Receptor complexes cotransported via polarized endocytic pathways form clusters with distinct organizations.

Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands. We show that pIgA-R and TFR complexes form increasingly organized clusters during cotransport from basolateral to perinuclear endosomes. The organization of these receptor clusters in basolateral versus perinuclear/apical endosomes is significantly different; the former showing a mixed random/clustered distribution while the latter highly organized clusters. Our results indicate that although both perinuclear and apical endosomes comprise pIgA-R and TFR clusters, their E% levels are significantly different suggesting that these receptors are packed into clusters in a distinct manner. The quantitative FRET-based assay presented here suggests that different receptor complexes form clusters, with diverse levels of organization, while being cotransported via the polarized endocytic pathways.

Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.

Verapamil and cyclosporin A modulate doxorubicin toxicity by distinct mechanisms.

Cyclosporin A has been reported to enhance the sensitivity of cells displaying multiple drug resistance to anthracyclines. However, the mechanism of action of cyclosporin A in modulating drug resistance is still controversial. This study compares the effects of cyclosporin A and verapamil on doxorubicin resistance in chinese hamster ovary cells (CHRC5) using several criteria including in vitro cytotoxicity, drug accumulation, intracellular distribution by video microscopy, and nuclear DNA damage. Our results demonstrate that verapamil modulation of doxorubicin resistance was paralleled by cellular accumulation of doxorubicin, altered intracellular distribution of doxorubicin from cytoplasm to nucleus, and an increase in the formation of doxorubicin related DNA strand breaks. In contrast, the modulating effect of cyclosporin was qualitatively different. High concentrations of cyclosporin (5 micrograms/ml) increased doxorubicin accumulation and caused partial redistribution to the nucleus. However, with low concentrations of cyclosporin (1 microgram/ml) increased doxorubicin sensitivity was observed without changes in net accumulation of doxorubicin or intracellular distribution, and without enhanced doxorubicin induced DNA breakage. These results suggest that cyclosporin A can modulate doxorubicin cytotoxicity by means other than interference with the P-glycoprotein drug efflux system.

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis.

The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two-photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two-photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two-photon excitation and confocal microscopy also provide improved rejection of "out-of-focus" noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two-photon images. We have also found that the two-photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers.

Fluorescence resonance energy transfer microscopy: a mini review.

Fluorescence resonance energy transfer (FRET) microscopy is a better method than the x-ray diffraction, nuclear magnetic resonance, or electron microscopy for studying the structure and localization of proteins under physiological conditions. In this paper, we describe four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPalpha) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides two-dimensional spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleedthrough and photobleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. The fluorescence lifetime methods allow us to monitor FRET signals at the moment of the protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution. This paper will provide a brief review of the above-mentioned FRET techniques.

Fret imaging of pit-1 protein interactions in living cells.

The combined use of fluorescence resonance energy transfer (FRET) microscopy and expression of genetic vectors encoding protein fusions with green fluorescent protein (GFP) and blue fluorescent protein (BFP) provides an exceptionally sensitive method for detecting the interaction of protein partners in living cells. The acquisition of FRET signals from GFP- and BFP-fusion proteins expressed in living cells was demonstrated using an optimized imaging system and high sensitivity charge coupled device camera. This imaging system was used to detect energy transfer signals from a fusion protein containing GFP physically linked to BFP expressed in living HeLa cells. In contrast, the co-localization of noninteracting GFP- and BFP-fusion proteins was not sufficient for energy transfer. The FRET imaging system was then used to demonstrate dimerization of the pituitary-specific transcription factor Pit-1 within the living cell nucleus. © 1998 Society of Photo-Optical Instrumentation Engineers.

Reconstruction of cardiac displacement patterns on the chest wall by laser speckle interferometry.

Laser speckle interferometry has been used to determine the displacement pattern on the chest wall produced by the heart action during the systolic phase of the cardiac cycle. The time averaged specklegram, recorded in the image plane, is scanned by the pointwise method. From the values of the spacings of Young's fringes, measured at various points, the corresponding displacement pattern in the form of a 40 x 30 matrix, equal to that of the cardiac region, is reconstructed and color-coded by a PDP 11/23 image processor. This pattern shows significant variations in displacements at various locations. Depending on the clinical status of the subjects, further changes in the pattern are observed. The reconstructed cardiac displacement patterns, obtained by this new noncontact and noninvasive technique, provide information on the functioning of various parts of the heart in cardiac disorders which are in qualitative agreement with that of two-dimensional echocardiography.

Differential sensitivity of mouse neural crest cells to ethanol-induced toxicity.

Neural crest cells (NCCs) have been identified as an important target population relative to ethanol-induced teratogenicity in both mouse and avian models. Additionally, whole embryo culture mouse models have shown strain-related differences in sensitivity to ethanol-induced damage following acute exposure during early NCC development. That differential sensitivity of NCCs may contribute to these strain differences has been unexplored. For this purpose, cultured NCCs from an inbred mouse strain (C57BL/6J; C57) that is more sensitive to ethanol-induced teratogenicity than an outbred strain (ICR) were compared. This study showed that the incidence of cell death was significantly higher for the C57 NCCs than those from the ICR strain at all ethanol concentrations tested, and as early as 12 hours after initial exposure to 100 mM ethanol. The lateral mobility of the membrane lipids was faster and the membrane GM1 content was lower in C57 cells than ICR cells both under control conditions and at all doses and times tested. Ethanol exposure resulted in significant increases in the membrane lipid lateral mobility, and decreases in the membrane GM1 content that occurred in a dose and time-dependent manner in the NCCs from both strains. A significant correlation was found between the GM1 content and lateral mobility of the membrane lipids, the lateral mobility of membrane lipids and cell viability, as well as the GM1 content and cell viability in the NCCs from both strains. These results suggest that different strain sensitivities to ethanol-induced teratogencity may lie, at least in part, in the interstrain differential response of the NCC population and that the vulnerability of the NCCs to ethanol-induced death may be related to their endogenous membrane GM1 content.

Pauses, steps, and the mechanism of contraction.

Despite widespread controversy still surrounding the phenomenon, stepwise shortening has now been confirmed by five independent methods in this laboratory, and by several other methods in different laboratories. In this paper we offer preliminary evidence obtained with the most recent method--measurement of 'isotonic muscle length transients'. We find that the muscle length inflections observed after quick release to an isotonic load correspond to pauses and steps at the sarcomere level. Thus, pauses and steps are reflected not only in sarcomere length and segment length signals, but in the muscle length signal as well. We review several of the more illuminating features of stepwise shortening, as well as new ultrastructural observations which, taken together, point to an hypothesis for the generation of steps. The steps may be generated by shortening of one or another of the sarcomere's filaments: connecting filaments in the unactivated myofibril and thick filaments in the activated myofibril. Supporting evidence is considered.

Fluorescence Microscopy Study of Heterogeneity in Polymer-supported Luminescence-based Oxygen Sensors.

Despite the great potential of fluorescence microscopy, its application to date has largely been in the study of biological specimens. It will be shown that conventional fluorescence microscopy provides an invaluable tool with which to study the photophysics of polymer-supported luminescence-based oxygen sensors. The design of the imaging system, the measurement methods, and the data analysis used in the investigation of sensor systems are described. Fluorescence microscopic images of sensor films in which microheterogeneous regions exhibiting enhanced luminescence intensity and poorer oxygen quenching relative to the bulk response are shown. This is the first direct evidence that sensor molecules in various domains of the polymer support can exhibit different oxygen quenching properties. It will be shown that µ- and nano-crystallization of the sensor molecule are the probable source of both the observed heterogeneous microscopic responses and the microscopic and macroscopic nonlinear Stern-Volmer plots. The implications of these results in the rational design of luminescence-based oxygen sensors are discussed.

Potassium ion induced changes of crystal structure and fluorescence of a crown ether.

Luminescence properties and the x-ray structures of the fluorescent crown ether, 16-anthracen-ylmethyl-1,4,7,10,13-pentaoxa-16-aza-cyclooctadecane (CEA) and its complex with potassium hexafluorophosphate (CEAK) have been obtained. In the solid state CEAK gives a structured blue emission and CEA gives a broad structureless green emission. The differences in luminescence behavior are explained on the basis of crystal packing. X-ray analysis shows that every two adjacent anthracene moieties in CEA form a sandwich-like anti-parallel dimer; the green-structureless emission then arises from the pi-pi stack of the aromatic rings. In CEAK, disruption of the pi-pi stacking structure forces a large separation between the anthracene rings, which yields an anthracene monomer emission. Luminescence lifetime data support the assignments.

Effect of refraction on optical microscopic measurement of internal blood-vessel diameter and its correction.

In optical microscopic measurement of internal blood-vessel diameters, the effect of refraction must be taken into account to ensure accuracy of the result. This effect is discussed and an analytical correction formula derived. Phantom blood vessels with known internal and external diameters were used to test the validity of the correction formula. The errors obtained prior to correction were reduced significantly after correction.

Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus.

Cells respond to environmental cues by modifying protein complexes in the nucleus to produce a change in the pattern of gene expression. In this article, we review techniques that allow us to visualize these protein interactions as they occur in living cells. The cloning of genes from marine organisms that encode fluorescent proteins provides a way to tag and monitor the intracellular behavior of expressed fusion proteins. The genetic engineering of jellyfish green fluorescent protein (GFP) and the recent cloning of a sea anemone red fluorescent protein (RFP) have provided fluorescent tags that emit light at wavelengths ranging from the blue to the red spectrum. Several of these color variants can be readily distinguished by fluorescence microscopy, allowing them to be used in combination to monitor the behavior of two or more independent proteins in the same living cell. We describe the use of this approach to examine where transcription factors are assembled in the nucleus. To demonstrate that these labeled nuclear proteins are interacting, however, requires spatial resolution that exceeds the optical limit of the light microscope. This degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). The application of FRET microscopy to detect the interactions between proteins labeled with the color variants of GFP and the limitations of the FRET approach are discussed. The use of different-color fluorescent proteins in combination with FRET offers the opportunity to study the complex behavior of key regulatory proteins in their natural environment within the living cell.

Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell.

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity-based FRET microscopy is limited by spectral bleed-through and fluorophore concentration. Fluorescence lifetime imaging (FLIM) microscopy and lifetime measurements are independent of change in fluorophore concentration or excitation intensity, and the combination of FRET and FLIM provides high spatial (nanometre) and temporal (nanoseconds) resolution. Because only the donor fluorophore lifetime is measured, spectral bleed-through is not an issue in FRET-FLIM imaging. In this paper we describe the development of a nanosecond FRET-FLIM microscopy instrumentation to acquire the time-resolved images of donor in the presence and the absence of the acceptor. Software was developed to process the acquired images for single and double exponential decays. Measurement of donor lifetime in two different conditions allowed us to calculate accurately the distance between the interacting proteins. We used this approach to quantify the dimerization of the transcription factor CAATT/enhancer binding protein alpha in living pituitary cells. The one- and two-component analysis of the donor molecule lifetime in the presence of acceptor demonstrates the distance distribution between interacting proteins.

A-band shortening in single fibers of frog skeletal muscle.

The question of whether A-bands shorten during contraction was investigated using two methods: high-resolution polarization microscopy and electron microscopy. During shortening from extended sarcomere lengths in the passive state, sarcomere-length changes were essentially accounted for by I-band shortening. During active shortening under otherwise identical conditions, the sarcomere length change was taken up approximately equally by A- and I-bands. Several potential artifacts that could give rise to apparent A-band shortening were considered and judged unlikely. Results obtained with polarization microscopy were similar to those obtained with electron microscopy. Thus, modest but significant thick filament shortening appears to occur during active sarcomere shortening under physiological conditions.

Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.

The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and fluorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occurring between 15 and 20 degrees C. Most of the label which becomes cell associated at 37 degrees C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an endosomal/lysosomal marker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonucleotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be blocked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.

Conventional, confocal and two-photon fluorescence microscopy investigations of polymer-supported oxygen sensors.

Luminescence-based, polymer-supported oxygen sensors, particularly those based on platinum group complexes, continue to be of analytical importance. Commercial applications range from the macroscopic (e.g. aerodynamic investigations in wind tunnels, monitoring of oxygen concentration during fermentation, and measurement of biological oxygen demand) to the microscopic (e.g. imaging of oxygen in blood, tissue, cells and other biological samples). Problems hindering the design of improved oxygen sensors include non-linear Stern-Volmer calibration plots and the multi-exponentiality of measured lifetime decays, both of which are attributed primarily to heterogeneity of the sensor molecule in the polymer support matrix. Conventional, confocal and two-photon fluorescence microscopy have proven to be invaluable tools with which the microscale heterogeneity and response of luminescence-based oxygen sensors can be investigated and compared to the macroscopic response. Results obtained for three ruthenium(II) alpha-diimine complexes in polydimethylsiloxane polymer supports indicate the presence of unquenched microcrystals within the polymer matrix that probably degrade oxygen quenching sensitivity and linearity of the Stern-Volmer quenching plot. Two-photon fluorescence microscopy proved most useful for imaging microcrystals within sensor films, and conventional microscopy allowed direct comparison between microscopic and macroscopic sensor response. The implications of the results in the rational design and mass production of luminescence-based oxygen sensors are significant.

Truncated estrogen receptor product-1 suppresses estrogen receptor transactivation by dimerization with estrogen receptors alpha and beta.

The estrogen receptor (ER) is a ligand-activated transcription factor that acts as a homodimer. Truncated estrogen receptor product-1 (TERP-1) is a pituitary-specific, estrogen-induced, isoform of rat ERalpha that is transcribed from a unique start site and contains only the C-terminal region of the full-length receptor. TERP-1 does not affect transcription directly but suppresses ligand-activated ERalpha and ERbeta activity. Because TERP-1 contains a dimerization domain and part of the coactivator binding pocket, we hypothesized that it modulates ER function by direct interactions with full-length ER or the steroid receptor coactivator, SRC-1. Localization studies demonstrate that TERP-1 is present in the cytoplasm and nucleus of transfected cells and colocalizes with nuclear ER. Protein binding studies show that TERP-1 forms heterodimers with both ERalpha and ERbeta and inhibits ERalpha binding to its cognate DNA response element. TERP-1 also binds SRC-1, and increasing levels of SRC-1 decrease the TERP-1-ERalpha interactions, in agreement with the rescue of TERP-1-suppressed ERalpha transcriptional activity by SRC-1. Mutational analysis of TERP-1 and ERalpha in the activation helix and the AF-2 dimerization helix indicates that TERP-1 acts predominantly through dimerization with ERalpha. Therefore, TERP-1 suppression of ER transcription occurs primarily by formation of inactive heterodimers and secondarily by competition for coactivators.