RESUMEN
During cancer therapy with DNA-damaging drug-agents, the development of secondary resistance to apoptosis can be observed. In the search for novel therapeutic approaches that can be used in these cases, we monitored chemotherapy-induced apoptosis resistance in a syngenic mouse tumor model. For this, syngenic murine colorectal carcinoma cells, which stably expressed a FRET-based caspase-3 activity sensor, were introduced into animals to induce peritoneal carcinomatosis or disseminated hepatic metastases. This syngenic system allowed in vitro, in vivo and ex vivo analysis of chemotherapy induced apoptosis induction by optically monitoring the caspase-3 sensor state in the tumor cells. Tumor tissue analysis of 5-FU treated mice showed the selection of 5-FU-induced apoptosis resistant tumor cells. These and chemo-naive fluorescent tumor cells could be re-isolated from treated and untreated mice and propagated in cell culture. Re-exposure to 5-FU and second line treatment modalities in this ex-vivo setting showed that 5-FU induced apoptosis resistance could be alleviated by imatinib mesylate (Gleevec). We thus show that syngenic mouse systems that stably express a FRET-based caspase-3 sensor can be employed to analyse the therapeutic efficiency of apoptosis inducing chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Microscopía Fluorescente/métodos , Animales , Secuencia de Bases , Caspasa 3/metabolismo , Neoplasias Colorrectales/enzimología , Cartilla de ADN , Transferencia Resonante de Energía de Fluorescencia , Fluorouracilo/farmacología , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la PolimerasaRESUMEN
Global analysis of fluorescence lifetime image microscopy (FLIM) data can be used to obtain an accurate fit of multi-exponential fluorescence decays. In particular, it can be used to fit a bi-exponential decay to single frequency FLIM data, which is not possible with conventional fitting techniques. Bi-exponential fluorescence decay models can be used to analyse quantitatively single frequency FLIM data from samples that exhibit fluorescence resonance energy transfer (FRET). Global analysis algorithms simultaneously fit multiple measurements acquired under different experimental conditions to achieve higher accuracy. To demonstrate that bi-exponential models can indeed be fitted to single frequency data, we derive an analytical solution for the special case of two measurements and use this solution to illustrate the properties of global analysis algorithms. We also derive a novel global analysis algorithm that is optimized for single frequency FLIM data, and demonstrate that it is superior to earlier algorithms in terms of computational requirements.
RESUMEN
Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.
Asunto(s)
Movimiento Celular/fisiología , Integrina beta1/fisiología , Isoenzimas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , Cicatrización de Heridas/fisiología , Sustitución de Aminoácidos , Neoplasias de la Mama , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Movimiento Celular/efectos de los fármacos , Cromonas/farmacología , Proteínas del Citoesqueleto , Inhibidores Enzimáticos/farmacología , Femenino , Proteínas Fluorescentes Verdes , Humanos , Cinética , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Microscopía Confocal , Morfolinas/farmacología , Mutagénesis Sitio-Dirigida , Forbol 12,13-Dibutirato/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Fosfotreonina/metabolismo , Proteína Quinasa C-alfa , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Células Tumorales CultivadasRESUMEN
The deconvolution of fluorescence lifetime imaging microscopy (FLIM) data that were processed with global analysis techniques is described. Global analysis of FLIM data enables the determination of relative numbers of molecules in different protein reaction states on a pixel-by-pixel basis in cells. The three-dimensional fluorescence distributions of each protein state can then be calculated and deconvolved. High-resolution maps of the relative concentrations of each state are then obtained from the deconvolved images. We applied these techniques to quantitatively image the phosphorylation state of ErbB1 receptors tagged with green fluorescent protein in MCF7 cells.
Asunto(s)
Microscopía Fluorescente/métodos , Proteínas/metabolismo , Línea Celular , Receptores ErbB/aislamiento & purificación , Receptores ErbB/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes , FosforilaciónRESUMEN
Proteins provide the building blocks for multicomponent molecular units, or pathways, from which higher cellular functions emerge. These units consist of either assemblies of physically interacting proteins or dispersed biochemical activities connected by rapidly diffusing second messengers, metabolic intermediates, ions or other proteins. It will probably remain within the realm of genetics to identify the ensemble of proteins that constitute these functional units and to establish the first-order connectivity. The dynamics of interactions within these protein machines can be assessed in living cells by the application of fluorescence spectroscopy on a microscopic level, using fluorescent proteins that are introduced within these functional units. Fluorescence is sensitive, specific and non-invasive, and the spectroscopic properties of a fluorescent probe can be analysed to obtain information on its molecular environment. The development and use of sensors based on the genetically encoded variants of green-fluorescent proteins has facilitated the observation of 'live' biochemistry on a microscopic level, with the advantage of preserving the cellular context of biochemical connectivity, compartmentalization and spatial organization. Protein activities and interactions can be imaged and localized within a single cell, allowing correlation with phenomena such as the cell cycle, migration and morphogenesis.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Genes Reporteros , Proteínas Luminiscentes/metabolismo , Espectrometría de Fluorescencia , Animales , Genes erbB-1 , Proteínas Fluorescentes Verdes , Humanos , Cinética , Proteínas Luminiscentes/genética , Microscopía Fluorescente/métodos , Unión Proteica , Transporte de Proteínas , Espectrometría de Fluorescencia/métodosRESUMEN
Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.
Asunto(s)
Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/química , Animales , Apoptosis , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Caspasa 3 , Caspasas/análisis , Caspasas/genética , Línea Celular , Transferencia de Energía , Variación Genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Mamíferos , Microscopía Fluorescente/métodos , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodos , TransfecciónRESUMEN
FRET microscopy enables the detection of different biochemical states of proteins in cells. The use of fluorescence in the detection of proteins, by chemical modification, by immunofluorescence, or by genetic encoding of a green fluorescent protein fusion protein, provides more information than just the location of the protein in the cell. The properties of the fluorophore can be exploited to extract information on protein-protein interactions. A straightforward, quantitative imaging approach is presented to measure FRET that is based on internal calibration by acceptor photobleaching.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas/metabolismo , Coloración y Etiquetado/métodos , Animales , Proteínas Fluorescentes Verdes , HumanosRESUMEN
Fluorescence microscopy has played a tremendous role in uncovering the morphological features of cells and the expression pattern of proteins by immunofluorescence. Since the discovery of green-fluorescent proteins (GFPs), this technique has undergone a revival in the life sciences as the spatial distribution of ectopically expressed fusion proteins inside living cells can now be followed more easily. By further exploiting the photophysical properties of the emitted fluorescence with microspectroscopic methods, spatial information on the biochemical parameters of intracellular processes and reactions can be obtained. This possibility will not only play an important role in the understanding of biochemical reactions in signal processing and fidelity but also help to uncover the molecular mechanisms of organelle and cell morphogenesis.
Asunto(s)
Enzimas/metabolismo , Biología Molecular/métodos , Proteínas/análisis , Proteínas/metabolismo , Enzimas/análisis , Fluorescencia , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)-tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime imaging microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.
Asunto(s)
Membrana Celular/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Transducción de Señal , Arsenicales/farmacología , Carbocianinas , Difusión , Dimerización , Endocitosis , Transferencia de Energía , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Fluorescencia , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Humanos , Fragmentos Fab de Inmunoglobulinas , Ligandos , Proteínas Luminiscentes , Microscopía Confocal , Microscopía Fluorescente , Microesferas , Fosforilación , Fosfotirosina/inmunología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Células Tumorales CultivadasRESUMEN
Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.
Asunto(s)
Microscopía Fluorescente/métodos , Microscopía Fluorescente/estadística & datos numéricos , Algoritmos , Animales , Proteínas Bacterianas/metabolismo , Chlorocebus aethiops , Interpretación Estadística de Datos , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Células VeroRESUMEN
We describe an extremely simple method by which optically sectioned fluorescence images may be obtained with conventional microscopes using laser illumination. A one-dimensional grid pattern is introduced into the illumination system, together with a rotating ground glass diffuser. This causes an image of the grid pattern to be projected into the specimen. Images taken at three spatial positions of the grid are processed in a simple manner to provide optically sectioned images of fluorescent specimens.
Asunto(s)
Aumento de la Imagen , Microscopía Fluorescente/métodos , Animales , Células COS/citología , Células COS/metabolismo , Isoenzimas/metabolismo , Rayos Láser , Microscopía Confocal/métodos , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfaRESUMEN
The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set-up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto-optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons 'on' and 'off' as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the 'on' state of the intensifier relative to its 'off' state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square-pulse modulation. A phase-dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel-by-pixel basis using a non-linear fit to the dispersion relationships. The fitting algorithms were tested on a simulated data set and were successful in disentangling two populations having 1 ns and 4 ns fluorescence lifetimes. Spatial invariance of the lifetimes was exploited to improve the accuracy significantly. Multiple frequency fluorescence lifetime imaging microscopy was then successfully applied to resolve the fluorescence lifetimes and fluorescence intensity contributions in a rhodamine dye mixture in solution, and green fluorescent protein variants co-expressed in live cells.
Asunto(s)
Fluorescencia , Microscopía Fluorescente/métodos , Análisis de Fourier , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/metabolismo , Rodaminas/análisisRESUMEN
We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1] [2] [3] [4] and concomitant activation of its intracellular tyrosine kinase domain [5] [6] [7]. Trans-autophosphorylation of the receptor [8] [9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.
Asunto(s)
Microscopía Fluorescente/métodos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Confocal/métodos , Fosforilación , Fosfotirosina/metabolismo , Pruebas de Precipitina , Factores de TiempoRESUMEN
Protein kinase C (PKC) has been implicated in integrin-mediated spreading and migration. In mammary epithelial cells there is a partial co-localization between beta1 integrin and PKCalpha. This reflects complexes between these proteins as demonstrated by fluorescense resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy and also by coprecipitation. Constitutive complexes are observed for the intact PKCalpha and also form with the regulatory domain in an activation-dependent manner. Expression of PKCalpha causes upregulation of beta1 integrin on the cell surface, whereas stimulation of PKC induces internalization of beta1 integrin. The integrin initially traffics to an endosomal compartment in a Ca(2+)/PI 3-kinase/dynamin I-dependent manner and subsequently enters an endocytic recycling pathway. This induction of endocytosis by PKCalpha is a function of activity and is not observed for the regulatory domain. PKCalpha, but not PKCalpha regulatory domain expression stimulates migration on beta1 integrin substrates. This PKCalpha-enhanced migratory response is inhibited by blockade of endocytosis.
Asunto(s)
Movimiento Celular , Integrina beta1/metabolismo , Proteína Quinasa C/metabolismo , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Endocitosis , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Activación Enzimática/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/química , Proteína Quinasa C/genética , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.
Asunto(s)
Proteínas Luminiscentes/genética , Microscopía Fluorescente/métodos , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Fluorescencia , Proteínas Fluorescentes Verdes , Datos de Secuencia Molecular , Células VeroRESUMEN
Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The nanosecond excited-state lifetime is independent of probe concentration or light path length but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell.
Asunto(s)
Microscopía Fluorescente/métodos , Animales , Procesamiento de Imagen Asistido por Computador , Fosforilación , ProteínasRESUMEN
Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.
Asunto(s)
Isoenzimas/metabolismo , Microscopía Fluorescente , Proteína Quinasa C/metabolismo , Células 3T3 , Animales , Neoplasias de la Mama/enzimología , Células COS , Catálisis , Citoplasma/enzimología , Retículo Endoplásmico/enzimología , Transferencia de Energía , Activación Enzimática , Fluorescencia , Colorantes Fluorescentes , Aparato de Golgi/enzimología , Proteínas Fluorescentes Verdes , Humanos , Sueros Inmunes , Isoenzimas/inmunología , Proteínas Luminiscentes , Ratones , Fosforilación , Fosfotreonina/inmunología , Fosfotreonina/metabolismo , Proteína Quinasa C/inmunología , Proteína Quinasa C-alfa , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
A microscope set-up and numerical methods are described which enable the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high-frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase-dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase-dependent data gives three-dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov-Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Microscopía FluorescenteRESUMEN
Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 = 6 +/- 4 microM). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.
Asunto(s)
Proteínas de Unión al Calcio , Calcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Secuencia de Aminoácidos , Animales , Corteza Cerebral/metabolismo , Dimerización , Exocitosis , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Ratas , Espectrometría de Fluorescencia , Sinaptotagmina II , SinaptotagminasRESUMEN
We previously identified the ZNF85 (HPF4) KRAB zinc finger gene, a member of the human ZNF91 family. Here, we show that the ZNF85 gene is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line. Immunocytochemical localization of a panel of beta-Gal/ZNF85 fusion proteins revealed that ZNF85 contains at least one nuclear localization signal located in the spacer region connecting the KRAB domain with the zinc finger repeats. Bacterially expressed ZNF85 zinc finger domain bound strongly and exclusively to DNA in vitro in a zinc-dependent manner. The KRAB(A) domain of the ZNF85 protein and of several other members of the ZNF91 family exhibited repressing activity when tested in Gal4 fusion protein assays. The repression was significantly enhanced by the addition of the KRAB (B) domain, whereas further addition of other conserved regions had no effect. The ZNF85 KRAB(A) and (B) domains in vitro bound several nuclear proteins that might constitute critical cofactors for repression.