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Correspondence to: Morten P. Oksvold, Center for Cellular Stress Responses, Inst. of Pathology, U. of Oslo, Rikshospitalet, N-0027, Oslo, Norway. E-mail: m.p.oksvold@labmed.uio.no
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Summary |
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Intracellular signaling relies on the orchestrated cooperation of signaling proteins and modules, their intracellular localization, and membrane trafficking. Recently, a repertoire of fluorescence-based techniques, which significantly increases our potential for detailed studies of the involved mechanisms, has been introduced. Microscopic techniques with increased resolution have been combined with improved techniques for detection of signaling proteins. Transfections of fluorescently tagged proteins have allowed in vivo microscopy of their trafficking and interactions with other proteins and intracellular structures. We present an overview of general signaling principles and a description of techniques based on fluorescent microscopy suited for studies of signaling mechanisms.
(J Histochem Cytochem 50:289303, 2002)
Key Words: fluorescence microscopy, extended resolution, protein interactions, intracellular signaling
DURING THE INCEPTION of the postgenomic era, protein interactions and regulatory mechanisms have been emphasized in cell biology and biomedical research. This also includes investigations of cell signaling. Diverse applications of fluorescence microscopy have earned increasing interest for such studies. Multi-staining techniques with new fluorochromes, confocal microscopy, transfections with probes expressing fluorescently labeled proteins, and in vivo microscopy have all contributed to the increasing merits of fluorescence microscopy. In particular, these techniques have contributed to the notion that intracellular signaling is not the result of disordered chemical reactions of freely diffusible components but rather is spatially highly regulated. Transduction of signals is highly dependent on protein interactions, subcellular localization, and trafficking of signaling modules. Specific signaling emanates from intracellular organelles and structures, such as plasma membrane rafts, endosomes, and mitochondria. Currently, a painstaking mapping of the functions, interplay, and crosstalk among the diverse proteins and structures participating in cell regulation, and how these control specific cell functions, is taking place. Here we discuss applications of fluorescence microscopy for studies of intracellular signaling.
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Concepts of Signal Transduction |
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Intracellular signaling is initiated by activation of receptors in the cytoplasm and the plasma membrane. This initiates cascades of protein interactions, ultimately leading to altered activities of transcription factors in the nucleus. This review focuses on four families of plasma membrane receptors to discuss fluorescence microscopy methods in signal transduction studies: G-protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), cytokine receptors (CRs), and the immunoreceptor (IR) family. Despite striking differences in receptor structure and function, the different transmembrane receptor families share several downstream signal transducers. Often, each receptor type can initiate several signaling chains, and different receptors can use the same or similar chains. There is ubiquitous crosstalk among the different signaling pathways, and signaling along one chain often modifies transduction along other chains. Our current knowledge of these signaling chains has been examined in several contemporary reviews (
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After ligand binding, plasma membrane receptors transmit signals through chains of signal transducers. Receptor or adaptor protein kinases become activated, and protein phosphorylations create docking sites for downstream transducers containing SH2 or PTB domains. Other pathways rely on activation of enzymes that produce second messengers such as cAMP, inositol-1,4,5-trisphosphate (IP3), and diacylglycerol. Not only receptors but also many of their downstream effectors are located in or close to the plasma membrane. GPCRs are situated together with heterotrimeric G-proteins, the effector enzyme adenylyl cyclase or phospholipase C (PLC), whereas adaptor proteins such as Shc and Src and small G-proteins such as Ras are found in the vicinity of RTKs. Cytokine receptors are without intrinsic kinase activities but are complexed with tyrosine kinases of the Jak family. The immunoreceptors, T-cell receptor, B-cell receptor, and Fc-receptors, are multimeric complexes containing ligand-binding subunits, co-receptors, and chains involved in signal transduction. Signals from plasma membrane receptors are transmitted to cytoplasmic modules, often via G-protein-mediated mechanisms. Some of the most studied cytoplasmic modules are the MAP kinase module consisting of an MAP kinase kinase kinase, an MAP kinase kinase, and an MAP kinase. Examples include the Raf, MEK1, and Erk1/2 modules. There are strong indications that these modules are physically integrated by specific scaffolding proteins, as has been demonstrated for JIP-1 integrating the MLK, MKK7, and JNK modules (
The significance of subcellular localization and activation-induced relocation of signaling proteins and modules for signaling efficiency and specificity has recently been realized. Plasma membrane receptors are probably not floating randomly in the lipid bilayer, as previously hypothesized by the SingerNicolson fluid mosaic model, but are distributed in specific regions that differ in their composition of lipids and structural proteins (, PDGF receptor, Rap1, and PKC
are examples from a growing list of signaling proteins that have been found enriched in caveolae (
Therefore, different principles can be identified for maintenance of signaling specificity, efficiency, and modulation. The phenotype of expressed receptors, signal transducers, regulating proteins, and transcription factors determines which pathways are available in a particular cell. Signaling proteins possesses several proteinprotein (e.g., SH2 and SH3) and proteinphospholipid (e.g., FYVE) domains for specific docking along signaling cascades of pre-formed and activation-induced complexes. Scaffolding several signaling components into functional signal modules is another way of maintaining signaling specificity and efficiency. Finally, the location of signaling modules in subcellular organelles or specialized organelle regions and their activation-induced routing along membrane compartments modify the cellular effects. These principles prompt the requirement for accurate techniques to determine intracellular distribution, activation-induced translocations, and co-localization of signaling proteins in their native and activated state.
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Detection of Signaling Molecules |
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Probes for Signal Proteins
Since the discovery of oncogenes, antibodies have been used for detection of their products, oncoproteins (
Recently, antibodies to activated forms of signaling proteins have become available. Initially, antibodies to phosphorylated tyrosine were utilized (1, which docks to autophosphorylated EGFR, was conjugated to Cy5 and used as a probe for fluorescence microscopy. Antibodies have also been made to other activation-induced conformation alterations (
Tagging of Transfected Gene Products
A different approach to study the functions of signal transducing proteins has been to transfect cells or animals with probes containing the corresponding genes. This can be the full-length gene, the wild-type gene, or the gene containing deletions or mutations in specific domains for functional studies (
Recently, genes encoding fluorescent proteins were introduced as tags for transfected proteins (
A prerequisite for the use of a reporter gene is to establish that it does not interfere with the localization or function of the protein. The size of GFP (238 amino acids) suggests that it can interfere with the normal functions of a fused protein. Whereas an EGFR construct fused to GFP was shown to function normally (-helical conformation has been designed for genetic incorporation into proteins of interest (
It should be noted that transfected genes are ectopically expressed, often at high levels. The localization and functions of the proteins may therefore be corrupted. It has been shown that overexpression of the putative scaffold protein KSR inhibits Ras signal transduction, although it was identified as a mediator of Ras signaling by genetic means (
Detection of Second Messengers
In some cases, intracellular signaling can be detected because a signaling intermediate binds to and thus alters the fluorescent properties of a fluorochrome. In particular, such techniques have been used to study Ca2+ fluxes in cells after growth signals or other stimuli. Ca2+ signaling depends on Ca2+ influx from outside the cell or from intracellular stores in the ER. Voltage-operated Ca2+ channels and receptor-operated Ca2+ channels regulate influx over the plasma membrane, whereas IP3 binding to its receptor releases Ca2+ from the ER (
Recently it has been realized that reactive oxygen species (ROS) (e.g., O-2, H2O2, OH-) are signaling intermediates (
Demonstration of Gene Activation
Several techniques have been applied to detect gene activation, the end point in signaling cascades emanating from the plasma membrane, at a cellular level. For example, immunocytochemistry (ICC) has been used to detect c-fos protein induction in specific cholinergic and non-cholinergic neurons of rat basal forebrain after ventricular instillation of NGF (
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High-resolution Localization of Signaling Proteins |
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Multicolor Overlaying
The increasing versatility of fluorescence microscopy for signaling studies relies partly on the ability to detect and compare several independent probes in the same cell sample or tissue section. Multicolor staining techniques can be used to identify cell populations expressing a particular signal transducer, to define which subcellular organelles contain a signal transducer, and to determine if different signal transducers co-localize and may therefore interact. Such techniques have been used to show that pre-neoplastic liver lesions and unaltered hepatocytes contain similar levels of EGFR, whereas bile duct cells and putative stem cells contain considerable lower levels (
Immunological multistaining recquires crossover controls to ensure absence of immunological crossreactions (
Analysis of multistained cells requires accurate overlaying of the images representing each fluorochrome. Often it is also necessary to quantify fluorescence intensities, the degree of fluorochrome co-localization, or the intensity ratios of two or more fluorochromes. Images captured by video or digital cameras or by confocal laser scan microscopes are well suited for computerized image analysis for such purposes (
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Markers of Intracellular Organelles and Structures |
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For functional characterization of signaling mechanisms, it is necessary to precisely define the intracellular localization of the participating signaling transducers. It was early realized that oncogene protein functions relied on their intracellular localization (
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It is now also possible to identify specific organelles in living cells transfected with organelle-targeted GFP (
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Microscopic Resolution and Optical Sectioning |
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Confocal microscopy offers increased horizontal and, in particular, vertical resolution compared to conventional fluorescence microscopy (
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Different techniques have been developed to extend the resolution of fluorescence microscopy. Images from confocal microscopy and digital fluorescence microscopy can be digitally deconvoluted (
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Dynamics of Protein Interactions and Trafficking |
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In Vivo and Two-photon Microscopy
By in vivo fluorescence microscopy, the movement of signaling molecules and their allocations to cellular organelles can be studied in cultured cells, or in some cases in cultured organ slices (
In vivo microscopy of thick multicellular preparations, such as brain slices and embryos, has gained from the development of two-photon microscopy (350 nm) can also be excited by two red photons (
700 nm) if they reach the fluorophore at the same time. To obtain this, the photon density must be approximately a million times what is required for one-photon absorption. Pulsed lasers are used to obtain this. The peak laser power is very high but the average power is only slightly higher than in confocal microscopy. Because two-photon excitation is obtained only at the focal point of the microscope, photobleaching and cell damage are minimized, which is crucial for in vivo microscopy. The sample penetration is also increased, often two- to threefold what is obtainable with confocal microscopy. The resolution of two-photon microscopy may be somewhat poorer than that of confocal microscopy because longer excitation wavelengths are used. In particular, Ca2+ signaling has been studied with this technique, but GFP-tagged AMPA receptor distribution in living neurons has also been investigated with this method (
Measurements of Protein Mobility In Vivo
Fluorescence recovery after photobleaching (FRAP) was introduced 30 years ago as a technique to monitor the mobility and dynamics of fluorescent proteins in living cells. With the introduction of GFP its usefulness has increased, and FRAP is now widely used in studies of intracellular signaling (reviewed by (
Whereas FRAP measures recovery of fluorescence, a technique called fluorescence loss in photobleaching (FLIP) characterizes the reduction of fluorescence in cellular regions when a small region is photobleached. With this technique,
Detection of Protein Binding in Intracellular Organelles
Very low levels of a fluorescently labeled molecule can be measured, and its binding to other structures determined, by fluorescence correlation spectroscopy (1 femtoliter or less) can be measured in living cells, allowing studies of plasma membrane regions and intracellular organelles. FCM measures the fluctuations in emission when fluorescently labeled molecules diffuse in and out of a defined volume. These fluctuations reflect the average number of labeled molecules in the volume, as well as the characteristic diffusion time of each molecule across the defined volume. Binding of a labeled molecule with another (unlabeled) molecule will slow down the diffusion and is readily detectable. This technique has just recently been adapted to microscopy and its feasibility is still not fully explored. However, it was used to characterize tagged EGFR diffusion in different cellular structures (
Molecule Interactions by Fluorescence Resonance Energy Transfer (FRET)
Protein interactions, either among components of a signaling cascade or among signaling and scaffolding proteins, are essential for signal transduction. Demonstrations of such interactions are therefore useful verifications of signal activation or other regulatory mechanisms. Traditionally, co-immunoprecipitation analyses have been used to detect such protein interactions. These assays are often difficult to perform because signaling complexes easily break up during protein extraction from cells. Furthermore, proteins may co-precipitate on the basis of their presence in the same membrane and not because of direct interactions. Similarly, induction of signaling protein co-localization can be monitored by fluorescence multi-staining techniques. For example, EGFR and Shc co-localize in early endosomes after EGF stimulation. The resolution limit of conventional microscopy is not good enough to prove direct molecular interaction by co-localization. Fluorescence resonance energy transfer (FRET) is a method that can detect protein co-localization in the 5-nm range and thus can provide a much better basis for proving direct interactions. The technique is based on the fact that when two overlapping fluorochromes are closely located, excitation of the lower-wavelength fluorochrome induces energy transfer to the higher-wavelength fluorochrome (5 nm apart. Energy can also be transferred to a closely situated non-fluorescent molecule, and this can be detected as attenuation of emission. Energy transfer also protects against fading, which can be exploited to detect protein interaction. FRET and related techniques can be adapted to microscopic examinations and have been particularly useful for studies of cultured cells doubly transfected with genes tagged to GFP mutants of overlapping fluorescence. The significance of these methods is their potential to directly detect protein interactions in living cells. Recently, visualization of receptor-mediated activation of G-proteins by monitoring FRET between
- and ß-subunits fused to cyan and yellow fluorescent proteins was described in living Dictyostelium discoideum cells (
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Conclusions and Perspectives |
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During the post-genomic era, emphasis is turning from the identification of genes and proteins towards characterization of their functions. Multicolor fluorescence techniques have proved highly versatile for such studies. In particular, this is due to the significance of accurate intracellular localization and co-localization of signal transducers with other proteins. Recent publications have presented the prospect of increasing the confocal microscopic resolution even beyond the resolution limit of light. At present, the intracellular localization of signaling proteins has much to gain from a better supply and characterization of markers for intracellular organelles and subregions. The development of user-friendly, general-purpose image analysis programs integrating image overlaying, co-localization analysis, fluorometry, 3D reconstruction, image ratioing, and time-lapse analysis will further this progress. Furthermore, the inherent possibility of crossreactions, which in many cases is difficult to control for, underscores the need to supplement fluorescence microscopy with other techniques including Western blotting, co-immunoprecipitation, subcellular fractionation techniques, and immunoelectron microscopy.
Particularly for studies of transfected, wild-type, and mutated signal proteins, fluorescence microscopy has become indispensable for characterization of specific molecular mechanisms. In vivo fluorescence, in which trafficking of signaling proteins can be examined in living cells under controlled conditions and after specific stimuli, is receiving increasing attention. Such techniques are particularly suited for FRET analysis for direct demonstration of protein interactions. It should be kept in mind, however, that the effects of ectopically expressed proteins do not necessarily reflect physiological processes. Overexpression of a protein may titer out binding proteins, as observed for AKAP proteins, or may lead to non-physiological intracellular localization (
The prospect of "imaging biochemistry inside cells" has been introduced (
Received for publication October 16, 2001; accepted October 24, 2001.
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