Liver tumors distinguished by immunofluorescence microscopy with antibodies to proteins of intermediate-sized filaments.

Antibodies against constitutive proteins of different types of intermediate-sized filaments were used in immunofluorescence microscopy on frozen sections of normal rat liver and various rat liver tumors induced by treatment with nitrosamines. Antibodies to tonofilament prekeratin stained bile duct epithelia and hepatocytes of normal liver and hepatocellular carcinoma cells and ductal cells of cholangiofibromas. These cells were not significantly stained by antibodies to vimentin. By contrast, antibodies to vimentin stained mesenchymal cells of normal liver and cells of early and advanced angiosarcomas and of undifferentiated spindle cell sarcoma. These mesenchymal tumor cells were not stained with antibodies to prekeratin. The presence of intermediate-sized filaments in these tumors, often in large whorl-like aggregates, was also demonstrated by electron microscopy. The results show that immunofluorescence microscopy with antibodies to cytoskeletal proteins is a powerful tool for the classification and differential diagnosis of mesenchymal and epithelial liver tumors. We propose that staining with antibodies to proteins of different types of intermediate filaments can be used to improve the identification of tumors of other organs, including metastases, as well as non-neoplastic proliferative lesions.     

Lenticular intermediate-sized filaments: biosynthesis and interaction with plasma membrane.

Electron microscopical features of the lens fiber plasma membrane-cytoskeleton complex are suggestive of an intimate association between the intermediate-sized filaments (IF) and the lipid bilayer. Biochemical analysis of this complex reveals the occurrence of an appreciable amount of vimentin as a protein subunit of lenticular IF. Additional evidence for association between IF and membranes is provided by the observation that newly synthesized vimentin is associated with plasma membranes added to a reticulocyte lysate programmed with lens polyribosomes. Concomitantly alpha-crystallin polypeptide chains (alpha A2) are also found associated with the plasma membrane together with a hitherto unidentified 47-kilodalton protein. Once associated with the lipid bilayer, the vimentin polypeptide resists urea treatment, suggesting that it has become an integral constituent associated with part of the membrane.     

Estimation of changes in vimentin filaments induced by etoposide and doxorubicin in human leukemia cell line K-562 by using immunofluorescence microscopy.

This study was undertaken to examine the influence of etoposide and doxorubicin on the distribution of vimentin in cells of human leukemia cell line K-562 by using immunofluorescence microscopy. The cells were cultured with 5 different doses of etoposide: 0.02, 0.2, 2, 20, 200 microM/l and three doses of doxorubicin: 0.5, 5, 10 microM/l. Changes in vimentin filaments were dependent on concentration of drugs compared to untreated control cells. Cells treated with 20 microM/l, especially with 200 microM/l etoposide were much bigger from other cells exposed to lower doses of etoposide and control cells, and their number decreased. In most control cells vimentin was seen as a ring with the increased concentration on one pole of the cells. In 20 microM/l and 200 microM/l etoposide the cells showed rather a diffuse cytoplasmic staining pattern. Vimentin filaments were organized as a dense network in cytoplasm of these cells. Immunofluorescence studies on K-562 cells treated with doxorubicin showed that cells incubated with 5 microM/l doxorubicin have much diffuse staining pattern of vimentin with delicate reticular structure and with intense staining near one pool of the cells. Addition of 10 microM/l doxorubicin to cells resulted in increasing of fluorescence staining, which appeared in the cells as enough dense network with intense staining rather in the centre of the cell.     

Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

The electron optical analog of immunofluorescence microscopy combines three developments: (i) photo-electron microscopy to produce a high-resolution image of exposed components of the cell, (ii) site-specific antibodies, and (iii) photoemissive markers coupled to the antibodies to make the distribution of sites visible. This approach, in theory, provides a way to extend the useful immunofluorescence microscopy technique to problems requiring much higher resolution. The resolution limit of fluorescence microscopy is limited to about 200 nm by the wavelength of the light used to form the image, whereas in photoelectron microscopy the image is formed by electrons (current resolution: 10-20 nm; theoretical limit: 5 nm or better depending on the electron optics). As a test system, cytoskeletons of CV-1 epithelial cells were prepared under conditions that preserve microtubules, and the microtubule networks were visualized by both indirect immunofluorescence and immunophotoelectron microscopy using colloidal gold coated with antibodies. Colloidal gold serves as a label for immunophotoelectron microscopy, providing enhanced photoemission from labeled cellular components so that they stand out against the darker background of the remaining unlabeled structures. In samples prepared for both immunofluorescence and immunophotoelectron microscopy, individual microtubules in the same cells were visualized by both techniques. The photoemission of the colloidal gold markers is sufficiently high that the microtubules are easily recognized without reference to the immunofluorescence micrographs, indicating that this approach can be used, in combination with antibodies, to correlate structure and function in cell biological studies.     

Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics.

Subunit complexes of cytokeratin polypeptides from intermediate-sized filaments (IF) of various tissues and cultured cells from rat, cow, and man were solubilized in low-salt buffer containing 4 M urea and exposed to increasing concentrations of urea, followed by urea gradient electrophoresis or two-dimensional gel electrophoresis at different urea concentrations. Correspondingly, cytokeratin polypeptides dissociated in 9.5 or 10 M urea were dialyzed into lower concentrations of urea and allowed to reassociate into specific complexes. It was found that the polypeptide constituents of a given cytokeratin complex dissociate in the form of a rather sharp "melting curve" and that dissociated polypeptides reassociate in the same mode of dependence on urea concentration. The midpoint of melting in urea (Um) is a characteristic property of a given complex of cytokeratin polypeptides. Um values differ markedly between different cytokeratin complexes, ranging from 5.9 to 9.0 M urea. The results also show that cytokeratins do not form complexes with vimentin, another type of IF protein. The data suggest that certain cytokeratin polypeptides are complementary and contain sequences that direct their association into specific complexes forming IF subunits.     

Association of microtubules and intermediate filaments in chicken gizzard cells as detected by double immunofluorescence.

By double indirect immunofluorescence, using guinea pig and rabbit antibodies to tubulin and to desmin, we have simultaneously labeled microtubules and intermediate filaments in cultured chicken embryo gizzard cells. At the resolution of the light microscope there was extensive but not complete superposition of the labeling patterns for the two filamentous structures within cells in interphase and an essentially complete dissociation of the two labeling patterns in cells in mitosis. These results indicate that there is an extensive association of microtubules and intermediate filaments in these interphase muscle cells and suggest that this association is regulated metabolically.     

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.     

Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy

Electron microscopy and indirect immunofluorescence microscopy using monospecific tubulin antibodies were performed in parallel on glutaraldehyde-fixed tissue culture cells without osmium fixation. In order to reduce the excess aldehyde groups of the strongly crosslinked cellular matrix, which normally interfere with subsequent immunofluorescence microscopy, a mild NaBH(4) treatment was introduced during or after the dehydration steps. Cells processed through the NaBH(4) step show, in transmission electron microscopy, normal cytoplasmic microtubules approximately 250 A in diameter. When such cells are subjected to indirect immunofluorescence microscopy using monospecific tubulin antibody they reveal a complex system of unbroken, fine, fluorescent fibers traversing the cytoplasm between the perinuclear space and the plasma membrane. Thin sections of cells processed through the indirect immunofluorescence procedure show antibody-decorated microtubules with a diameter of approximately 600 A. This decoration is not obtained when non-immune IgGs are used instead of monospecific antitubulin IgGs. Thus, a direct comparison of cytoplasmic microtubules in glutaraldehyde-fixed cells by both electron microscopy and immunofluorescence microscopy can be obtained.     

Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy.

During our studies with antibodies against structural proteins of the cytoskeleton of eukaryotic cells we have observed that sera from many normal rabbits decorate a fiber system in cells of the established rat kangaroo cell line Pt K2. The display and organization of these fibers are different from those of microfilament bundles (decorated by antibody to actin) and microtubules (decorated by antibody to tubulin). This new fiber system can be further distinguished by its resistance to reorganization when cells are treated with Colcemid or cytochalasin B. The decoration of this fiber system is not detected if Pt K2 cells are fixed with formaldehyde. Such sera also appear to decorate swirls of perinuclear fibers in mouse Neuro 2a cells, and in mouse 3T3 cells treated with mitotic drugs. Comparison of the immunofluorescence pictures with electron microscopic data suggests that the sera are visualizing bundles of intermediate 7- to 10-nm filaments.     

Instrumentation technology problems in immunofluorescence microscopy

The influence of construction units of the microscope such as filters, lamps and objectives upon fluorescent intensity and contrast is demonstrated. Further, the bleaching of fluorescence and the possibilities of contrast illumination are dealth with. Of crucial importance to the evaluation of the fluorescent images is their contrast rather than the intensity of fluorescence. Narrow band excitation is beneficial and brightening of the background, strong bleaching as well as too great a secondary magnification are disadvantageous to the contrast.     

Identification of a subspecies-specific capsular antigen from Bacteroides melaninogenicus subspecies asaccharolyticus by immunofluorescence and electron microscopy.

An indirect fluorescent antibody test was developed with the use of hyperimmune rabbit antiserum to a purified capsular polysaccharide of Bacteroides melaninogenicus subspecies asaccharolyticus. All of 23 strains of B. melaninogenicus subspecies asaccharolyticus were fluorescence-positive in this test. All 11 strains of Bacteroides melaninogenicus subspecies intermedius tested and three strains of Bacteroides melaninogenicus subspecies melaninogenicus were fluorescence-negative. Thirty-one strains of other bacterial species were also fluorescence-negative. The indirect fluorescent antibody test demonstrated the presence of a subspecies-specific capsular antigen from B. melaninogenicus subspecies asaccharolyticus. The capsular antigen was further demonstrated by electron microscopy with ruthenium red, a polysaccharide-staining material.     

Vessel size-dependent expression of intermediate-sized filaments, calponin, and h-caldesmon in smooth muscle cells of human coronary arteries

Summary The arterial media is composed of a heterogeneous population of smooth muscle cells (SMCs). Recently, the properties of SMCs were observed to be heterogeneous not only among individual cells but also among arteries of the same vascular bed. To test the hypothesis that a site-specific heterogeneity exists in the SMCs of human coronary arteries, we examined the expression of desmin, vimentin, calponin, and high-molecular-weight (h-) caldesmon in arteries of various sizes. Specimens of arteries were obtained at autopsy from 12 patients: 6 adults (67 ± 4 years old); 3 younger adults (26 ± 2 years old); and 3 neonates. The size of the arteries was estimated by the number of SMC layers of the media. The expression was compared in SMCs of large arteries (>10 layers in adults, >5 layers in neonates), medium-sized arteries (5–10 layers in adults, 3–5 SMC layers in neonates), and small arteries (<3 layers). In adults, the percentage of arteries positive for desmin was lower in the small (17% ± 3%) and medium-sized arteries (44% ± 12%) than in the large arteries (94% ± 6%) (P < 0.01). The percentage of arteries positive for calponin was also lower in the small (18% ± 2%) and medium-sized arteries (66% ± 5%) than in the large arteries (100%) (P < 0.01). The percentage for vimentin and h-caldesmon did not differ among large, medium-sized, and small arteries. These observations in adults were similar to those in younger adults or neonates. The phenotypes of medial SMCs are vessel sizedependent in human coronary arteries. This finding should be important for understanding the site-specific characteristics of vascular function in the regulation of myocardial perfusion or those of vascular responses to environmental changes.This study was partly supported by Grant-in-Aid for Scientific Research No. 10670626 from the Ministry of Education, Science and Culture of Japan.     

Cell kinetic analysis of intact rat colonic crypts by confocal microscopy and immunofluorescence

BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS: Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5- bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS: S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS: Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision. (Gastroenterology 1996 Dec;111(6):1493-500)     

Structure of fibroblastic intermediate filaments: analysis of scanning transmission electron microscopy.

The structure of fibroblastic intermediate filaments from Chinese hamster ovary cells has been investigated by scanning transmission electron microscopy. Freshly extracted (native) filaments were compared with filaments reassembled in vitro from purified decamin. From digital micrographs of unstained specimens, direct measurements of linear mass density were performed on many individual filaments. Native filaments beyond a certain minimal length constitute a homogeneous population, averaging 38 +/- 4 kilodaltons (kDal)/nm. A minor but distinct polymorphic variant (23 +/- 4 kDal/nm) was also present as very short filaments or end-segments; these may represent breakdown products or assembly intermediates. Analysis of reassembled filaments demonstrates that the in vitro assembly reaction is--in the main--faithful, although the distribution of their mass measurements is appreciably broader than that of the native data. In addition to the predominant type at 37 +/- 4 kDal/nm and a minor component at 26 +/- 4 kDal/nm, small amounts of a third, more massive, polymorphic variant at 52 +/- 5 kDal/nm were also present. Micrographs of negatively stained specimens clearly demonstrate that the filaments are composed of bundles of protofilaments--each 2-3 nm in diameter--and also reveal an axial periodicity of about 46 nm. The implications of these findings are discussed for three classes of model previously proposed for the structure of intermediate filaments.     

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.Biological processes are inherently driven by the molecular-scale organization of biomolecular assemblies, which arrange in precise structures that are essential for biological functions in cells and tissues. The extent to which the biological function depends on the underlying molecular-scale organization is particularly evident in filamentous assemblies, such as DNA filaments and cytoskeletal protein filaments. Changes in the local higher-order organization of DNA filaments is tightly linked to essential DNA-mediated processes including control of gene expression, DNA replication, and DNA repair. However, how specific DNA-binding proteins affect DNA filament architecture and thus DNA-mediated functions is poorly understood (1). Similarly, the spatial organization of cytoskeletal filaments in cells and tissues is also weakly explored, despite their central role in generating forces and driving cell motility, cell division, and tissue morphogenesis (2). Electron microscopy has been widely used to provide molecular-scale images of the structure of such filament assemblies; however, it typically involves several daylong sample preparation and ultrathin sectioning of the biological material, thus limiting investigations in whole cells and tissues.Polarized fluorescence imaging is a powerful approach for elucidating the structural organization of filament assemblies because it is compatible with a wide variety of microscopy techniques, thus enabling studies across multiple spatial and temporal scales. Polarized fluorescence imaging allows not only retrieval of structural information from molecular orientation measurements (3, 4) but also quantification of depolarization-induced processes such as in homo-fluorescence resonant energy transfer, which serve as indicators for biomolecular clustering or polymerization (5, 6). Molecules act as oriented absorption/emission dipoles, whose pointing direction can be monitored, exploiting the polarized nature of light. This property has been used to determine the organization of molecular assemblies whose orientation is constrained, in particular in the cell membrane (7–11). Recent works using polarized microscopy have also evidenced microscopic-scale organization of septin filaments in budding yeast (12, 13) and actin filaments in Drosophila tissues (14), opening a path for in vivo structural imaging. Mapping the local organization of complex filament assemblies using polarized fluorescence imaging thus appears as a key approach to understand fundamental biological functions as diverse as DNA-mediated processes and animal cell mechanics. Although significant advances have been made toward orientational order imaging using polarized fluorescence, investigations are still limited to the diffraction-scale resolution, which hampers single filament-scale observation that is required for a quantitative structural analysis of local disorder. Moreover, the retrieved information does not directly report the orientational organization of protein filaments, but rather the mixture between filament orientations and the intrinsic rotational mobility of the attached fluorescent probes (wobbling), which depends on the rigidity of their linker (10, 15).It is possible to extract information on orientational mobility using single-molecule detection, which can reveal processes that are often missed in ensemble averaging. Seminal single-molecule studies have used light polarization to measure single-molecule orientations, using excitation polarization modulation (16), analyzed direction (17), or more refined schemes to access out-of-plane tilt information (for a review, see ref. 18). Another great advantage of single-molecule imaging is the possibility to achieve superresolution imaging, which relies on single-molecule localization to reconstruct images at nanometer-scale precision, providing that emitters emit temporally independently (19–21). Combining superresolution imaging with single-molecule orientation measurements would provide an ultimate way to image the structural organization of filamentous assemblies at high spatial resolution in vivo. Although a similar combination has been applied to probe the rotational mobility of single molecules in isotropic environments (22), its use to quantify orientations in ordered systems (e.g., filamentous structures) presents several challenges. First, molecular orientation can itself affect localization properties (23–25), and therefore the quality of image reconstruction. This effect has been shown, however, to be less dramatic when rotational mobility occurs in 2D in the sample plane, or in a large angular range (26). Second, the measurement of molecular orientation involves splitting the signal into polarized detection/excitation channels, which can decrease localization precision if not properly processed. At last, although in-plane components of single-molecule orientations are relatively simple to extract, the measurement of out-of-plane orientations of molecules requires more sophisticated experimental schemes and detection algorithms (18, 27). Several signal analysis solutions have been proposed for both orientation and localization monitoring (25, 28–30); however, there is no report yet on quantitative superresolution imaging of molecular orientations in ordered systems.In this work, we present a quantitative method combining steady-state in-plane single-molecule orientation measurements and superresolution imaging, using polarization-resolved direct stochastic optical reconstruction microscopy (polar-dSTORM). We propose a simple experimental scheme, which is compatible with high signal-to-noise ratio conditions and which provides structural information in filamentous assemblies in 2D, which is sufficient for order interpretation. We present a dedicated algorithm for polarized single-molecule localization and quantification, and show that the imaging of orientational behaviors can be achieved with high accuracy, providing that stringent signal analysis is performed. We further show how to exploit the measured polarized signals to retrieve information on both the fluorescence label wobbling and the local disorder of biological filaments, two parameters that are not discernible in diffraction-limited ensemble methods. We illustrate the use of polar-dSTORM in microtubule networks in fixed cells and double-stranded DNA (dsDNA) in vitro, and evidence the effect of the fluorophore structure on its angular wobbling when linked to actin stress fibers in fixed cells via phalloidin conjugates. These results reveal that ensemble polarization-resolved methods generally overestimate molecular order, illustrating that single-molecule approaches are needed for determining the structural organization of biomolecular assemblies in an unbiased manner.     

Nucleoside kinases in T and B lymphoblasts distinguished by autoradiography.

Nucleoside kinases catalyze the initial step leading to the accumulation of deoxypurine nucleotides that occurs in patients with inherited deficiencies of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This accumulation is thought to interfere with DNA synthesis in lymphocytes and, thus, to cause the immune defects associated with these enzymopathies. However, there is controversy about the identity of the nucleoside kinases that are responsible for intracellular phosphorylation of deoxyadenosine in adenosine deaminase deficiency and deoxyguanosine in purine nucleoside phosphorylase deficiency. To distinguish the nucleoside kinases present in T and B lymphoblastoid cells, we have coupled discontinuous PAGE with autoradiography. This procedure showed that deoxycytidine kinase (NTP:deoxycytidine 5'-phototransferase, EC 2.7.1.74), deoxyadenosine kinase (ATP:deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76), and adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) are all present in both T and B lymphoblasts. While adenosine kinase is expressed at nearly equal levels in B and T cells, the deoxynucleoside kinases are expressed at much lower levels in B cells than in T cells. The autoradiographic data agreed with assays of the nucleoside kinase activities. Molecular weights were determined by using 5-10% polyacrylamide gels. Mr values were 29,000 for adenosine kinase, 41,000 for deoxyadenosine kinase, and 53,000 for deoxycytidine kinase and its isozyme. The reduced expression of deoxycytidine and deoxyadenosine kinases in B lymphoblasts may account for the lower accumulation of deoxypurine nucleotides in B cells as compared with T cells.     

G-cell counts in antral endoscopic biopsies by immunofluorescence.

Antral gastrin-producing cell (G-cells) were counted by an immunofluorescence technique in the antral biopsies obtained at endoscopy from 67 subjects; they included patients with duodenal ulcer, gastritis, and individuals with a normal gastric mucosa. The G-cell count was significantly lower (P less than 0.01) in patients with duodenal ulcer (142 G cells per mm2) in comparison to normal subjects (327 G cells per mm2). No statistically significant correlation was found between the G-cell number and any of the other parameters tested (pentagastrin test, basal serum gastrin and its response to a standard meal).