No abstract

No abstract

Abstract. Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESE where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dosedependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.number of studies indicate that fibronectin (FN) ~ and certain FN fragments are potent chemoattractants for fibroblastic cells (5,17,27,34,41,44,47). Presumably this activity is important in wound healing where fibroblasts from the surrounding tissues are recruited to the site of damage to produce the matrix molecules necessary for full repair (48). The rate of repair is believed to be related to the numbers of cells recruited to the wound site and thereby to the level of chemoattractants and the duration of their production. Chemotaxis to FN may also be involved in pathological situations including fibrosis and tumor cell metastasis (2,22,28,30,31,44,(52)(53)(54). The chemotactic region of FN has been localized within a large cell-binding chymotryptic 160-kD peptide (47) and in a cathepsin D-derived 140-kD peptide which lacks gelatin-binding activity (3, 5, 41).1. Abbreviations used in this paper: FN, fibronectin; PDGE plateletderived growth factor.Studies with various chemoattractants in eukaryotic and prokaryotic cells show that the responding cells have receptors which bind the attractant and elicit the intracellular events that direct cell movement (6,12,22,24,36,56). Here we have attempted to determine if the chemotactic response of 3T3 cells and their SV40-transformed counterpart to FN occurs through the RGD-attachment site of the molecule (37, 40) and an FN receptor (Mr 140,000) (18,19,51). Studies with antibodies to the cell-binding site in FN, with antibodies...

An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/ p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

No abstract