Recapitulating cell-cell adhesion using N-cadherin biologically tethered to substrates.
Intercellular adhesion modulated by cadherin molecules plays an important role in diverse cellular functions including tissue morphogenesis, regeneration, and pathogenesis. However, it is a challenging task to decipher the effects of cell-cell adhesion in vitro because of difficulty in controlling the extent and numbers of cell-cell contacts. In this study, we hypothesize that tethering recombinant extracellular domains of neural cadherin with a C-terminal immunoglobulin Fc domain (N-Cad-Fc) to a substrate with an immobilized anti-Fc antibody (Fc-antibody) and a bifunctional polymer, which is reactive to both protein and substrate, would allow us to recapitulate cell-cell adhesion, independent of the number of cells plated on the substrate. To examine this hypothesis, we first immobilized Fc-antibody to a polyacrylamide hydrogel and a methacrylate-substituted glass using poly(amino-2-hydroxyethyl-co-2-methacryloxyethyl aspartamide)-g-poly(ethylene glycol)-N-hydroxysuccinimide ester (PHMAA-g-PEGNHS) and then incubated the gel in medium containing defined concentrations of the recombinant N-Cad-Fc. The resulting N-Cad-conjugated substrate enabled us to modulate adhesion of bone marrow stromal cells to the gel surface by varying the surface density of N-Cad-Fc. In contrast, direct chemical conjugation of N-Cad-Fc to the gel surface did not support cell adhesion. Additionally, the glass substrate biologically tethered with N-Cad-Fc promoted neuronal adhesion significantly more than substrates coated with poly-l-lysine. We suggest that this novel biological tethering method could be broadly applicable for modifying substrates with a variety of classical cadherins to enable the systematic study of the effects of cadherin-modulated cell-cell adhesion on cellular activities.
Neuroscience Program, §Department of Chemical and Biomolecular Engineering, ⊥Department of Molecular and Integrative Physiology, #Department of Chemistry, and &Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois, United States.
Johana C M
JC
Vega L
Min Kyung
MK
Lee
Jae Hyun
JH
Jeong
Cartney E
CE
Smith
Kwan Young
KY
Lee
Hee Jung
HJ
Chung
Deborah E
DE
Leckband
Hyunjoon
H
Kong
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000378:metabolism*; D015820:Cadherins / Q000737:chemistry / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D013379:Substrate Specificity / Q000502:physiology
D015703:Antigens, CD; C050361:CDH2 protein, human; D015820:Cadherins