Biomedical subjects

Benjamin Geiger

Publications and source records attributed to Benjamin Geiger.

At least 37 records · Page 2Linked to original sources

PubMed2005

Resolving and classifying haematopoietic bone-marrow cell populations by multi-dimensional analysis of flow-cytometry data.

The study of normal or malignant haematopoiesis requires the analysis of heterogeneous cell populations using multiple morphological and molecular criteria. Flow cytometry has the capacity to acquire multi-parameter information of large haematopoietic cell populations, utilizing various combinations of >200 molecular markers (clusters of differentiation, CD). However, current flow cytometry analyses are based on serial gating of two-parametric scatter plots--a process that is inherently incapable to discriminate all subgroups of cells in the data. Here we studied the cellular diversity of normal bone marrows (BM) using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on the myeloid lineage. Twenty-three subclasses of cells were resolved, many of them inseparable even when examined in all possible two-parametric scatter plots. The multi-dimensional analysis could distinguish the haematopoietic progenitors according to International Society of Haematotherapy and Graft Engineering criteria from other types of immature cells. Based on the defined clusters, we designed a classifier that assigns BM cells in samples to subclasses based on robust six-dimensional position and extended shape. The analysis presented here can manage successfully both the increasing numbers of haematopoietic cellular markers and sample heterogeneity. This should enhance the ability to study normal haematopoiesis, and to identify and monitor haematopoietic disorders.

Antigens, CD↗

PubMed2005

Measurement of protein tyrosine phosphorylation in cell adhesion.

This chapter describes biochemical, immunochemical, and microscopic approaches to measure protein tyrosine phosphorylation after cell adhesion. We have outlined detailed procedures to biochemically examine the phosphotyrosine content of cellular proteins by Western blotting, which in some cases can be performed using phospho-specific antibodies. Furthermore, we have described in detail the examination of subcellular localization of phosphotyrosine-containing proteins in focal adhesions using immunofluorescence. Finally, a quantitative fluorescence microscopic technique using an SH2-containing phosphotyrosine reporter to monitor tyrosine phosphorylation in focal adhesions in live cells is described.

3T3 Cells↗

PubMed2005

Application of microscope-based FRET to study molecular interactions in focal adhesions of live cells.

This chapter describes the use of microscope-based fluorescence resonance energy transfer to follow dynamic interaction of molecules localized at focal adhesions. We first outline the significance of studying dynamic interactions in focal adhesions of living cells, and second provide an overview of the method itself. This is followed by a protocol for fluorescence resonance energy transfer measurements in live cells.

Animals↗

PubMed2004

Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton.

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.

Actins↗

PubMed2004

Spatial and temporal sequence of events in cell adhesion: from molecular recognition to focal adhesion assembly.

A new concept that attributes a pivotal role to the pericellular coat in the regulation of the early stages of cell adhesion is presented. Quick, adaptable, and transient adhesion through multiple cooperative weak interactions provides the cell with an additional level of modulation in the decision-making process that precedes the commitment to adhesion at a particular site. Hyaluronan emerges as a modulator of cell adhesion in certain cells, mediating binding or repulsion through its polyelectrolyte character, in addition to its chirality and molecular-recognition properties. The biophysical properties of hyaluronan as well as its ultrastructural organization are analyzed in relation to this proposed function.

Animals↗

PubMed2004

Cell mechanosensitivity controls the anisotropy of focal adhesions.

Cellular adhesions are modulated by cytoskeletal forces or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity can be driven at least in part by the elastic, cell-contractility-induced deformations of protein molecules that form the adhesion. The model accounts for observations of anisotropic growth and shrinkage of focal adhesions in the direction of the force and predicts that focal adhesions only grow within a range of force that is determined by the composition and matrix properties. This prediction is consistent with the observations of a force threshold for the appearance of elongated focal adhesions and the disruption of adhesions into fibrils on a mobile extracellular matrix. The growth dynamics is calculated and the predicted sliding of focal adhesions is consistent with several experiments.

Anisotropy↗

PubMed2004

Induction of apoptosis in cultured endothelial cells by a cadherin antagonist peptide: involvement of fibroblast growth factor receptor-mediated signalling.

Cadherins are a family of transmembrane glycoproteins mediating calcium-dependent, homophilic cell-cell adhesion. In addition, these molecules are involved in signaling events, regulating such processes as cell motility, proliferation, and apoptosis. Members of the cadherin subfamily, called either classical or type I cadherins, contain a highly conserved sequence at their homophilic binding site consisting of the three amino acids--histidine-alanine-valine (HAV). Previous studies have shown that peptides containing the HAV motif inhibit cadherin-dependent events such as cell aggregation, compaction, and neurite outgrowth. We report here that a cyclic peptide, N-Ac-CHAVC-NH2 can perturb cadherin-mediated endothelial cell interactions, resulting in a progressive apoptotic cell death. This effect depends on cell density, as it is only observed when dense cultures are treated with the peptide. Adherens junction (AJ)-associated cadherin and catenins are differentially affected by the N-Ac-CHAVC-NH2 treatment, as judged by double immunofluorescence labeling followed by immunofluorescence-ratio imaging. However, cell-cell adhesions are largely retained during the first few hours after addition of the peptide. It was also observed that following treatment, actin filaments partially lose their plasma membrane anchorage at AJs and translocate towards the cell center. Interestingly, addition of basic fibroblast growth factor to confluent, peptide-treated, endothelial cell cultures, completely blocks apoptosis and the inhibitory peptide reduce the phosphorylation of the FGF receptor target protein FRS2, suggesting that the peptide exerts its effect by inhibiting cadherin-mediated activation of fibroblast growth factor receptor signaling. We propose that cadherin-mediated signaling is essential for maintaining viability of confluent endothelial cells, and that its perturbation by N-Ac-CHAVC-NH2 drives these cells to apoptosis.

Actin Cytoskeleton↗

PubMed2004

Attenuation of the p53 response to DNA damage by high cell density.

The p53 tumor suppressor is critical for preventing cancer progression. Numerous observations suggest that p53 function can be modulated by the cells' microenvironment. We addressed specifically the impact of cell crowding on the induction of p53 by DNA damage. We report that cell crowding attenuates markedly p53 upregulation, transcriptional activation and subsequent p53-dependent apoptosis following exposure to genotoxic stress. The p53 protein remains short-lived in confluent cultures regardless of the extent of DNA damage, even though it undergoes efficient phosphorylation on the mouse equivalent of human p53 serine 15. This inhibitory effect of cell crowding is not a secondary consequence of density-dependent cell cycle arrest (contact inhibition). Microscopic examination indicates that dense cultures display prominent cadherin-mediated cell-cell junctions, and only poor cell-matrix focal adhesions, whereas sparse cells possess conspicuous matrix adhesions and essentially no cell-cell contacts. High-density cell culture might recapitulate the microenvironment of cells in a living organism, where the response of p53 to DNA damage is reported to be low in some organs and ages. The impact of cell density on p53 activation may have important bearings on the involvement of p53 in tumor suppression and the cellular response to anticancer therapy.

Animals↗

PubMed2004

Early molecular events in the assembly of the focal adhesion-stress fiber complex during fibroblast spreading.

Cell adhesion to the extracellular matrix triggers the formation of integrin-mediated contact and reorganization of the actin cytoskeleton. Examination of nascent adhesions, formed during early stages of fibroblast spreading, reveals a variety of forms of actin-associated matrix adhesions. These include: (1). small ( approximately 1 microm), dot-like, integrin-, vinculin-, paxillin-, and phosphotyrosine-rich structures, with an F-actin core, broadly distributed over the ventral surfaces of the cells; (2). integrin-, vinculin-, and paxillin-containing "doublets" interconnected by short actin bundles; (3). arrays of actin-vinculin complexes. Such structures were formed by freshly plated cells, as well as by cells recovering from latrunculin treatment. Time-lapse video microscopy of such cells, expressing GFP-actin, indicated that long actin cables are formed by an end-to-end lining-up and apparent fusion of short actin bundles. All these structures were prominent during cell spreading, and persisted for up to 30-60 min after plating. Upon longer incubation, they were gradually replaced by stress fibers, associated with focal adhesions at the cell periphery. Direct examination of paxillin and actin reorganization in live cells revealed alignment of paxillin doublets, forming long and highly dynamic actin bundles, undergoing translocation, shortening, splitting, and convergence. The mechanisms underlying the assembly and reorganization of actin-associated focal adhesions and the involvement of mechanical forces in regulating their dynamic properties are discussed.

Actin Cytoskeleton↗

PubMed2004

Functional and structural reversibility of H-7 effects on the conventional aqueous outflow pathway in monkeys.

To determine the mechanism of H-7-induced outflow resistance decrease, the reversibility of H-7 effects on outflow pathway was studied physiologically and morphologically in live monkey eyes. Total outflow facility was measured by two-level constant pressure perfusion before (baseline measurement) and after (post-drug measurement) anterior chamber (AC) exchange with 300microM H-7 or vehicle in opposite eyes of eight monkeys. H-7 was then removed by AC exchange with drug-free vehicle in both eyes, followed by a 2.5hr waiting period, after which outflow facility was measured again with (Group 2; n=4) or without (Group 1; n=4) another preceding drug-free AC exchange. For morphological study, five monkeys were initially perfused similarly to Group 1 in physiology, but the facility measurement beginning 2.5hr after drug removal was either omitted or replaced by gold solution infusion. Following baseline measurement, two of the five monkeys received H-7 or vehicle in opposite eyes, while three monkeys received H-7 in both eyes 2.5hr apart, contributing one H-7-treated 'recovery' eye and one H-7-treated 'acute' eye. After perfusion, both eyes of all five monkeys were studied by light and electron microscopy. Outflow facility during post-drug measurement in the H-7-treated eye was significantly increased by two-fold. However, the facility increase was reduced when measured beginning 2.5hr after drug removal, with the reduction being greater in Group 1. 'Recovered' outflow facility after drug removal gradually increased again under continuous AC infusion with drug-free vehicle. Morphologically, major changes in and around Schlemm's canal (SC) in the H-7-treated 'acute' eye included protrusion of the entire inner wall (IW) into SC, relaxation of the IW cells and reorganization of the IW cytoskeleton. The changes in IW cells and juxtacanalicular region of the H-7-treated 'recovery' eye were non-uniform, with areas resembling the vehicle-treated eye ('contracted areas') and areas resembling the H-7-treated 'acute' eye ('relaxed areas'). The average junction-to-junction distances in the IW cells of the H-7-treated 'recovery' eye were intermediate between the vehicle-treated eye and the H-7-treated 'acute' eye. In conclusion, H-7's effect on outflow facility seems reversible, but AC exchange or continuous infusion with drug-free vehicle can re-elevate the 'recovered' outflow facility. Major morphological changes in the TM immediately after H-7 include IW protrusion, cellular relaxation and cytoskeleton reorganization. The decrease in 'relaxed areas' in the TM, in conjunction with the reversed outflow facility, 2.5hr after drug removal suggests that cellular relaxation in the TM is the structural basis for H-7-induced increase in outflow facility.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2004

Halofuginone, an inhibitor of collagen synthesis by rat stellate cells, stimulates insulin-like growth factor binding protein-1 synthesis by hepatocytes.

BACKGROUND/AIMS: Halofuginone, an inhibitor of collagen synthesis, prevented and caused resolution of established hepatic fibrosis. A genomic approach in vivo was used to search for additional genes responsible for halofuginone mode of action. METHODS: Fibrosis was induced in rats by thioacetamide (TAA) and evaluated by collagen type I gene expression and the levels of collagen, tissue inhibitors of metalloproteinases-2 and smooth-muscle actin. Halofuginone was given in the diet. cDNA from liver biopsies was hybridized on Atlas arrays comprising of 588 genes. The results were confirmed by Northern blots and in situ hybridization. RESULTS: Insulin-like growth factor binding protein-1 (IGFBP-1) was one of the 13 genes differentially expressed in the fibrotic liver after halofuginone treatment. After 2 and 4 weeks, halofuginone prevented the TAA-induced down-regulation of IGFBP-1 gene expression. Halofuginone also prevented the TAA-dependent changes in IGFBP-3 gene expression. Halofuginone affected IGFBP-1 synthesis in rat hepatocytes and cells of hepatocyte origin and caused time- and dose-dependent increases in the IGFBP-1 gene expression and synthesis by HepG2 cells. The IGFBP-1 secreted by HepG2-inhibited stellate cell motility. CONCLUSIONS: Halofuginone is an anti-fibrotic drug that inhibits collagen synthesis by stellate cells and preventing alteration in the synthesis of IGFBPs by hepatic cells.

Animals↗

PubMed2003

Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells.

Cellular locomotion is driven by repeated cycles of protrusion of the leading edge, formation of new matrix adhesions and retraction of the trailing edge. In this study we addressed the molecular composition and dynamics of focal complexes, formed under the leading lamellae of motile cells, and their maturation into focal adhesions. We combined phase-contrast and fluorescence microscopy approaches to monitor the incorporation of phosphotyrosine and nine different focal adhesion proteins into focal complexes in endothelial cells, migrating into an in vitro 'wound'. We show that newly formed complexes are located posterior to an actin-, VASP- and alpha-actinin-rich region in the lammelipodium. They are highly tyrosine phosphorylated, contain beta3-integrin, talin, paxillin and low levels of vinculin and FAK, but are apparently devoid of zyxin and tensin. The recruitment of these proteins into focal complexes occurs sequentially, so that their specific protein composition depends on their age. Interestingly, double color, time-lapse movies visualizing both paxillin and zyxin, indicated that the transition from paxillin-rich focal complexes to definitive, zyxin-containing focal adhesions, takes place only after the leading edge stops advancing or retracts. These observations illuminate, for the first time, early stages in focal complex assembly and the dynamic process associated with its transformation into focal adhesion.

Actinin↗

PubMed2003

Targeting membrane-localized focal adhesion kinase to focal adhesions: roles of tyrosine phosphorylation and SRC family kinases.

In the present study, we examined regulation of activated focal adhesion kinase localization in focal adhesions. By using focal adhesion kinase fused to an inert transmembrane anchor, we found that the focal contact targeting region within focal adhesion kinase was preserved in the membrane-targeted fusion protein. However, upon tyrosine phosphorylation, full-length focal adhesion kinase became excluded from focal adhesions. This negative regulation of localization could be abolished by mutating key amino acid residues of focal adhesion kinase shown previously to be involved in adhesion-mediated signal transduction. Hyper-phosphorylation of endogenous focal adhesion kinase induced by pervanadate resulted in a similar reduction of localization at focal adhesions. We also show here that Src family kinases are essential for the phosphorylation-dependent exclusion of focal adhesion kinase from focal adhesions. We propose here a molecular model for the tyrosine phosphorylation-dependent regulation of focal adhesion kinase organization involving Src kinases and an inhibitory phosphorylation of the C-terminal (Tyr-925) tyrosine residue.

3T3 Cells↗

PubMed2003

Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption.

Tyrosine phosphorylation of focal adhesion components is involved in the regulation of focal adhesion formation and turnover, yet the underlying molecular mechanisms are still poorly defined. In the present study, we have used quantitative fluorescence microscopy to investigate the dynamic relationships between the incorporation of new components into growing focal adhesions and tyrosine phosphorylation of these sites. For this purpose, a new approach for monitoring phosphotyrosine levels in live cells was developed, based on a 'phosphotyrosine reporter' consisting of yellow fluorescent protein fused to two consecutive phosphotyrosine-binding Src-homology 2 (SH2)-domains derived from pp60(c-Src). This YFP-dSH2 localized to cell-matrix adhesions and its intensity was linearly correlated with that of an anti-phosphotyrosine antibody labeling. The differential increase in vinculin and phosphotyrosine levels was examined in live cells by two-color time-lapse movies of CFP-vinculin and YFP-dSH2. In this study, focal adhesion growth was triggered by microtubule disruption, which was previously shown to stimulate focal adhesion development by inducing cellular contraction. We show here that, 2 minutes after addition of the microtubule-disrupting drug nocodazole, the local densities of the focal adhesion-associated proteins vinculin, paxillin and focal adhesion kinase (FAK) are significantly elevated and the focal adhesion area is increased, whereas elevation in tyrosine phosphorylation inside the growing adhesions occurs only a few minutes later. Phosphotyrosine and FAK density reach their maximum levels after 10 minutes of treatment, whereas vinculin and paxillin levels as well as focal adhesion size continue to grow, reaching a plateau at about 30 minutes. Our findings suggest that protein recruitment and growth of focal adhesions are an immediate and direct result of increased contractility induced by microtubule disruption, whereas tyrosine phosphorylation is activated later.

Amino Acid Sequence↗

PubMed2003

Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy.

BACKGROUND: The cytoskeleton consists of complex arrays of fibers that play indispensable roles in cell structure and function. The cytoskeletal fibers are concertedly involved in numerous cellular processes, including cell adhesion, locomotion, intracellular transport, and cell division. The organization of the cytoskeleton was extensively studied, mainly by immunofluorescence microscopy, yet these studies were mostly qualitative, and a reliable quantitative approach for determining fiber structure and distribution is still missing. METHODS: In this study we developed algorithms for filament feature extraction, based on fluorescence microscopy. These algorithms are robust against blurring by slight defocus, high background, and noise, and are applicable to both fixed, immunolabeled cells and live cells expressing fluorescently tagged cytoskeletal proteins. The implemented FiberScore program is used in order to recognize, segment, and quantify various structural parameters of the cytoskeleton, including total fiber-associated fluorescence, as well as fiber length and orientation. Furthermore, these parameters can be determined for different cytoskeletal proteins in the same sample tagged with multiple-fluorescent labels, and the results can be correlated with other cellular parameters. RESULTS: FiberScore was used here for the quantification of simultaneous changes in microtubule and actin filaments induced by the microtubule-disrupting drug nocodazole. Actin filaments, which are reported to respond reciprocally to microtubule disruption, are found to be affected by both immediate and delayed signals. CONCLUSIONS: Analysis of the organization of fibers by the FiberScore algorithm allows quantification of the cytoskeletal signature of cells and offers reliable multiparametric functional assays for effects of drugs and other perturbations evaluated on a cell-by-cell basis.

3T3 Cells↗

PubMed2003

Organization and adhesive properties of the hyaluronan pericellular coat of chondrocytes and epithelial cells.

Hyaluronan is a megadalton glycosaminoglycan composed of repeating units of D-N-acetylglucosamine-beta-D-Glucuronic acid. It is known to form a highly hydrated pericellular coat around chondrocytes, fibrosarcoma, and smooth muscle cells. Using environmental scanning electron microscopy we detected fully hydrated hyaluronan pericellular coats around rat chondrocytes (RCJ-P) and epithelial cells (A6). Hyaluronan mediates early adhesion of both chondrocytes and A6 cells to glass surfaces. We show that chondrocytes in suspension establish early "soft contacts" with the substrate through a thick, hyaluronidase-sensitive coat (4.4 +/- 0.7 microm). Freshly-attached cells drift under shear stress, leaving hyaluronan "footprints" on the surface. This suggests that chondrocytes are surrounded by a multilayer of entangled hyaluronan molecules. In contrast, A6 cells have a 2.2 +/- 0.4- microm-thick hyaluronidase-sensitive coat, do not drift under shear stress, and remain firmly anchored to the surface. We consider the possibility that in A6 cells single hyaluronan molecules, spanning the whole thickness of the pericellular coat, mediate these tight contacts.

Animals↗

PubMed2003

Heparanase mediates cell adhesion independent of its enzymatic activity.

Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate and is implicated in diverse physiological and pathological processes. In this study we report on a novel direct involvement of heparanase in cell adhesion. We demonstrate that expression of heparanase in nonadherent lymphoma cells induces early stages of cell adhesion, provided that the enzyme is expressed on the cell surface. Heparanase-mediated cell adhesion to extracellular matrix (ECM) results in integrin-dependent cell spreading, tyrosine phosphorylation of paxillin, and reorganization of the actin cytoskeleton. The surface-bound enzyme also augments cell invasion through a reconstituted basement membrane. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating that heparanase functions as an adhesion molecule independent of its endoglycosidase activity. The combined feature of heparanase as an ECM-degrading enzyme and a cell adhesion molecule emphasizes its significance in processes involving cell adhesion, migration, and invasion, including embryonic development, neovascularization, and cancer metastasis.

Animals↗

PubMed2003

Adhesion-dependent cell mechanosensitivity.

The conversion of physical signals, such as contractile forces or external mechanical perturbations, into chemical signaling events is a fundamental cellular process that occurs at cell-extracellular matrix contacts, known as focal adhesions. At these sites, transmembrane integrin receptors are associated via their cytoplasmic domains with the actin cytoskeleton. This interaction with actin is mediated by a submembrane plaque, consisting of numerous cytoskeletal and signaling molecules. Application of intrinsic or external forces to these structures dramatically affects their assembly and triggers adhesion-mediated signaling. In this review, we discuss the structure-function relationships of focal adhesions and the possible mode of action of the putative mechanosensor associated with them. We also discuss the general phenomenon of mechanosensitivity, and the approaches used to measure local forces at adhesion sites, the cytoskeleton-mediated regulation of local contractility, and the nature of the signaling networks that both affect contractility and are affected by it.

Animals↗