Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue.However,stripe...Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue.However,stripes offer only a choice between extremes,while for gradients,cell receptor saturation,migration history,and directional persistence confound the interpretation of cellular responses.Here,we introduce nanodot stripe assays(NSAs)formed by adjacent stripes of nanodot arrays with different surface coverage.Twenty-one pairwise combinations were designed using 0,1,3,10,30,44 and 100%stripes and were patterned with 200×200,400×400 or 800×800 nm 2 nanodots.We studied the migration choices of C2C12 myoblasts that express neogenin on NSAs(and three-step gradients)of netrin-1.The reference surface between the nanodots was backfilled with a mixture of polyethylene glycol and poly-D-lysine to minimize nonspecific cell response.Unexpectedly,cell response was independent of nanodot size.Relative to a 0%stripe,cells increasingly chose the high-density stripe with up to~90%of cells on stripes with 10%coverage and higher.Cell preference for higher vs.lower netrin-1 coverage was observed only for coverage ratios>2.3,with cell preference plateauing at~80%for ratios≥4.The combinatorial NSA enables quantitative studies of cell haptotaxis over the full range of surface coverages and ratios and provides a means to elucidate haptotactic mechanisms.展开更多
基金We acknowledge financial support from the NSERC Discovery Grant programM.D.received funding from a NeuroEngineering NSERC-CREATE.
文摘Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue.However,stripes offer only a choice between extremes,while for gradients,cell receptor saturation,migration history,and directional persistence confound the interpretation of cellular responses.Here,we introduce nanodot stripe assays(NSAs)formed by adjacent stripes of nanodot arrays with different surface coverage.Twenty-one pairwise combinations were designed using 0,1,3,10,30,44 and 100%stripes and were patterned with 200×200,400×400 or 800×800 nm 2 nanodots.We studied the migration choices of C2C12 myoblasts that express neogenin on NSAs(and three-step gradients)of netrin-1.The reference surface between the nanodots was backfilled with a mixture of polyethylene glycol and poly-D-lysine to minimize nonspecific cell response.Unexpectedly,cell response was independent of nanodot size.Relative to a 0%stripe,cells increasingly chose the high-density stripe with up to~90%of cells on stripes with 10%coverage and higher.Cell preference for higher vs.lower netrin-1 coverage was observed only for coverage ratios>2.3,with cell preference plateauing at~80%for ratios≥4.The combinatorial NSA enables quantitative studies of cell haptotaxis over the full range of surface coverages and ratios and provides a means to elucidate haptotactic mechanisms.
