Fluorescent probes for living cells

The functional characteristics of fluorescent probes used for imaging and measuring dynamic processes in living cells are reviewed. Initial consideration is given to general design requirements for delivery, targeting, detectability and fluorescence readout, and current technologies for attaining them. Discussion then proceeds to the more application-specific properties of intracellurion indicators, membrane potential sensors, probes for proteins and lipids, and cell viability markers. 1998 © Chapman & Hall     

Fluorescent probes for bioimaging applications

Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of the molecules of interest, without the need of genetic engineering of the sample. In this review, following a brief outline of the principle of fluorescence imaging, we recount some recent achievements in the field of small-molecular fluorescent probes. First, probes for metal cations, including those suitable for two-photon imaging, are introduced. Next, methodologies to visualize proteases are discussed, with special emphasis on activity-based probes for use in vivo. All these probes have been confirmed to be applicable to cellular or in vivo imaging.     

Fluorescent probes for the structure and function of metallothionein

Fluorescence methods have been instrumental in demonstrating that the structure of human metallothionein in vivo depends on the availability of metal ions and the redox environment. Differential chemical modifications of its cysteine thiols with fluorescent probes allowed determination of three states: metallothionein (zinc-bound thiolate), thionein (free thiols), and thionin (disulfides). Interrogation of its zinc-binding properties with fluorescent chelating agents revealed that the affinities for the seven zinc ions vary over four orders of magnitude. Attachment of fluorescent labels generated metallothionein FRET (fluorescence resonance energy transfer) sensors for investigating its structure and function in living cells.     

Fluorescent lipid probes in the study of viral membrane fusion

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.     

Fluorescent dyes as probes to study lipid-binding proteins

We studied the equilibrium binding of two hydrophobic fluorescent dyes, ANS and bisANS, to four members of a family of intracellular lipid-binding proteins: IFABP, CRABP I, CRABP II, and ILBP. The spectral and binding parameters for the probes bound to the proteins were determined. Typically, there was a single binding site on each protein for the ligands. However, IFABP cooperatively bound a second bisANS molecule in the binding pocket. Comparative analysis of affinities and spectral characteristics for the two probes allowed us to examine the contributions of electrostatic and hydrophobic interactions to the binding process, and to address some aspects of the internal structure of the studied proteins.     

Fluorescent probes for detection of protozoan parasites

Fluorescent probes generally provide a rapid and simple staining technique, valuable for the rapid diagnosis of protozoal infections. However, many of these staining techniques have disadvantages for clinical tests: (I) they require a fluorescence microscope which is not always available in clinical laboratories; (2) the preparations are not permanent because the fluorescent probes do not withstand dehydration; (3) variable quenching of the fluorescence may occur, unless proper preventive measures are taken. In this article, Fumihiko Kawamoto and Nobuo Kumodo explain some of the most widely used fluorescent probes, and discuss how problems in their use can be minimised.     

Synthesis and activity of C11-modified wortmannin probes for PI3 kinase

The key role played by PI3 kinase in cancer, hormone action, and a host of other biological functions suggests that specific inhibitors whose disposition could be ascertained in vivo would be useful in biological research or, potentially, for imaging PI3K in a clinical setting. Wortmannin (Wm, 1) is an inhibitor of PI3 kinase with high specificity for this enzyme. We synthesized three modified Wm probes, a biotinylated Wm (7a), a 4-hydroxy-3-iodophenylated Wm, which was obtained both unlabeled (7b) and labeled with (125)I (8), and a fluoresceinated Wm (7c), through modification at C-11, and evaluated their inhibitive activity as inhibitors of PI3 kinase. Biotinylated (7a) and 4-hydroxy-3-iodophenylated Wm's (7b) had IC(50)s for PI3K of 6.11 and 11.02 nM, respectively, compared to an IC(50) for Wm of 1.63 nM. Fluoresceinated Wm (7c) lost considerably more activity than the other derivatives, with an IC(50) of 64.9 nM. The (125)I labeled 4-hydroxy-3-iodophenylated Wm (8) could be detected after reaction with an immunoprecipitate of PI3 kinase. The activity of these reporter Wm's is discussed in relationship to earlier findings on the pharmacological activity of Wm derivatives and the ability of inhibitors to fit into the ATP pocket of PI3 kinase.     

Review: Fluorescent probes for living cells

The functional characteristics of fluorescent probes used for imaging and measuring dynamic processes in living cells are reviewed. Initial consideration is given to general design requirements for delivery, targeting, detectability and fluorescence readout, and current technologies for attaining them. Discussion then proceeds to the more application-specific properties of intracellurion indicators, membrane potential sensors, probes for proteins and lipids, and cell viability markers. 1998 © Chapman & Hall     

Fluorescent probes for bacterial cytoplasmic membrane research

Fluorescent methods in biological and medical research are extremely useful at the cellular and molecular levels. This is due to sensitive and affordable detection equipment and a variety of specific and more general fluorescent probes, and analytical procedures. In this article, I examine the use of fluorescence membrane probes to study the fluidity (membrane polarization) of the bacterial cytoplasmic membrane, central to energy transduction, ion and nutrient transport and diffusion of water and gases.     

Activation of cGMP-dependent protein kinase by protein kinase C

The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.     

Small-molecule FRET probes for protein kinase activity monitoring in living cells

In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Förster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.     

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.     

Fluorescent and spin label probes of the environments of the sulfhydryl groups of porcine muscle adenylate kinase.

The environments of the two sulfhydryl groups of procine muscle adenylate kinase have been investigated by chemical modification reactions. The results indicate that the environments of the two-SH groups of procine muscle adenylate kinase are markedly different and that substrates induce conformational changes in the enzyme in the region of the sulfhydryl groups. The fluorogenic reagent 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-chloride) reacts specifically with the -SH groups of the enzyme at pH 7.9. One thiol group reacts with NBD-chloride approximately 40-fold faster than the other one, and the fast reacting group has been identified as Cys-25 in the amino acid sequence. The similarity of the rate of the more slowly reacting Cys-187 with NBD-chloride to that of glutathione with the same reagent is consistent with its location on the surface of the enzyme as determined by x-ray crystallography structure. The fast reacting Cys-25 in the interior of the structure can be approached by compounds such as NBD-chloride via a cleft. Reaction of Cys-25, presumably located close to the catalytic center, leads to complete inactivation of the enzyme. Substrates such as ATP, MgATP, and ADP which bind to the triphosphate subsite of the enzyme decrease the rate of reaction of Cys-25 by factors up to 3.5 but have only a small effect (approximately equal to 10%) on the reactivity of Cys-187. AMP, however, has a pronounced effect on the reactivity of Cys-187, the slowly reacting group. The multisubstrate analogue P-1, P-5-di-(adenosine-5)pentaphosphate (Ap-5A) decreases the rate of reaction of the fast reacting thiol group by a factor of 300. The behavior of Cys-25 toward NBD-chloride, i.e. super-reactivity in the absense of Ap-5A and slow reactivity in the presence of the multisubstrate inhibitor, was characteristic for both porcin and carp adenylate kinase. In the presence of Ap-5A adenylate kinase can be selectively modified at Cys-187; the introduction of the fluorescent NBD group at this position has no effect on enzymatic activity. A slow transfer of the NBD group occurs from the third groups to the epsilon-amino group of Lys-31. This transfer reaction is further evidence that the structure of adenylate kinase in dilute solution is similar to that of the crystalline enzyme since the x-ray data have shown that the sulfur of Cys-187 and the epsilon-nitrogen of Lys-31 are less than 4 A apart. The strongly fluorescent NBD-NH-enzyme possesses full activity and binds substrates as. cont'd     

Nuclear protein kinase C

Protein kinase C (PKC) isozymes constitute a family of ubiquitous phosphotransferases which act as key transducers in many agonist-induced signaling cascades. To date, at least 11 different PKC isotypes have been identified and are believed to play distinct regulatory roles. PKC isoforms are physiologically activated by a number of lipid cofactors. PKC is thought to reside in the cytoplasm in an inactive conformation and to translocate to the plasma membrane or cytoplasmic organelles upon cell activation by different stimuli. However, a sizable body of evidence collected over the last 20 years has shown PKC to be capable of translocating to the nucleus. Furthermore, PKC isoforms are resident within the nucleus. Studies from independent laboratories have to led to the identification of quite a few nuclear proteins which are PKC substrates and to the characterization of nuclear PKC-binding proteins which may be critical for finely tuning PKC function in this cell microenvironment. Several lines of evidence suggest that nuclear PKC isozymes are involved in the regulation of biological processes as important as cell proliferation and differentiation, gene expression, neoplastic transformation, and apoptosis. In this review, we shall highlight the most intriguing and updated findings about the functions of nuclear PKC isozymes.     

Comprehensive determination of protein tyrosine pKa values for photoactive yellow protein using indirect 13C NMR spectroscopy

Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated ¹³C_γ resonances in sensitive and well-resolved two-dimensional ¹³C-¹H spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK_a values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41–11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state.     

Yeast protein kinase C

The mammalian protein kinase C (PKC) superfamily plays regulatory roles in many different cellular processes. However, due to the many members that exist in cells, it is very complicated to present experimental evidence of the particular function of each member. In contrast, yeasts have only one or two PKC members and genetic tools have unveiled their role as main regulators of cell integrity. In this review, we will discuss the function of yeast protein kinase C homologues, their mechanism of activation and the signalling pathways that they regulate in two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe.