DNA-mediated cell-cell attachment studied using supported lipid bilayers

Abstract

Event Abstract Back to Event DNA-mediated cell-cell attachment studied using supported lipid bilayers Yusuke Arima1 and Hiroo Iwata1 1 Kyoto University, Institute for Frontier Medical Sciences, Japan It is important to understand and control the cell microenvironment in order to develop methods for tissue engineering and the expansion or differentiation of stem cells. Engineering of cell surface with natural and synthetic molecules is expected to provide new approaches for biomedical applications[1]. We have used single-stranded DNA, which was conjugated both to poly(ethylene glycol) and to a terminal phospholipid (ssDNA-PEG-lipid, Fig. 1a) for cell surface engineering[2]. The lipid moiety is inserted in the lipid bilayer of living cells through hydrophobic interaction, resulting in presentation of ssDNA on the cells surface. This allows for modification of cell surface with biomolecules[2] and attachment of cells to either substrates or different cells[3], all of which present the complementary ssDNA. For the development of cell surface engineering, it is important to understand the behavior of ssDNA-PEG-lipids on cell surface and its structural role on cell attachment. In this study, we employed supported lipid bilayers (SLBs) as a cell surface model to examine behavior of ssDNA-PEG-lipids during cell attachment (Fig. 1b). SLBs consisting of egg L-α-phosphatidylcholine were prepared by vesicle fusion to glass. ssDNA-PEG-lipids consisting of 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE) were synthesized according to our previous study [3]. SLBs were then modified with a mixture of fluorescein-labeled SeqA-PEG-DPPE (FAM-SeqA-PEG-DPPE) and non-labeled SeqB-PEG-DPPE with varying ratio in order to mimic cell membrane which carries multiple cell surface receptors. Human T cell lymphoblast-like cell line (CCRF-CEM) was modified with either SeqA’-PEG-DPPE or SeqB’-PEG-DPPE. Fluorescence images before and after seeding cells were obtained by a total internal reflection fluorescence microscope. The ssDNA-PEG-DPPE was homogeneously distributed over both the cell membrane and the SLB and exhibited lateral fluidity as examined using a fluorescence recovery after photobleaching (FRAP). When cells modified with SeqA’-PEG-DPPE were seeded, the cell attached region became brighter (Fig. 2a, left), indicating that FAM-SeqA-PEG-DPPEs were recruited at the cell attached region. This result suggests that lateral diffusion of FAM-SeqA-PEG-DPPEs are slowed when associated with their complementary SeqA’-PEG-DPPEs at the opposite lipid bilayer. In contrast, cell attached region became darker when cells modified with SeqB’-PEG-DPPE attached (Fig. 2a, right), indicating that FAM-SeqA-PEG-DPPEs were excluded from the cell attached region. The recruitment and exclusion were observed regardless of density and fraction of FAM-SeqA-PEG-DPPE on SLB (Fig. 2b). In summary, our results demonstrate that cell attachment induces localization of ssDNA-PEG-lipids. The recruitment/exclusion of ssDNA-PEG-lipids effectively increases the local density of interacting ssDNA-PEG-lipids at contacting region and will lead to an increase in the work of adhesion. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular Science” (No. 2306) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan