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ABSTRACT: Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined.
We present here a compositional imaging approach for the analysis and display of multi-component compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focal-adhesion-associated complexes to Rho-kinase inhibition.
Multicolor compositional imaging resolves "molecular signatures" characteristic to focal-adhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional "contents-resolved" dimensions. We propose that compositional imaging can serve as a powerful tool for studying complex multi-molecular assemblies in cells and for mapping their distribution at sub-micron resolution.
PLoS ONE 02/2008; 3(4):e1901. · 4.09 Impact Factor
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ABSTRACT: 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.
British Journal of Haematology 05/2005; 129(3):420-31. · 4.94 Impact Factor
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ABSTRACT: 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.
Experimental Cell Research 05/2004; 294(2):366-78. · 3.58 Impact Factor
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ABSTRACT: Heparanase is an endo-beta-D-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme's biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis.
Experimental Cell Research 12/2002; 281(1):50-62. · 3.58 Impact Factor
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ABSTRACT: Heparanase is a heparan-sulfate-degrading endoglycosidase that has important roles in various biological processes, including angiogenesis, wound healing and metastatsis. Human heparanase is synthesized as a 65 kDa latent precursor, which is proteolytically processed into a highly active 50 kDa form. Extracellular heparanase is found in various tissues and is utilized by both normal cells and metastatic cancer cells to degrade heparan sulfate moieties in basement membranes and extracellular matrices. This study characterizes the processing and trafficking events associated with cellular activation of extracellular heparanase. We show that primary human fibroblasts are capable of binding and converting the 65 kDa heparanase precursor into its highly active 50 kDa form, concomitantly with its cytoplasmic accumulation. Heparanase uptake depends on the actin cytoskeleton integrity, resulting in a prolonged storage of the enzyme, mainly in endosomal structures. Heparanase endocytosis and its proteolytic activation are independent processes, indicating that heparanase cleavage is a cell surface event. Heparin completely inhibits heparanase endocytosis but only partially inhibits its association with the cells, suggesting that cell surface heparan sulfate moieties play a specific role in its endocytosis. Cellular binding and uptake of extracellular heparanase control its activation, clearance rate and storage within the cells.
Journal of Cell Science 06/2002; 115(Pt 10):2179-87. · 6.11 Impact Factor
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ABSTRACT: The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as
focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target
known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent
induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing
dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of
these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent
protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed
this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology
protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced
formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1
is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal
contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional
assembly in response to local force.
The Journal of Cell Biology 06/2001; 153(6):1175-1186. · 10.26 Impact Factor
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Eli Zamir,
Menachem Katz,
Yehudit Posen,
Noam Erez,
Kenneth M. Yamada,
Ben-Zion Katz,
Shin Lin,
Diane C. Lin,
Alexander Bershadsky,
Zvi Kam,
Benjamin Geiger
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ABSTRACT: Here we use time-lapse microscopy to analyse cell–matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP–paxillin to analyse focal contacts and GFP–tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometres per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain 51 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometres per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell–matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.
Nature Cell Biology 02/2000; 2(4):191-196. · 19.49 Impact Factor
