Formation Of Adhesion Structures Research Articles

Determining the roles of membrane tension in breast cancer cell migration

Within the past two years, breast cancer has exceeded lung cancer as the most frequently diagnosed cancer, ranking first for incidence and mortality in most of the world. Cancer reaches its deadliest stage when metastatic spread of cells occurs from tumor sites. Cell metastasis relies, in part, on 1) endocytic membrane trafficking and 2) the formation of adhesive contacts with the extracellular matrix. These processes are regulated by mechanical tension on the cell membrane, which varies spatially within migrating cells. While tension gradients assist in maintaining polarity of the cell during migration, recently it has been shown that these gradients create an asymmetry in endocytic dynamics across the plasma membrane, termed as polarized membrane trafficking. Our preliminary data have shown that changes in membrane tension additionally result in the heterogeneous expression of two distinct adhesive structures during proliferation and nondirectional migration of breast cancer cells. Although membrane tension, endocytosis, adhesion, and cell migration have been extensively studied individually, it is unknown how these processes are collectively regulated by breast cancer cells to metastasize. We thus aim to develop experimental and analytical approaches that will allow us to monitor polarized membrane trafficking, the formation of adhesive structures, and membrane tension in several migrating breast epithelial cell lines. This involves using a microfluidic platform that simultaneously allows the leading and trailing edge of a cell to be separately treated during migration and enables the application of inputs native to the cellular microenvironment. Examination of the various regulatory pathways on the cell membrane that contribute to breast cancer metastasis will significantly impact the development of therapeutic targets in breast cancer drug discovery.

How does plasticity of migration help tumor cells to avoid treatment: Cytoskeletal regulators and potential markers.

Tumor shrinkage as a result of antitumor therapy is not the only and sufficient indicator of treatment success. Cancer progression leads to dissemination of tumor cells and formation of metastases - secondary tumor lesions in distant organs. Metastasis is associated with acquisition of mobile phenotype by tumor cells as a result of epithelial-to-mesenchymal transition and further cell migration based on cytoskeleton reorganization. The main mechanisms of individual cell migration are either mesenchymal, which depends on the activity of small GTPase Rac, actin polymerization, formation of adhesions with extracellular matrix and activity of proteolytic enzymes or amoeboid, which is based on the increase in intracellular pressure caused by the enhancement of actin cortex contractility regulated by Rho-ROCK-MLCKII pathway, and does not depend on the formation of adhesive structures with the matrix, nor on the activity of proteases. The ability of tumor cells to switch from one motility mode to another depending on cell context and environmental conditions, termed migratory plasticity, contributes to the efficiency of dissemination and often allows the cells to avoid the applied treatment. The search for new therapeutic targets among cytoskeletal proteins offers an opportunity to directly influence cell migration. For successful treatment it is important to assess the likelihood of migratory plasticity in a particular tumor. Therefore, the search for specific markers that can indicate a high probability of migratory plasticity is very important.

Osteoprotegerin exposure at different stages of osteoclastogenesis differentially affects osteoclast formation and function.

This study aimed to investigate the effects of osteoprotegerin (OPG), a decoy receptor for receptor activator for nuclear factor κB ligand (RANKL), during the various stages of osteoclast differentiation, and additionally investigate its effects on osteoclast adhesion and activity. RAW264.7 murine monocytic cells were incubated with macrophage colony-stimulating factor and RANKL for 1, 3, 5, or 7days, followed by an additional 24-h incubation in the presence or absence of OPG (80ng/mL). We examined osteoclast differentiation and adhesion capacity using the tartrate-resistant acid phosphatase (TRAP) assay and immunofluorescence microscopy, and additionally examined cell growth in real time using the xCELLigence system. Furthermore, the expression levels of TRAP, RANK, integrin β3, matrix metalloproteinase 9, cathepsin K, carbonic anhydrase II, and vesicular-type H(+)-ATPase A1 were examined using western blotting. OPG exposure on day 1 enhanced the osteoclast growth curve as well as adhesion, and increased RANK and integrin β3 expression. In contrast, exposure to OPG at later time points (days 3-7) inhibited osteoclast differentiation, adhesion structure formation, and protease expression. In conclusion, the biological effects of OPG exposure at the various stages of osteoclast differentiation were varied, and included the enhanced adhesion and survival of preosteoclasts, the block of differentiation from the early to the terminal stages of osteoclastogenesis, and suppression of mature osteoclast activation following OPG exposure during the terminal differentiation stage, suggesting that the effects of OPG exposure differ based on the stage of differentiation.

Osteoprotegerin induces podosome disassembly in osteoclasts through calcium, ERK, and p38 MAPK signaling pathways

Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca2+ concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30ng/mL) and M-CSF (25ng/mL). The cells were pretreated with Bapta-AM (5μM), U0126 (5μM), or SB202190 (10μM) for 30min, followed by 40ng/mL OPG for 3h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca2+ concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca2+ concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix.

Selective modulation of cell response on engineered fractal silicon substrates

A plethora of work has been dedicated to the analysis of cell behavior on substrates with ordered topographical features. However, the natural cell microenvironment is characterized by biomechanical cues organized over multiple scales. Here, randomly rough, self-affinefractal surfaces are generated out of silicon,where roughness Ra and fractal dimension Df are independently controlled. The proliferation rates, the formation of adhesion structures, and the morphology of 3T3 murine fibroblasts are monitored over six different substrates. The proliferation rate is maximized on surfaces with moderate roughness (Ra ~ 40 nm) and large fractal dimension (Df ~ 2.4); whereas adhesion structures are wider and more stable on substrates with higher roughness (Ra ~ 50 nm) and lower fractal dimension (Df ~ 2.2). Higher proliferation occurson substrates exhibiting densely packed and sharp peaks, whereas more regular ridges favor adhesion. These results suggest that randomly roughtopographies can selectively modulate cell behavior.

Development and Pathogenicity of the Fungus Crinipellis perniciosa on Interaction with Cacao Leaves

ABSTRACT We investigated developmental changes in the primary mycelium of Crinipellis perniciosa upon its interaction with immature and mature leaves of Theobroma cacao. On nutritive medium, the primary mycelium grew significantly slower in the presence of host tissue than without host tissue. In the absence of the cacao leaves, incomplete phase transition occurred after 5 days, wherein older hyphae progressed to the dikaryotic state and growing tips remained monokaryotic. Phase transition occurred between 3 and 5 days on mature leaves, 10 and 12 days on meristematic leaves, and required 2 weeks on T. cacao callus tissue. The biotrophic mycelia were able to invade immature and mature cacao leaves without open wounds or stomata. Club-shaped hyphal tips and the formation of adhesive structures were induced by cuticle extracts and suggest host recognition. The initial cuticular disintegration at the site of penetration was followed by blister formation and complete digestion of leaves by the primary mycelium. The data suggest specific interactions between host and pathogen that control the onset of the necrotrophic phase of the fungus. The data further indicate that primary mycelium rather than spores can be used to study C. perniciosa pathogenicity.

Elevated c-Src is linked to altered cell-matrix adhesion rather than proliferation in KM12C human colorectal cancer cells.

Elevated expression and/or activity of c-Src, the prototype of the Src family of protein tyrosine kinases, is associated with the development of human colon cancer. However, despite the known pleiotropic effects of these kinases in promoting (a) cell growth downstream of growth factor receptors, and (b) the dynamic regulation of integrin adhesions in fibroblast model systems, their precise role in epithelial cancer cells is unknown. Here we addressed whether elevated expression and activity of cellular Src alters cell proliferation and/or cell–matrix adhesion in cancer cells from the Fidler model of colorectal metastasis. Although elevated Src correlates with ability to metastasise to the liver after intrasplenic injection, we found that this was not linked to enhanced growth, either in vitro or in vivo as sub-cutaneous tumours. However, elevated Src was associated with enhanced attachment to extracellular matrix. In addition, adhesion to fibronectin, was suppressed by agents that inhibited Src activity, while enforced elevation of Src in non-metastatic cells was sufficient to stimulate adhesion to fibronectin and enhanced assembly of adhesion complexes, without influencing cell growth. Thus, we conclude that one role of elevated Src in human colon cancer cells is to modulate integrin-dependent cell–matrix attachment and formation of adhesion structures, which may, in turn, influence cell motility and integrin-dependent cellular responses.British Journal of Cancer (2002) 87, 1128–1135. doi:10.1038/sj.bjc.6600594 www.bjcancer.com© 2002 Cancer Research UK