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For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes independently of their catalytic activity, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.

Ultraviolet (UV)-photofunctionalization of titanium substantially enhances the strength and quality of osseointegration by promoting osteogenic cellular attachment and proliferation. However, the mechanism underlying the initial interaction between the cells and the surface of the material remains to be elucidated, especially where the influence of surface roughness is excluded as a factor. The effect of UV-photofunctionalization on the adhesive strength and cellular stiffness of a single osteoblast and its association with the extent of cell spread, cytoskeletal development and focal adhesion assembly on a very smooth titanium surface was evaluated. Rat bone marrow-derived osteoblasts were cultured on UV-treated or untreated mirror-polished titanium disks. The mean critical shear force required to initiate detachment of a single osteoblast (n=10) was >2000nN on a UV-treated surface at 3h incubation, which was 17 times greater than that on an untreated surface. The mean total energy required to complete the detachment of osteoblasts (n=10) was consistently >60pJ on a UV-treated titanium surface after 24h culture, which was up to 42 times greater than that on an untreated surface. Cellular shear modulus, which represents cellular stiffness, was consistently greater on a UV-treated surface than on an untreated surface after 24h incubation (n=10). This strengthening of cell adhesion and cellular mechanical properties on UV-treated titanium was accompanied by enhanced cell spread and actin fiber development and increased levels of vinculin expression. These results indicate that UV-photofunctionalization substantially strengthens osteoblast retention on titanium bulk material with no topographical features, and that this is associated with enhancement of intracellular structural development during the cell adhesion process.

It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed. Fil: Di Venosa, Gabriela Mariana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Perotti, Christian. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. University of Calgary. Southern Alberta Research Institute; Canadá Fil: Batlle, Alcira Maria del C.. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Investigacion Sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina

Nature provides cleverly concerted mechanisms which are able to control specific and non-specific interactions between cells and biological surfaces in a dynamic environment.[1] The successful imitation of this ability is of crucial importance for the understanding of cell adhesion to man-made surfaces,[2] and will aid in making significant impact in the future development and fabrication of new, functional biomaterials,[3] long-term antifouling surfaces[4] and novel in vitro diagnostic tools.[5] Researchers have previously fabricated surfaces, typically using self-assembled monolayers (SAMs)[6] in an attempt to control and understand cell adhesion, via the introduction of specific terminal moieties to the SAM to elicit specific surface properties.[7] However, despite the advancements achieved by investigating the interface as a ‘static’ environment, the mechanism behind the cell adhesion process remain unclear[8] representing one of the biggest challenges for many scientific areas ranging from tissue engineering,[9] medicine,[10] cell biology,[11] immunology[12] and marine biofouling.[13] In particular, researchers have tended to use cells to classify surfaces (i.e., bactericidal, repellent) while the ideal approach would be using the surface to understand cell behaviour. Towards this aim, scientists have discovered the possibility to control surface properties by switching their characteristics in response to particular needs.[1b, 14] Surfaces presenting tuneable moieties have been widely used in the study of specific protein-repellent/adhesion phenomena.[15] However, there are only a few reports on the control of non-specific cell adhesion[16] due to the lack of specific targeting on the cell surface, and the difficulties encountered in the interpretation of the outcome of end-point assays.[17] To date, dynamic control over cell adhesion has been achieved by using “smart” surfaces mostly formed by stimuli-responsive polymers.[18] For instance, the thermo-responsive polymer poly(N-isopropylacrylamide) and the closely related polyacrylamides have been widely investigated for preparing biologically relevant switchable surfaces,[19] as well as polymeric materials that can tune their properties from bacteriostatic to non-fouling and antimicrobial.[20] However, compared to such polymer systems, SAMs will allow higher precision of distribution and faster changes in surface properties.[21] Therefore, utilising such dynamic SAMs, the possibility exists to monitor the first interaction between a cell and a surface in real-time. Additionally, the passage between reversible and non-reversible cell adhesion can be ascertained. SAMs with stimuli-responsive characteristics have been used for the adhesion of smaller analytes such as proteins,[15a-d] and DNA,[22] and control specific interactions with cell surface receptors[23] but there has been no attempt to study non-specific cell adhesion on such smart surfaces.[24] In this context, the development of a reversible and fast electrochemical switchable surface, based on homogeneously distributed two-component SAMs represents a promising tool for studying cell-surface interactions in vitro and in real-time, and is a significant step forward for the fabrication of similar systems in vivo. The use of an applied potential for changing the surface properties will allow the study of bacterial interactions without perturbing their natural environment, and therefore their viability (as it may happen by changing the pH and temperature).

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Calpha (PKCalpha) and its subsequent interaction with beta(1) integrin since disruption of PKCalpha binding to beta(1) integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCalpha leading to its association with beta(1) integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.

Arginine-specific ADP-ribosyltransferase present in secretory granules of chicken polymorphonuclear leukocytes (so-called heterophils) was shown to be released into the extracellular space by secretagogues (Terashima et al., J. Biochem. 120 (1996) 1209–1215). In the present work, we examined fibronectin as an extracellular target protein of the released transferase. Fibronectin was ADP-ribosylated by purified transferase and stoichiometry of ADP-ribose incorporation into fibronectin was 1.0 mol/mol of fibronectin. Cell adhesion and spreading assays revealed that ADP-ribosylation of fibronectin markedly inhibited the adhesion activity of fibronectin. A proteolytic peptide map of ADP-ribosylated fibronectin demonstrated that the modification occurs in the cell binding domain of fibronectin. ADP-ribosylation of the RGD peptide suggests that the RGD sequence is the modification site in the domain. ADP-ribosylation of fibronectin in plasma means that fibronectin can probably serve as the substrate for extracellularly released ADP-ribosyltransferase in vivo. Thus, in the extracellular space, ADP-ribosyltransferase released from polymorphonuclear leukocytes may perhaps be involved in regulation of cell adhesion process by interfering with the activity of fibronectin.

CD98 is a multifunctional heterodimeric membrane protein involved in the regulation of cell adhesion as well as amino acid transport. We show that CD98 cross-linking persistently activates Rap1 GTPase in a LFA-1-dependent manner and induces LFA-1/ICAM-1-mediated cell adhesion in lymphocytes. Specific phosphatidylinositol-3-kinase (PI3K) inhibitors suppressed both LFA-1 activation and Rap1GTP generation, and abrogation of Rap1GTP by retroviral over-expression of a specific Rap1 GTPase activating protein, SPA-1, totally inhibited the LFA-1/ICAM-1-mediated cell adhesion. These results suggest that CD98 cross-linking activates LFA-1 via the PI3K signaling pathway and induces accumulation of Rap1GTP in a LFA-1-dependent manner, which in turn mediates the cytoskeleton-dependent cell adhesion process.

Studies were carried out to learn more about the critical SH groups involved in cell spreading. Pretreatment of suspended baby hamster kidney (BHK) cells with 3 mM-iodoacetate or iodoacetamide for 10 min at 4 degrees C completely inhibited the ability of the cells to spread on fibronectin-coated substrata. If, however, BHK cells were permitted to attach and spread before being treated with the SH-binding reagents, and then harvested by trypsinization and assayed for spreading on fibronectin-coated substrata, there was no inhibition of cell spreading. The extent of prior attachment required before the cells became insensitive to the SH-binding reagents was tested and was found to occur early during the cell adhesion process, before any cell spreading was observed. In analytical experiments, there did not appear to be any difference in the total number of SH groups between suspended or spread cells as determined with 5,5′-dithiobis-(2-nitrobenzoic acid). The uptake of radiolabelled iodoacetate into intact spread cells, however, was found to be 3.5 times less than that found with suspended cells. On the other hand, the distribution of incorporated radioactivity into suspended and spread cells was similar. Most of the radioactivity (approximately 70%) was incorporated into small molecules (e.g. glutathione and cysteine), less (approximately 20%) was incorporated into cytoplasmic proteins, and the least incorporation (approximately 10%) was into the cell cytoskeleton. The data are interpreted to indicate there is a decreased permeability of spread cells to the SH-binding reagents.