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PubMedDate not supplied

The relationship between [Ca2+]i and cell death using an in vivo model: a study using the ced-1 mutant strain of C. elegans.

The ced-1 mutant of the free-living nematode, Caenorhabitis elegans, was used to study cell injury and cell death in relation to changes in intracellular ionized calcium ([Ca2+]i). This animal, which is being genetically characterized, may prove to be extremely useful for certain toxicologic studies because of its small size, optical transparency, rapid generation time, and the morphologic and genetic data currently available. During the development of this animal, 131 of 1,090 ultimate somatic cells undergo programmed cell death. Using mutagenesis techniques, several genes responsible for this death have been identified. In this study, we have taken advantage of the ced-1 mutant in which dead cells accumulate, as they cannot be phagocytized and removed. Although changes in [Ca2+]i have been studied in relation to cell injury and cell death, observations have been essentially restricted to in vitro monolayer cultures because of the methodology involved. To study the relationship between changes in [Ca2+]i and injury in vivo, we selected this animal model for further study and report here the morphological changes following the effects of ionomycin treatment in relation to increases of [Ca2+]i and cell death as measured using the fluorescent probes Fluo-3/AM and propidium iodide, respectively. The technique of confocal laser scanning microscopy is ideally adapted to such measurements in these living animals, and the results can be readily correlated with those made with Nomarski differential interference contrast microscopy as well as with transmission electron microscopy. The results support previous in vitro observations and show that early increases of [Ca2+]i accompany early reactions to injury. Furthermore, the results also show that changes in this small invertebrate metazoan parallel those seen in mammalian systems, including human. Thus, the current study indicates that ced-1 C. elegans can potentially serve as an in vivo model not only for evaluating the possible temporal relationship of [Ca2+]i elevation with cell death but also for evaluating the [Ca2+]i elevation observed in relation to other phenomena and in evaluating toxic agents.

Animals↗
PubMed1982

[Immunohistochemical observations on tubulin and alpha actinin during mitosis of cultured fibroblasts].

In this work we studied alpha-actinin and tubulin sites in rabbit fibroblasts in culture. Antibodies anti-alpha-actinin were used for indirect PAP-reaction while antibodies anti-tubulin were used for indirect immunofluorescence method. The observations were carried out by light microscopy, phase-contrast and interference-contrast microscopy, with regard to actinin, and by fluorescence microscopy, with regard to tubulin. During the early mitotic phase, alpha-actinin is localized all over the cell membrane of the fibroblasts, forming a sort strong protective cap, while during diacinesis it forms only rings, localizing below the cell membrane and the philopodia. Thus tubulin forms the bundle fibres during mitotic phases.

Actinin↗
PubMed2000

Computational model of DIC microscopy for reconstructing 3-D specimens.

Differential Interference Contrast (DIC) microscopy is a powerful visualization tool used to study live biological cells. Its use, however, has been limited to qualitative observations. The inherent non-linear relation between the object properties and the image intensity makes quantitative analysis difficult. As a first step towards measuring optical properties of objects from DIC images, we develop a model for the image formation process using methods consistent with energy conservation laws. We verify our model by comparing real image data of manufactured specimens to simulated images of virtual objects. As the next step, we plan to use this model to reconstruct the three-dimensional properties of unknown specimens.

Computer Simulation↗
PubMed1987

Fibroblast adhesion to RGDS shows novel features compared with fibronectin.

As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.

Actins↗
PubMed2003

Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy.

Calmodulin (CaM), a small calcium-binding protein, is the key mediator of numerous calcium-induced changes in cellular activity. Its ligands include enzymes, cytoskeletal proteins and ion channels, identified in large part by biochemical and cell biological approaches. Thus far it has been difficult to assess the function of CaM genetically, because of the maternal supply in Drosophila and the presence of at least three nonallelic genes in vertebrates. Here we use the unique possibility offered by the C. elegans model system to inactivate the single CaM gene (cmd-1) through RNA interference (RNAi). We show that the RNAi microinjection approach results in a severe embryonic lethal phenotype. Embryos show disturbed morphogenesis, aberrant cell migration patterns, a striking hyperproliferation of cells and multiple defects in apoptosis. Finally, we show that RNAi delivery by the feeding protocol does not allow the efficient silencing of the CaM gene obtained by microinjection. General differences between the two delivery methods are discussed.

Amino Acid Sequence↗
PubMed1992

Contractile proteins in the hyaline cells of the chicken cochlea.

Hyaline cells are a single layer of epithelial cells found at the inferior edge of the sensory epithelium in the chick cochlea. They rest directly above a specialized region of the basilar membrane at a point where it connects to the fibrocartilaginous skeleton of the cochlear duct. The basal cytoplasm of the hyaline cells contains a bundle of linearly aligned actin filaments that resemble stress fibers in their organization. The actin filaments are anchored in the basal plasma membranes of the cells, which are, in turn, associated with the underlying basal lamina and the extracellular matrix of the basilar membrane. We have used a combination of transmission electron microscopy, differential-interference-contrast and epifluorescence light microscopy, and confocal laser scanning microscopy to study the composition and organization of these actin bundles within the hyaline cells. The bundles are arranged into triangular wedges that are oriented radially across the basilar membrane. Each cell contains one or two actin wedges. Adjacent cells can have them aligned in opposite directions so that in a whole-mount surface preparation they appear as interdigitations. Immunofluorescent staining of the hyaline cells has shown that smooth muscle myosin and alpha-actinin are co-localized to the actin bundles. Smooth muscle myosin is also found throughout the cytoplasm of the cells. The fact that hyaline cells in the chick cochlea are contacted by efferent nerve fibers suggests that these cells may regulate tension on the basilar membrane via the specialized bundle of actin filaments.

Animals↗
PubMed1999

Rearrangement of the F-actin cytoskeleton in estradiol-treated MCF-7 breast carcinoma cells.

In response to treatment with 17beta-estradiol, MCF-7 human breast carcinoma cells undergo a marked rearrangement of the F-actin cytoskeleton. The most conspicuous aspect of this rearrangement is the formation of an extensive array of lamellipodial structures which are situated beneath cell clusters. Treatment of cells with 17beta-estradiol in the presence of the anti-estrogen ICI182,780 suppressed the development of the lamellipodial structures, indicating that this cytoskeletal rearrangement is mediated by the estrogen receptor. Time-lapse, video-enhanced, differential interference contrast microscopy reveals that the lamellipodial structures are actively motile beneath cell clusters. Furthermore, the lamellipodial structures form few focal contacts with the underlying substrate of the coverslip, as evidenced by either interference reflection microscopy or staining for the focal contact protein talin, indicating that these structures are not strongly adhered to the substratum. Immunofluorescence localization of E-cadherin indicates that this cell-cell adhesion receptor is present within these structures as either adhesion plaque- or point contact-like depositions. These findings implicate the cadherin-based cell-cell adhesion system in supporting tumor cell motility over adjacent cell surfaces via discrete adhesive structures which are associated with motile lamellipodia.

Actins↗
PubMed1976

Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique.

The closeness of contact between cultured chick heart fibroblasts and glass substrates has been examined by interference reflexion microscopy. Evaluation of the optical technique demonstrated that both the film of medium between the cell and substrate and the cell itself act as two superimposed thin films which generate a complex interference pattern in reflected light or low illuminating numerical aperture (I.N.A.). The interference pattern generated by the cell consists of first and higher orders of interference and can be eliminated from the image by increasing the I.N.A. to greater than 1.0. The residual zero-order interference pattern at high I.N.A. originates from the thin film of medium between cell and substrate and corresponds to the closeness of contact between cell and substrate. Based on the zero-order interference pattern and a comparison of the same cells with differential interference optics, the following features were recognized in living chick heart fibroblasts. Focal contacts, 0.25-0.5 mum wide, 2-10 mum long, separated by 10-15 nm from the substrate, are located under the peripheral regions of the leading lamellae and near the edge of extended non-spreading regions of the cell margin in moving and stationary cells. Individual focal contacts are coincident with cytoplasmic fibres of the same dimensions as the contact or with the peripheral ends of longer fibres that extend from the focal contact toward the centre of the cell. In spreading cells a second type of contact is present under the peripheral regions of the leading lamellae. This close contact consists of a broad area of uniform cell-to-substrate separation distance (ca. 30 nm). Focal contacts are distributed within the close contact under the leading lamellae. Lamellipodia extend ahead of the close contact and are separated by 100 nm or more from the substrate. Depending on the previous motile history of the cell, the close contact extends to varying degrees under the centre of the cell but it is typically dissected here by discrete areas of distinctly greater cell-to-substrate separation (100-140 nm). Evidence for the adhesive nature of the focal contacts is considered. The common identity is discussed of the focal contacts and associated cytoplasmic fibres described here in living cells with the regions of closest apposition to the substrate and associated cytoplasmic plaques and bundles of microfilaments seen in EM studies.

Cell Adhesion↗
PubMed1993

Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.

The movements of E-cadherin, epidermal growth factor receptor, and transferrin receptor in the plasma membrane of a cultured mouse keratinocyte cell line were studied using both single particle tracking (SPT; nanovid microscopy) and fluorescence photobleaching recovery (FPR). In the SPT technique, the receptor molecules are labeled with 40 nm-phi colloidal gold particles, and their movements are followed by video-enhanced differential interference contrast microscopy at a temporal resolution of 33 ms and at a nanometer-level spatial precision. The trajectories of the receptor molecules obtained by SPT were analyzed by developing a method that is based on the plot of the mean-square displacement against time. Four characteristic types of motion were observed: (a) stationary mode, in which the microscopic diffusion coefficient is less than 4.6 x 10(-12) cm2/s; (b) simple Brownian diffusion mode; (c) directed diffusion mode, in which unidirectional movements are superimposed on random motion; and (d) confined diffusion mode, in which particles undergoing Brownian diffusion (microscopic diffusion coefficient between 4.6 x 10(-12) and 1 x 10(-9) cm2/s) are confined within a limited area, probably by the membrane-associated cytoskeleton network. Comparison of these data obtained by SPT with those obtained by FPR suggests that the plasma membrane is compartmentalized into many small domains 300-600 nm in diameter (0.04-0.24 microns2 in area), in which receptor molecules are confined in the time scale of 3-30 s, and that the long-range diffusion observed by FPR can occur by successive movements of the receptors to adjacent compartments. Calcium-induced differentiation decreases the sum of the percentages of molecules in the directed diffusion and the stationary modes outside of the cell-cell contact regions on the cell surface (which is proposed to be the percentage of E-cadherin bound to the cytoskeleton/membrane-skeleton), from approximately 60% to 8% (low- and high-calcium mediums, respectively).

Animals↗