Abstract
On traditional cell culture substrates cells adhere to a planar 2D surface where ligands are presented immobile. A more realistic presentation of cell adhesion ligands which can account for lateral mobility and a more tissue-like 3D presentation would allow studies addressing fundamental questions of significant importance for applications such as tissue engineering and implant intregration. To study the effect of lateral mobility of cell membrane interaction cues in three dimensions, we have developed and characterized a platform which generically enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Here, we describe its application in presenting a soluble cell adhesive ligand coupled through streptavidin-antibody linkage to lipids in a supported lipid bilayer (SLB) coated microwell. The lateral mobility of the presented ligands was controlled through a small change in temperature. The SLB phospholipid composition was choosen such that below its melting transition at 30 °C the ligands are immobile, while above 30 °C they are laterally mobile. The platform thus enables the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligand presented on the same cell membrane mimetic surface.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kusumi A, Suzuki K, Koyasako K (1999) Mobility and cytoskeletal interactions of cell adhesion receptors. Curr Opin Cell Biol 11:582–590
Yap AS, Brieher WM, Gumbiner BM (1997) Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol 13:119–146
Cavallaro U, Christofori G (2004) Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 4:118–132
Mossman KD, Campi G, Groves JT, Dustin ML (2005) Altered TCR signaling from geometrically repatterned immunological synapses. Science 310:1191–1193
Takeichi M (1991) Cadherin cell-adhesion receptors as a morphogenetic regulator. Science 251:1451–1455
Salaita K, Nair PM, Petit R, Neve R, Das D, Grey J, Groves J (2010) Restriction of receptor movement alters cellular response: physical force sensing by EphA2. Science 327:1380–1385
Tozeren A, Sung KL, Sung LA, Dustin ML, Chan PY, Springer TA, Chien S (1992) Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules. J Cell Biol 116:997–1006
Gumbiner BM (1996) Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345–357
Takeichi M (1995) Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7:619–627
Yap AS (1998) The morphogenetic role of cadherin cell adhesion molecules in human cancer: a thematic review. Cancer Invest 16:252–261
Mege R-M, Gavard J, Lambert M (2006) Regulation of cell-cell junctions by the cytoskeleton. Curr Opin Cell Biol 18:541–548
Groves JT, Dustin ML (2003) Supported planar bilayers in studies on immune cell adhesion and communication. J Immunol Methods 278:19–32
Berat R, Remy-Zolghadry M, Gounou C, Manigand C, Tan S, Salto C, Arenas E, Bordenave L, Brisson AR (2007) Peptide-presenting two-dimensional protein matrix on supported lipid bilayers: an efficient platform for cell adhesion. Biointerphases 2:165–172
Perez TD, Nelson WJ, Boxer SG, Kam L (2005) E-Cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion. Langmuir 21:11963–11968
Svedhem S, Dahlborg D, Ekeroth J, Kelly J, Hook F, Gold J (2003) In situ peptide-modified supported lipid bilayers for controlled cell attachment. Langmuir 19:6730–6736
Nam J-M, Nair PM, Neve RM, Gray JW, Groves JT (2006) A fluid membrane-based soluble ligand-display system for live-cell assays. Chembiochem 7:436–440
Thid D, Holm K, Eriksson PS, Ekeroth J, Kasemo B, Gold J (2008) Supported phospholipid bilayers as a platform for neural progenitor cell culture. J Biomed Mater Res A 84:940–953
Tsai J, Kam LC (2010) Lateral mobility of E-Cadherin enhances rac1 response in epithelial cells. Cell Mol Bioeng 3:84–90
Andreasson-Ochsner M, Romano G, Håkanson M, Smith ML, Reimhult E, Vogel V, Textor M (2011) Single cell 3-D platform to study ligand mobility in cell–cell contact. Lab Chip 11:2876–2883
Dusseiller M, Smith ML, Vogel V, Textor M (2006) Microfabricated three-dimensional environments for single cell studies. Biointerphases 1:P1–P4
Ochsner M, Dusseiller MR, Grandin HM, Luna-Morris S, Textor M, Vogel V, Smith ML (2007) Micro-well arrays for 3D shape control and high resolution analysis of single cells. Lab Chip 7:1074–1077
Ochsner M, Textor M, Vogel V, Smith ML (2010) Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells in response to rigidity and shape. PLoS One 5:e9445
MacDonald RC, MacDonald RI, Menco BPM, Takeshita K, Subbarao NK, Hu LR (1991) Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta 1061:297–303
Niessen CM, Gumbier BM (2002) Cadherin-mediated cell sorting not determined by binding or adhesion specificity. J Cell Biol 156:389–399
Chien Y-H, Jiang N, Li F, Zhang F, Zhu C, Leckband D (2008) Two-stage cadherin kinetics require multiple extracellular domains but not the cytoplasmic region. J Biol Chem 283:1848–1856
Zhu B, Chappuis-Flament S, Wong E, Jensen IE, Gumbiner BM, Leckband D (2003) Functional analysis of the structural basis of homophilic cadherin adhesion. Biophys J 84:4033–4042
Sorribas H, Padeste C, Tiefenauer L (2002) Photolithographic generation of protein micropatterns for neuron culture applications. Biomaterials 23:893–900
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Andreasson-Ochsner, M., Reimhult, E. (2013). Mobile and Three-Dimensional Presentation of Adhesion Proteins Within Microwells. In: Coutts, A. (eds) Adhesion Protein Protocols. Methods in Molecular Biology, vol 1046. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-538-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-538-5_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-537-8
Online ISBN: 978-1-62703-538-5
eBook Packages: Springer Protocols