Abstract
Nectins are Ca2+-independent cell adhesion molecules found at cadherin-based adherens junctions. We used a dual pipette assay that measures the forces required to separate cell doublets to determine how nectins affect the formation and strength of cell-cell adhesion. Less force was required to separate doublets of L cells expressing nectin-1 or nectin-3 than to separate doublets of E-cadherin-expressing cells. Heterodimers formed between cells expressing nectin-1 or nectin-3 adhered more strongly than homodimers. Nectin-3 that does not trans-interact with nectin-1 inhibited E-cadherin-mediated adhesion. However, the extracellular fragment of nectin-1 did not have an agonistic effect on E-cadherin-dependent cell adhesion when it trans-interacted with nectin-3, expressed at high levels in cells. In contrast, the extracellular fragment of nectin-3 had a significant agonistic effect on cadherin-based adhesion when it interacted with endogenous nectin-1, expressed at low levels in cells. Our results indicate that E-cadherin is the key molecule involved in cell adhesion and that the regulation of E-cadherin-based adhesion involving cellular nectin-1 trans-interacting with nectin-3 is qualitatively different from that involving cellular nectin-3 trans-interacting with nectin-1 and depends on the nectin levels expressed by cells.
Highlights
Cadherin-based cell adhesion is a critical determinant of tissue architecture in developing and adult metazoan organisms [1,2,3]
- and ␥-catenins interact with the cytoplasmic tail of E-cadherin, forming a complex that is coupled to the actin cytoskeleton through ␣-catenin ( an actin-binding protein [8, 17]) via its interactions with a number of actin-binding proteins, such as ␣-actinin and vinculin, or by directly binding to actin itself (18 –21)
Recent studies have shown that the structural organization of cadherin-based adherens junctions (AJs) and tight junctions in polarized epithelia is disrupted in afadin Ϫ/Ϫ mice [45]
Summary
Antibodies and Reagents—DECMA-1 (rat anti-E-cadherin) antibody and phalloidin-fluorescein isothiocyanate were purchased from Sigma. Cell Lines and Reagents—N1L, N1⌬CL, and N3L clones are mouse L fibroblasts stably transfected clones expressing nectin-1, cytoplasmic deleted nectin-1⌬C (carrying the first 514aa), or nectin-3, respectively L fibroblasts stably transfected to express E-cadherin (EL cells) were produced as described previously [47]. N3EL (#2) cells expressing E-cadherin and FLAG-nectin-3 were produced as described in a previous study [48]. Microscopy—The expression of E-cadherin and nectins was monitored by immunofluorescence microscopy in cells cultured on glass coverslips as described previously [50]. Cell suspensions were prepared in a way to preserve E-cadherin at their surface and incubated in cold Dulbecco’s modified Eagle’s medium with a specific antibody directed against the extracellular domain of E-cadherin or against the cytoplasmic domain of nectins.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
