Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins

The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick β_1 subunit of integrin, suggesting that β_(1-)integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (>1 µg ml^(-1); Low-LM), but not at high coating concentration of laminin (10 µg ml^(-1); High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required >0.1 mM Ca^(2+) or Mn^(2+). Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165,000 Mr which is also found in immunoprecipitates using antibodies against the β_1 subunit of integrin and is likely to be an integrin alpha subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of β_(1-)integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin

Controlled surface initiated polymerization of N-isopropylacrylamide from polycaprolactone substrates for regulating cell attachment and detachment

Poly(ε-caprolactone) (PCL) substrates were modified with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) brushes to direct and control cellular attachment and detachment. Prior to brush growth, the surface of PCL was activated by a diamine to allow for initiator coupling. Infrared spectra taken before and after cell culturing demonstrated the covalently attached nature of the PNIPAM brushes. PCL is a biocompatible polymer and to prove that the modifications described above did not change this characteristic property, a cell attachment/detachment study was carried out. The modified substrates showed a lower cell attachment when compared to PCL alone and to PCL films modified with the initiator. The possibility to detach the cells in the form of a sheet was proved using PNIPAM-modified PCL films by lowering the temperature to 25 °C. No relevant detachment was shown by the unmodified or by the initiator modified surfaces. This confirmed that the detachment was temperature dependent and not connected to other factors such as polymer swelling. These functionalized polymeric films can find applications as smart cell culture systems in regenerative medicine applications

Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibronectin: identification of integrin-related glycoproteins involved in attachment and process outgrowth.

Neuronal responses to extracellular matrix (ECM) constituents are likely to play an important role in nervous system development and regeneration. We have studied the interactions of a neuron-like rat pheochromocytoma cell line, PC12, with ECM protein-coated substrates. Using a quantitative cell attachment assay, PC12 cells were shown to adhere readily to laminin (LN) or collagen IV (Col IV) but poorly to fibronectin (FN). The specificity of attachment to these ECM proteins was demonstrated using ligand-specific antibodies and synthetic peptides. To identify PC12 cell surface proteins that mediate interactions with LN, Col IV, and FN, two different antisera to putative ECM receptors purified from mammalian cells were tested for their effects on PC12 cell adhesion and neuritic process outgrowth. Antibodies to a 140-kD FN receptor heterodimer purified from Chinese hamster ovarian cells (anti-FNR; Brown, P. J., and R. L. Juliano, 1986, J. Cell Biol., 103:1595-1603) inhibited attachment to LN and FN but not to Col IV. Antibodies to an ECM receptor preparation purified from baby hamster kidney fibroblastic cells (anti-ECMR; Knudsen, K. A., P. E. Rao, C. H. Damsky, and C. A. Buck, 1981, Proc. Natl. Acad. Sci. USA., 78:6071-6075) inhibited attachment to LN, FN, and Col IV, but did not prevent attachment to other adhesive substrates. In addition to its effects on adhesion, the anti-ECMR serum inhibited both PC12 cell and sympathetic neuronal process outgrowth on LN substrates. Immunoprecipitation of surface-iodinated or [3H]glucosamine-labeled PC12 cells with either the anti-FNR or anti-ECMR serum identified three prominent cell surface glycoproteins of 120, 140, and 180 kD under nonreducing conditions. The 120-kD glycoprotein, which could be labeled with 32P-orthophosphate and appeared to be noncovalently associated with the 140- and 180-kD proteins, cross reacted with antibodies to the beta-subunit (band 3) of the avian integrin complex, itself a receptor or receptors for the ECM constituents LN, FN, and some collagens

Giant FAZ10 is required for flagellum attachment zone stabilization and furrow positioning in Trypanosoma brucei

The flagellum and flagellum attachment zone (FAZ) are important cytoskeletal structures in trypanosomatids, being required for motility, cell division and cell morphogenesis. Trypanosomatid cytoskeletons contain abundant high molecular mass proteins (HMMPs), but many of their biological functions are still unclear. Here, we report the characterization of the giant FAZ protein, FAZ10, in Trypanosoma brucei, which, using immunoelectron microscopy, we show localizes to the intermembrane staples in the FAZ intracellular domain. Our data show that FAZ10 is a giant cytoskeletal protein essential for normal growth and morphology in both procyclic and bloodstream parasite life cycle stages, with its depletion leading to defects in cell morphogenesis, flagellum attachment, and kinetoplast and nucleus positioning. We show that the flagellum attachment defects are probably brought about by reduced tethering of the proximal domain of the paraflagellar rod to the FAZ filament. Further, FAZ10 depletion also reduces abundance of FAZ flagellum domain protein, ClpGM6. Moreover, ablation of FAZ10 impaired the timing and placement of the cleavage furrow during cytokinesis, resulting in premature or asymmetrical cell division

In vitro comparative study of fibroblastic behaviour on polymethacrylate (PMMA) and lithium disilicate polymer surfaces

Polymethyl methacrylate (PMMA) and lithium disilicate are widely used materials in the dental field. PMMA is mainly used for the manufacture of removable prostheses; however, with the incorporation of CAD-CAM technology, new applications have been introduced for this material, including as a provisional implant attachment. Lithium disilicate is considered the gold standard for definitive attachment material. On the other hand, PMMA has begun to be used in clinics as a provisional attachment until the placement of a definitive one occurs. Although there are clinical studies regarding its use, there are few studies on cell reorganization around this type of material. This is why we carried out an in vitro comparative study using discs of both materials in which human gingival fibroblasts (HGFs) were cultured. After processing them, we analyzed various cellular parameters (cell count, cytoskeleton length, core size and coverage area). We analyzed the surface of the discs together with their composition. The results obtained were mostly not statistically significant, which shows that the qualities of PMMA make it a suitable material as an implant attachment

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin.

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN

Toxoplasma gondii major surface antigen (SAG1): in vitro analysis of host cell binding

Previous studies have indicated that SAG1, the major surface molecule of the protozoan parasite Toxoplasma gondii, is an important attachment ligand for the host cell. However, the research data that supports this claim comes largely from studies investigating tachyzoite binding, and not SAG1 binding per se. In this study we successfully developed an in vitro attachment assay to directly evaluate the mechanism of SAG1-host cell binding. Competition experiments were then performed using SAG1 that had been pre-treated with the neoglycoprotein BSA-glucosamide or with antibody. Soluble BSA-glucosamide blocked SAG1 attachment to MDBK cells in a dose-dependent manner, implying that SAG1 binding is mediated, in part, via attachment to host cell surface glucosamine. Interestingly, pre-incubation of SAG1 in polyclonal sera from chronically infected mice failed to block binding. This challenges the assumption that anti-SAG1 antibodies block parasite attachment through the masking of SAG1 host cell binding domains. Taken together, this evidence presents new strategies for understanding SAG1-mediated attachment

Immobilization of Motile Bacterial Cells via Dip-pen Nanolithography

A strategy to bind bacterial cells to surfaces in a directed fashion via dip-pen nanolithography (DPN) is presented. Cellular attachment to pre-designed DPN generated microarrays was found to be dependent on the shape and size of the surface feature. While this observation is likely due in part to a dense, well formed mercaptohexadecanoic acid (MHA) monolayer generated via DPN, it may also simply be due to the physical shape of the surface structure. Motile Pseudomonas aeruginosa bacterial cells were observed to bind to DPN generated mercaptohexadecanoic acid/poly-L-lysine (MHA/PLL) line patterns, \u27blocks\u27 made up of eight lines with 100 nm spacings, with ~ 80% occupancy. Cellular binding to these \u27block\u27 surface structures occurs via an electrostatic interaction between negatively charged groups on the bacterial cell surface and positively charged poly-L-lysine (PLL) assemblies. These data indicate that these DPN generated \u27block\u27 surface structures provide a promising footprint for the attachment of motile bacterial cells that may find utility in cell based biosensors or single cell studies