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PubMed1984

Formation of myofibrils in spreading chick cardiac myocytes.

Cardiac myocytes were isolated from 5-6-day-old chick embryos and allowed to spread in culture. The distribution of alpha-actinin in the cells was followed for five days in culture by exposing permeabilized cells to rhodamine-labeled alpha-actinin and also by injecting the labeled alpha-actinin into living myocytes. In addition to labeling the Z bands of sarcomeres, the added alpha-actinin also labeled small particles that were usually arranged periodically in linear arrays with a spacing between particles of 0.3-2.0 micron. Actin was localized between the particles of alpha-actinin by means of fluorescein-labeled heavy meromyosin. The punctate localization of alpha-actinin was prominent in pseudopods, behind ruffles, and at the periphery of spreading cells. Long rows of particles of alpha-actinin were often parallel to one another with the alpha-actinin particles in register. These linear arrays appeared to merge laterally to form strands with broader concentrations of alpha-actinin. Other linear arrays were parallel to myofibrils in the cell and some extended outward from the ends of myofibrils. We conclude that during spreading of cardiac myocytes, myofibrils form at the cell periphery behind the extending margins of the cell, and that the aggregates of alpha-actinin found in these areas are nascent Z bands in the forming myofibrils.

Actinin↗
PubMed1986

Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading.

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.

Actin Cytoskeleton↗
PubMed2006

Selective spread of neurotropic herpesviruses in the rat hippocampus.

Transsynaptically spreading viruses are widely used for tracing neuronal circuits in both the central and peripheral nervous systems. However, viruses are capricious tools with selective spreading properties that can produce false-negative results. Using herpes simplex virus type 1 and two pseudorabies virus strains, we aimed at mapping quantitatively neuronal connections in the rat hippocampus. We found that none of the tested viruses infected CA3 pyramidal neurons across synapses following inoculation into the CA1 area. Combined injections of the viruses with the retrograde tracer cholera toxin B (CTB) resulted in CTB, but not virus labeling of CA3 pyramidal neurons. In contrast, other brain regions known to send inputs to the CA1 (the entorhinal cortex, medial septum and diagonal band of Broca, raphe nuclei) were transsynaptically infected. Our results indicate that Schaffer collaterals of CA3 pyramidal cells lack the appropriate cellular machinery for successful neurotropic herpesvirus infection. After injections of viruses into the dentate gyrus/CA3 area, we found labeling in commissurally projecting mossy cells and their afferent granule cells but not in contralateral CA3 pyramidal cells. Using this unique spreading property, we estimated that single mossy cells receive input from a compact cluster of 30-40 granule cells.

Animals↗
PubMed1991

Dissociation between syncytia formation and HIV spreading. Suppression of syncytia formation does not necessarily reflect inhibition of HIV infection.

In this study, we have observed that a dissociation may occur in vitro between syncytia formation and HIV spreading. Efficient HIV spreading and virus replication occurred either in HIV-infected LFA-1+ lymphocytes treated with anti-LFA-1 mAb or in HIV-infected lymphocytes genetically deficient in LFA-1, despite the fact that syncytia formation was completely suppressed. Therefore, these results indicate that syncytia formation cannot be used as the sole parameter to evaluate the spread of HIV in vitro.

Antibodies, Monoclonal↗
PubMed1997

Relapsing and remitting experimental allergic encephalomyelitis: a focused response to the encephalitogenic peptide rather than epitope spread.

The progression of experimental allergic encephalomyelitis (EAE) in certain mouse strains has been reported to involve a broadening of the response to myelin antigens, apparently resulting from priming to endogenous determinants of the myelin sheath. The phenomenon has been termed determinant spread. Interest in this effect has centered on the mechanism it offers to explain the progressive, relapsing and remitting course of EAE and indeed of multiple sclerosis. We have conducted a systematic, longitudinal study in SJL mice to look for determinant spread during relapsing and remitting EAE, correlating epitope recognition and cytokine production with disease severity. Disease was induced using three of the four encephalitogenic proteolipid protein or myelin basic protein epitopes, and responses to each of four epitopes recognized by SJL T cells were tracked through acute disease, remission and relapse. The responses of lymph node cells, splenocytes and central nervous system (CNS)-infiltrating T cells were analyzed. While marginal, transient responses to secondary epitopes were detectable in splenocytes, CNS-infiltrating cells showed a dominant response to the original disease-inducing epitope without evidence of a shift to other determinants during relapse. Disease relapse was correlated with an increase in CNS-infiltrating cells and a high proliferative and interferon (IFN)-gamma response to the disease-inducing peptide. During remission, there was a decrease in numbers of cells infiltrating the CNS. These cells were down-regulated, showing low if any response to the myelin peptides tested as measured by proliferation, production of IFN-gamma or production of IL-4. Our findings argue strongly against a causal role for determinant spread in disease relapse as observed in these models of EAE.

Amino Acid Sequence↗
PubMedDate not supplied

Extralaryngeal spread of cancer of the larynx: a study with whole-organ sections.

Thirty total laryngectomy and five pharyngolaryngoesophagectomy specimens were studied with the whole-organ sectioning technique. The horizontal plane of sectioning was chosen to study the pattern and degree of extralaryngeal spread in carcinoma of the larynx. There were 22 transglottic, 6 supraglottic, and 7 infraglottic tumors. Among them there were 1 T1, 2 T2, 5 T3, and 27 T4 lesions. Spreading anteriorly, the tumor was found to penetrate the thyrohyoid membrane in 7 (32%) out of 22 transglottic tumors. Among the entire group of 35 specimens the thyroid cartilage was penetrated in 18 cases (51%), and the infrahyoid strap muscles were invaded by tumor in 17 cases (49%). The cricothyroid membrane was penetrated in 19 cases (54%). Spreading posteriorly, the tumor reached or eroded the mucosa of the pyriform fossa in 15 cases (43%), including 10 total laryngectomy specimens. The postcricoid submucosa was involved by tumor in 13 cases (37%) among which 9 were total laryngectomy specimens. It is thus recommended that the wide-field technique should be used for laryngectomy. Awareness of the possibility of tumor reaching the mucosa of the pyriform fossa and the postcricoid area allows the surgeon to keep clear of tumor in performing total laryngectomy.

Carcinoma, Squamous Cell↗
PubMed1977

Detection of cellular immunity to tumor-associated antigen(s) in mice by the macrophage spreading inhibition test.

Soluble preparations of antigens from a methylcholanthrene-induced fibrosarcoma of C57BI mice were prepared by homogenization of tumor tissue and high-speed centrifugation of the homogenate. These preparations were able to sensitize syngeneic mice to tumor-associated antigens (TAA) of the fibrosarcoma, and to provoke a delayed hypersensitivity reaction when injected into the footpad of sensitized mice. Furthermore, the same soluble preparations could inhibit in vitro the spreading of peritoneal macrophages from mice sensitized to TAA. A similar inhibition of macrophage spreading was obtained when peritoneal cells from C57BI mice, bearing transplants of the fibrosarcoma, were incubated with the preparations. We conclude that the macrophage spreading inhibition test, like other in vitro assays, can detect cell-mediated immunity to tumor-associated antigens.

Animals↗
PubMed1985

Extracellular material secreted by human colonic adenocarcinoma cell lines promotes spreading in serum-free medium and induces neurite outgrowth of PC-12 cells.

A completely defined medium has been designed to promote cell proliferation of 2 colonic adenocarcinoma cell lines of epithelial origin (HT 29 and HRT 18). The spreading of both cell types, especially of HT 29 cells, was not possible in a serum-free medium supplemented with growth factors. Spreading was obtained in a defined medium (a 1/1 mixture of DMEM and F12 media supplemented with 5 micrograms/ml transferrin, 5 ng/ml EGF, 10 ng/ml selenite and 15 mM HEPES pH 7.3) with an extracellular matrix-like material (ECM) secreted by the cells themselves. The properties of the ECM have been studied: ECM secreted by the 2 cell lines induced very quick spreading of HT 29 and HRT 18 cells (1 to 2 hr vs. 12 to 24 hr in serum-supplemented medium). ECM induced morphological differentiation of a rat pheochromocytoma cell line (PC 12). PC-12 cells grown under these conditions began to develop neurite extensions as early as 2 hr after seeding.

Adenocarcinoma↗
PubMed1991

Dominance of metastatically competent cells in primary murine breast neoplasms is necessary for distant metastatic spread.

We have previously shown, using tumor cell populations genetically tagged by random integrations of plasmid DNA, that metastatically-competent clonal cell variants have a strong growth advantage within primary tumors over their non-metastatic counterparts. As a result, primary tumors can become overgrown by the progeny of such cells, a process referred to as "clonal dominance" of primary tumors by metastatically-competent cells. Because of the well-known "metastatic inefficiency" of the multi-step cascade process of spread and growth, clonal dominance within primary tumors may be necessary for distant metastatic spread or increase the probability of its occurrence. To examine this hypothesis mice were inoculated s.c. with mixture of non-metastatic and genetically tagged, metastatically-competent mouse mammary carcinoma cells in defined ratios, but always containing an excess of the unmarked non-metastatic population. Progressive overgrowth of the metastatic subpopulation was monitored as a function of time by Southern analysis of DNA obtained from mixed primary tumors. This allowed us to evaluate the effects that surgical removal of the primary tumor had before, during and after effective clonal dominance, and what influence this had on the subsequent formation of distant metastases. Surgical removal of primary tumors before metastatic clonal dominance resulted in a low (0.25%) frequency of lung metastases, whereas removal just 1 or 2 weeks later during or after clonal dominance was achieved resulted in a high (75-100%) frequency of such metastases. Our results support the hypothesis that dominance of primary tumors by metastatically competent cells may be necessary for distant metastatic spread, and also suggest that clonal interactions play a significant role in modulating the metastatic ability of tumor cells in vivo.

Adenocarcinoma↗
PubMed1988

Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules.

Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell-binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively-spliced type III homology-connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.

Animals↗
PubMed1993

Effect of the conformation and orientation of adsorbed fibronectin on endothelial cell spreading and the strength of adhesion.

The effect of surface hydrophobicity upon the conformation of the cell binding domain of fibronectin (Fn) and the influence of Fn conformation on bovine aortic endothelial cell (BAEC) adhesion were examined. The free sulfhydryl group of Fn located near the cell binding domain was selectively labeled with acrylodan, a polarity sensitive fluor. Fluorescence emission was monitored in solution and upon adsorption to hydrophilic glass and hydrophobic silanized glass. The acrylodan-labeled Fn emission maximum shifted to longer wavelengths upon adsorption and the shift was greater for acrylodan-labeled Fn adsorbed to hydrophilic glass than hydrophobic silane, suggesting that the acrylodan was in a more solvent accessible environment on glass than silane. BAEC, suspended in serum-free medium, attached for 15 or 120 min onto glass or silane surfaces containing preadsorbed Fn, after which cell spreading and the strength of adhesion in a parallel plate flow chamber were measured. Cell spreading was similar on both surfaces after 15 min attachment, but BAECs were more spread on glass than silane after 120 min. At low surface concentrations of Fn, BAECs were more adherent on glass than silane. At higher surfaces concentrations, adhesion was similar. After a 2-h incubation in serum-free medium, cells on glass showed more extensive development of focal contacts as determined by immunofluorescent staining for vinculin. Cell adhesion under flow was reduced on silane by inhibition of protein synthesis with cycloheximide, suggesting that cell attachment to silane was promoted by cellular synthesis of Fn. The results indicate that changes in the conformation of the Fn cell binding domain affect Fn affinity for its cell surface receptor.

Adsorption↗
PubMed1991

Extrachromosomal human immunodeficiency virus type-1 DNA can initiate a spreading infection of HL-60 cells.

In this report, we describe a human immunodeficiency virus type-1 (HIV-1)-infected promyelocytic cell line, OM, derived from HL-60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV-1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV-1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to less than 10% HIV-1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV-1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV-1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV-1 genome was less than 0.1%, indicating nonfaithful transmission of an unintegrated HIV-1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV-1 DNA can contribute to the spread of HIV-1 infection and give rise to cells which have stably integrated HIV-1 provirus.

Blotting, Southern↗
PubMed1977

Sulphonated polystyrene as an optimal substratum for the adhesion and spreading of mesenchymal cells in monovalent and divalent saline solutions.

Cell adhesion and spreading were studied on sulphonated polystyrene dishes in serum-free saline (Mn, Na, Cl, buffer) i.e., without an intervening protein layer. Spreading as a function of surface charge density, SCD, peaked around 2-10 negative charges per square nanometer, corresponding to a monomolecular layer of sulphonate ions. At optimal SCD, macrophages, BHK-C13 and whole mouse embryo secondary cells all showed considerable spreading, even in monovalent saline-more so than on a conventional tissue-culture surface. But outside this narrow range of SCD, or on protein-coated surfaces, the divalent cation was indispensable. The biphasic effect of sulphonation on cell adhesion is consistent with the theory that a substratum need not be biochemically specific, provided it is physiochemically polar, rigid and dense. According to this theory, polystyrene of sub-optimal SCD would not be sufficiently polar, while supra-optimal sulphonation would produce a hydrogel surface, lacking in local rigidity and density, due to osmotic swelling. The principle of polymer exclusion, by a surface hydrogel layer, is also consistent with observations on the inhibitory effects of adsorbed proteins-viz., albumin, collagen, serum and cellular exudate, respectively-contrasted with the ready attachment of cells to a bare, optimally charged substratum, in this minimal in vitro system.

Animals↗
PubMed1993

Effect of low extracellular Ca2+ on growth, spreading area, cytoplasmic Ca2+ concentration, and intracellular pH in normal and transformed human fibroblasts.

The transformation of certain cells reduces the requirement of extracellular Ca2+ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca2+ than normal WI-38 cells. Spreading area of the normal cells decreases when cultured in 10 microM Ca2+ medium. Intracellular calcium concentration ([Ca2+]i) of the normal and transformed cells cultured in 10 microM and 2 mM Ca2+ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca2+]i is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca2+]i of both normal and transformed cells. Although the total decrease in [Ca2+]i is the same for both cells, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca2+ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca2+]i value in both cells. In addition, we investigated, using BCECF as a fluorescent probe, the intracellular pH (pHi) of normal and transformed cells maintained at low and normal Ca2+. The low Ca2+ condition makes pHi acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attachment, followed by the reduced spreading area, induces the acidic pHi. These results suggest that the reduced Ca2+ requirement of transformed cells for growth is related to the mechanism of pHi regulation rather than Ca2+ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells.

Calcium↗
PubMed2005

Spreading of embryologically distinct urothelial cells is inhibited by SPARC.

The AON epitope of secreted protein acidic and rich in cysteine (SPARC) is a conserved motif expressed by human SPARC in a variety of human cell types. Through the use of a monoclonal antibody that recognizes this epitope, transitional epithelium was found to restrict expression of SPARC to the suprabasal and intermediate layer. Such intracellular expression was defined by immunoreactive signals that localized to the apical plasma membranes of suprabasal and intermediate cells. Polarization of SPARC to apical plasma membranes of suprabasal cells was retained in vitro by a subpopulation of cells that exhibited characteristics of suprabasal cells--cell-cycle quiescence, large cell volumes, and multiple nuclei. In contrast, the basal layer of transitional epithelium in vivo and cycling cells in vitro did not exhibit this apical staining pattern, but instead sequestered the SPARC polypeptide within urothelial cytoplasm and/or nuclei, as revealed by immunohistochemical analysis. Elution of soluble proteins and DNA from urothelial cells revealed the presence of SPARC within the nuclear matrix--and that SPARC colocalized with the nuclear matrix Ki-67 antigen. rSPARC activity was demonstrated and quantified with a rounding assay whereby the spreading of freshly plated cells was inhibited by recombinant SPARC in a concentration- and time-dependent manner. Inhibition of spreading was observed in urothelial cells derived from endoderm (bladder) and mesoderm (ureter) germ layers. Statistically significant differences were seen between urothelial cells from these two layers. Mesodermal cells recovered more slowly from the inhibitory effects of rSPARC, such that at hour 6 endodermal cells underwent significantly more spreading, as shown by a rounding index (RI). These experiments provide new insights about the matricellular trafficking of SPARC and suggest that intra- and extra-cellular localization patterns influence the development, homeostasis, and differentiation of transitional epithelium.

Cells, Cultured↗
PubMed1987

MSORTV imaging of electrotonic conduction in a syncitium: optical recording of polarization spread in a simple salivary gland.

The spread of hyperpolarizing current injected into the salivary gland of the snail Helisoma trivolvis was studied with the technique of multiple site optical recording of transmembrane voltage (MSORTV). Using MSORTV it was possible to monitor simultaneously the spread of current passed by an intracellular microelectrode in up to 124 separate gland regions. Our results demonstrate the feasibility of combining conventional intracellular injection techniques with MSORTV to visualize 2-dimensional current spread in an electrical syncitium.

Animals↗
PubMed1990

Role of adherence vs. spreading in the induction of membrane-associated interleukin-1 on mouse peritoneal macrophages.

Adherence of peritoneal exudate cells (PEC) to plastic has been shown to activate several PEC functions, such as tumor cell lysis and membrane-associated interleukin-1 (mIL-1) expression. Several studies have demonstrated that leukocyte adherence is dependent on divalent cations. In this study, ethylenediaminetetraacetic acid (EDTA), a known chelator of divalent cations, was used to evaluate the role of cell attachment vs. spreading in adherence-induced mIL-1 activity on resident C57BL/6 mouse PEC. Significant inhibition of PEC spreading on plastic and mIL-1 expression was noted when PEC were cultured in the presence of 10 mM EDTA. However, PEC remained adherent in the presence of EDTA and were able to express mIL-1 activity in response to a soluble stimulus lipopolysaccharide (LPS) 1 microgram/ml. These results suggest that the divalent cation-dependent spreading of PEC on plastic initiates or enhances the expression of mIL-1 activity. Additionally, adhesion and LPS stimulate mIL-1 expression by independent mechanisms.

Animals↗
PubMed1992

Interactions between HIV-infected monocytes and the extracellular matrix: increased capacity of HIV-infected monocytes to adhere to and spread on extracellular matrix associated with changes in extent of virus replication and cytopathic effects in infected cells.

Monocytes express cell surface receptors for extracellular matrix (ECM) proteins of basement membranes. These receptors are engaged during extravasation of cells through capillary endothelium into tissue. The number of human immunodeficiency virus (HIV)-infected monocytes that adhered to ECM over 2 h was threefold higher than that of uninfected control cells. This difference was ECM specific and was not observed with a bovine serum albumin substrate. Enhanced adhesion to ECM was evident in monocytes by 4 days after HIV infection and increased through 10 days. Monocytes exposed to a T cell-tropic HIV strain that binds to but does not replicate in monocytes showed no changes in adherence to ECM. Thus, productive infection of monocytes by HIV induces a significant increase in the capacity of these cells to interact with ECM. Enhanced adhesion of HIV-infected monocytes to ECM was associated with increased spreading: at 12 h, sixfold more HIV-infected monocytes were spread on ECM than were uninfected control cells. Cell processes of HIV-infected monocytes formed a complex network on ECM: many of these cells expressed HIV proteins as detected by indirect immunofluorescence. HIV-associated cytopathic effects and levels of virion-associated reverse transcriptase activity depended on the substrate to which monocytes were attached. Virus replication and cytopathic effects in monocytes adhered to ECM, fibronectin, or plastic alone were comparable. In contrast, HIV-infected monocytes attached to laminin showed a significant increase in virus replication and in extent of cytopathic effects through 2 weeks after infection. The lowest levels of HIV replication and cytopathic effects were in monocytes attached to collagen IV. Interactions between monocytes and ECM profoundly affect the manner in which these cells control HIV infection: HIV infection changes the capacity of infected monocytes to attach and spread on ECM; attachment to ECM alters the extent of virus replication in infected cells.

Cell Adhesion↗