Abstract
Micropipette manipulation measurements quantified the pre-steady state binding kinetics between cell pairs mediated by Xenopus cleavage stage cadherin. The time-dependence of the intercellular binding probability exhibits a fast forming, low probability binding state, which transitions to a slower forming, high probability state. The biphasic kinetics are independent of the cytoplasmic region, but the transition to the high probability state requires the third extracellular domain EC3. Deleting either EC3 or EC3-5, or substituting Trp(2) for Ala reduces the binding curves to a simple, monophasic rise in binding probability to a limiting plateau, as predicted for a single site binding mechanism. The two stage cadherin binding process reported here directly parallels previous biophysical studies, and confirms that the cadherin ectodomain governs the initial intercellular adhesion dynamics.
Highlights
The significant new findings of this study are that the initial, cadherin-mediated cell contact formation occurs by a complex kinetic process that requires the full ectodomain, and that this kinetic behavior is independent of the cytoplasmic domain
The two kinetic states are separated by a lag, the duration of which depends in part on the cadherin surface density and on the state of cadherin oligomerization
Measurements between RBCs coated identically with the truncated ectodomains demonstrate that, within the first 40 s, the cell adhesion kinetics are independent of the cytoplasmic domain
Summary
Cell Lines, Proteins, and Plasmids—The pEE14 plasmid containing the full-length Xenopus C-cadherin cDNA, the pEE14 plasmid containing the cDNA encoding the hexahistidinetagged C-cadherin extracellular domain with the W2A mutation, and the CHO cell line expressing the full-length C-cadherin with the W2A mutation were generously provided by B. To generate cell lines expressing the W2A mutant of soluble CEC1–5-His and the full-length C-cadherin, CHO-K1 cells were stably transfected with the plasmids encoding the different C-cadherin constructs. Sample Configuration and Preparation in the Micropipette Manipulation Assays—Fig. 1A exemplifies the configuration of the cells in the micropipette manipulation experiment. In this case, the CHO cell on the left expresses the full-length, wildtype C-cadherin (C-CHO) (Fig. 1B). B C-CHO Membrane Cytoplasmic domain oooooooo oooo o ooo oooooooo oooo o ooo EC5 EC4 EC3 EC2 EC1
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