Carbonic anhydrase localization by light and electron microscopy: a comparison of methods

The histochemical localization of carbonic anhydrase by Hansson's cobalt-salt method was compared with immunocytochemical localization in the proventriculus (glandular stomach), the chorioallantoic membrane, and in articular and growth-plate cartilages from the domestic hen. Numerous differences were observed. Staining was positive by Hansson's method and negative by immunocytochemistry for the submucosal glands of the proventriculus, articular cartilage cells, resting and proliferating cells of the growth plate, nuclei, and intercellular spaces. Red blood cells stained faintly and inconsistently by Hansson's method. Both methods were in agreement for the cytoplasm of the surface mucosal cells of the proventriculus, the cytoplasm of the villus cavity cells in the chorioallantoic membrane, and in hypertrophic cells of growth-plate cartilage. Acetazolamide usually inhibited the histochemical reaction, even in those sites that, according to other methods, did not contain enzyme. Consequently, acetazolamide inhibition appears to be an unreliable control for the histochemical reaction.     

Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.     

Distribution of a platinum anti-tumour drug in HeLa cells by analytical electron microscopy

The distribution of Pt in HeLa cell sections after cell treatment with cis-Pt(NH3)2Cl2 dissolved in dimethylsulphoxide was probed by analytical electron microscopy. Primary targets were the nucleolus and the inner side of the nuclear double membrane. Even after solvolysis in dimethylsulphoxide the drug reached similar sites. It is suggested that cell death may be due to Pt inhibition at the initiation sites of DNA synthesis.     

Fluorescence lifetime microscopy of the sodium indicator sodium-binding benzofuran isophthalate in HeLa cells

The behavior of the sodium indicator sodium-binding benzofuran isophthalate (SBFI) is investigated in HeLa cells by time-resolved fluorescence microscopy. The fluorescence relaxation of SBFI in HeLa cells can be described by a triexponential for intracellular sodium concentration ([Na(+)](i)) between 0 and 90 mM. Changes in [Na(+)](i) affect neither the fluorescence relaxation times (0.21, 0. 60, and 2.7 ns) nor the average decay time (2.2 ns). The preexponential factor of the shortest decay time is negative. However, the ratio of the fluorescence excitation signal at 340 nm to that at 380 nm increases with [Na(+)](i). To elucidate the behavior of SBFI in cells, experiments are performed on SBFI in buffer at various concentrations of sodium, potassium, and bovine serum albumin (BSA) and at various viscosities. The fluorescence decay is triexponential only in the presence of BSA. The relaxation times are independent of [Na(+)] and [BSA]. The preexponential factor of the shortest decay time is negative from a certain [BSA] on, which depends on [Na(+)]. The data indicate that interactions with intracellular components rather than microviscosity influence the SBFI behavior in cells. A model is suggested in which the fluorescence intensities are mainly determined by the signals from the Na(+) subsetSBFI and SBFI subsetprotein complexes.     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.     

Fluorescence lifetime microscopy of the Na+ indicator Sodium Green in HeLa cells

This study investigates the usefulness of lifetime measurements of Sodium Green for evaluating intracellular Na+ concentration ([Na+]i) in HeLa cells. Frequency-domain lifetime measurements are performed in HeLa cells and in different buffer solutions (with and without K+ and bovine serum albumin). In all cases, the fluorescence decays of Sodium Green are multiexponential, with decay times independent of [Na+]. Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin results in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponential. The ratio of the fractional intensity of the long decay time (tau2 = 2.4 +/- 0.2 ns) to that of the short component (tau1 = 0.4 +/- 0.1 ns) increases with [Na+]i. The changes in fluorescence decay with [Na+] are significantly less pronounced in cells as compared with the buffer solutions. Similar values for the resting [Na+]i were estimated from lifetime measurements of Sodium Green and from ratiometric measurements using SBFI. Alternatively, [Na+]i can be monitored by measuring only the phase angle at the modulation frequency of 160 MHz. The usefulness of this latter approach is demonstrated by following the changes in [Na+]i induced by reversible inhibition of the Na+/K+ pump.     

Exocytosis in colonic goblet cells visualized by video-enhanced light microscopy

In order to develop a method to quantify the mucus secretion, we observed the mucus epithelium of the rabbit colon under a video-enhanced differential interference contrast microscope. Upon stimulation with muscarinic agonists, secretory granules in individual goblet cells were found to undergo a rapid light intensity change. Simultaneously, the lumen was widened and filled with a cloudy material. In each cell, many of such responses were followed by formation of a large cavity which could be recovered after removal of the stimulant. We infer that the light intensity change of a granule arises from exocytosis. Direct counting of the frequency of these quantal responses would be very useful to monitor the secretory activity of single cell in real time at a high sensitivity.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

Analysis of replication factories in human cells by super-resolution light microscopy

Background  

DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution.     

Analysis of replication factories in human cells by super-resolution light microscopy

Background

Retinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 or retinoic-acid receptor responder 3, is a growth regulator, which induces apoptosis and differentiation. RIG1 is classified into the NC protein family. This study investigated functional domains and critical amino acids associated with RIG1-mediated cell death and apoptosis.

Results

Using enhanced green fluorescence protein (EGFP)-tagged RIG1 variants, RIG1 proteins with deletion at the NC domain significantly decreased cell death induced by RIG1, and fusion variants containing only the NC domain significantly induced apoptosis of HtTA cervical cancer cells. The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N¹¹²C¹¹³ motif double- (NC→FG) or triple- (NCR→FGE) mutated RIG1 variants. Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1_111–123 or Leu¹²¹-mutated RIG1_111–123:L→ C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1_111–123:NC→FG or RIG1_111–123:NCR→FGE, were cytoplasmically localized and did not induce apoptosis. The RIG1_111–123 also induced apoptosis of A2058 melanoma cells but not normal human fibroblasts.

Conclusion

The NC domain, especially the NC motif, plays the major role in RIG1-mediated pro-apoptotic activity. The RIG1_111–123 dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.     

Force microscopy of nonadherent cells: a comparison of leukemia cell deformability

Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E(infinity) = 855 +/- 670 Pa for HL60 cells, E(infinity) = 48 +/- 35 Pa for Jurkat cells, E(infinity) = 156 +/- 87 for neutrophils, mean +/- SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia.     

A comparison of methods for morphological studies of cultured cells

Summary A number of fixation methods for different types of cells in culture were compared, and the best preservation of nuclear and cytoplasmic details was obtained by fixation with Bouin's solution for 15 min, prior to staining with hematoxylin and eosin. All of the fixatives, including Bouin's solution, damaged various structures, notably the peripheral glas-attached cytoplasm and the intercellular connections. Micrographs obtained by bright field, phase contrast, and interference contrast (Nomarski) microscopy are presented. Much more realistic pictures, bringing out details not observed after fixation and staining, were obtained by Nomarski microscopy of living, unfixed cultures. Most conspicuous were numerous thin, cytoplasmic, cilia-like extensions, concentrated on the glass-attached peripheral margins, which were also visible on other cell surfaces and as intercellular connections. These structures were most characteristic of SV40-transformed human amnion cells. Although fixation and staining emphasize certain cell components (for example, inclusion bodies), many aspects of cellular morphology are better demonstrated by observing living cells by interference microscopy or by Nomarski interference contrast microscopy. Surface features of unfixed cells, seen by Nomarski interference contrast microscopy, were similar to the surface features of glutaraldehyde-osmium tetroxide-fixed cells studied as metallic replicas in the electron microscope. Supported in part by National Cancer Institute Research Grant CA-08748 and contributions from the Albert Soiland Cancer Foundation.     

Proteomic analysis of differential protein expression induced by ultraviolet light radiation in HeLa cells

Cells treated with ultraviolet (UV) radiation undergo cell cycle arrest at the S-phase and G1/S boundary, allowing DNA repair to occur. Several proteins such as replication protein A and DNA-dependent protein kinase have been suggested to be involved in UV-induced inhibition of DNA replication. However, the role of these proteins in inhibiting DNA replication remains unknown. Other proteins may play important roles in modulating functions of these proteins in response to UV-irradiation. To understand the broad range of proteins involved in this inhibition, we carried out a systematic study to identify specific proteins involved in UV-induced replication arrest using two-dimensional gel electrophoresis and mass spectrometry. Unique changes in protein expression level for 31 proteins were observed over a 24-hour time course, including calgizzarin, cyclophilin A, and macrophage migration inhibitory factor. The expression level changes of these proteins are dynamically correlated to DNA replication activity, suggesting involvement of these proteins in modulating DNA replication and repair activities. This proteomic approach provides opportunities to gain insights into the mechanism by which DNA replication is inhibited.