Mechanisms of Embryonic Limb Bud Intercellular Adhesion : Kinetic Analyses and Characterization of the Molecular Mechanism
When cultured in suspension, dissociated early limb bud cells segregate into aggregating and non-aggregating populations. While the non-aggregating cells die, resulting aggregates differentiate exclusively as cartilage. We are investigating the role of the cell surface in pre-chondrogenic condensation by analyzing the mechanism of aggregation in suspension culture. Immediately after their tryptic dissociation, limb bud cells undergo calcium-independent aggregation. This mechanism is completely and reversibly sensitive to cycloheximide, although this drug does not inhibit the re-appearance of all cell surface proteins. Fab’ fragments prepared from antisera directed against the surface of these cells fail to inhibit their aggregation. In contrast, after 16 h recovery equivalent cells exhibit a different, calcium-dependent aggregation mechanism and demonstrate distinct sensitivity to cycloheximide. This calcium-dependent aggregation mechanism can be maintained upon cells by the addition of exogenous calcium during trypsin dissociation. Fab fragments prepared from an antiserum directed against these recovered cells inhibit only calcium-dependent aggregation. A surface glycoprotein with an approximate molecular weight of 8.5 kD can be isolated directly from total solubilized limb bud membrane proteins by affinity chromatography against equivalent proteins covalently coupled to Sepharose in the presence of calcium. This same protein is recognized by the aggregation-inhibiting antiserum. We present evidence that this homophilic protein is the major limb bud cell adhesion molecule.
KeywordsCell Adhesion Molecule Intercellular Adhesion Aggregation Mechanism Recovered Cell Exogenous Calcium
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