticity and superimposed elasticity oscillations are caused by the collective action of myosin motor proteins. We characterized the cell as a mechanically multilayered structure, and followed cytoskeletal dynamics in the different layers with high time resolution. In conclusion, the collective activities of the myosin motor proteins define overall mechanical cell dynamics, reflecting specific changes of the chemical and mechanical environment.     

Bi-directional signal transduction by integrin receptors

The integrin family of cell surface glycoproteins functions primarily as receptors for extracellular matrix ligands. There are now many well characterized integrin-ligand interactions which are known to influence many aspects of cell behaviour including cell morphology, cell adhesion, cell migration as well as cellular proliferation and differentiation. However, in fulfilling these functions, integrins are not simple adhesion receptors that physically mediate connections across the plasma membrane. Rather, integrin function itself is highly regulated, largely through the formation of specific associations with both structural and regulatory components within cells. It is these intracellular interactions which allow integrin function to effect many biochemical signalling pathways and therefore to impinge upon complex cellular activities. Recently, much research has focused on elucidating the molecular mechanisms which control integrin function and the molecular processes which transduce integrin-mediated signalling events. In this review, we discuss progress in the field of integrin signal transduction including, where applicable, potential therapeutic applications arising from the research.     

Imaging molecular interactions in living cells

Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.