We show that the nanoscale adhesion geometry controls the spreading and differentiation of epidermal stem cells. We find that cells respond to such hard nanopatterns similarly to their behavior on soft hydrogels. Cellular responses were seen to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction rather than impaired recruitment of proteins involved in focal adhesion formation.
The effects of protein type and pattern size on cell adhesion, spreading, and focal adhesion development are studied. Fibronectin and vitronectin patterns from 0.1 to 3 μm produced by colloidal lithography reveal important differences in how cells adhere to and bridge focal adhesions across protein nanopatterns versus micropatterns. Vinculin and zyxin in focal adhesions but not integrins are seen to bridge ligand gaps. Differences in protein mechanical properties are implicated as important factors in focal adhesion development.
The importance of the lipid phase on the formation of supported lipid bilayers (SLBs) via vesicle fusion and on the resulting SLB homogeneity at SiO(2) surfaces has been studied by the quartz crystal microbalance with dissipation (QCM-D) monitoring technique. Physiologically relevant lipid compositions were chosen to correspond to different regions (l(d), l(o) and coexistence of phases) in established phase diagrams of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (PSM) and cholesterol. For most compositions, SLBs formed through vesicle rupture in a critical-surface-coverage dependent manner. Inclusion of PSM and cholesterol into POPC vesicles significantly impaired the vesicle rupture process such that a higher critical concentration of vesicles on the surface was needed before the rupture process started. When increasing the cholesterol content the vesicles formed SLBs containing more defects in the form of intact vesicles adsorbed on the surface up to a point (l(o) phase) where vesicles did not break at all but formed supported vesicular layers. The hampering of vesicle rupture is interpreted in terms of the ability of cholesterol to accommodate vesicle deformation. Experiments using elevated temperatures to alter the lipid membrane into a more fluid phase significantly improved the quality of the SLB showing the importance of both cholesterol content and the lipid phase on SLB homogeneity.
We have developed and characterized novel biomimetic membranes, formed at nanostructured sensor substrates with controlled curvatures, motivated by the many biological processes that involve membrane curvature. Model systems with convex nanostructures, with radii of curvatures (ROCs) of 70, 75, and 95 nm, were fabricated utilizing colloidal assembly and used as substrates for supported lipid bilayers (SLBs). The SLBs were formed via vesicle adsorption and rupture, and the vesicle deposition pathway was studied by means of quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. SLBs conforming to the underlying nanostructured surfaces, which exhibit increased surface area with decreased ROC, were confirmed from excess mass, monitored by QCM-D, and excess total fluorescence intensities. The formation of SLBs at the nanostructured surfaces was possible, however, depending on the ROC of the structures and the lipid vesicle charge the quality varied. The presence of nanostructures was shown to impair vesicle rupture and SLB formation was progressively hindered at surfaces with structures of decreasing ROCs. The introduction of a fraction of the positively charged lipid POEPC in the lipid vesicle membrane allowed for good quality and conformal bilayers at all surfaces. Alternatively, for vesicles formed from lipid mixtures with a fraction of the negatively charged lipid POPS, SLB formation was not at all possible at surfaces with the lowest ROC. Interestingly, the vesicle adsorption rate and the SLB formation were faster at surfaces with nanostructures of progressively smaller ROCs at high ratios of POPS in the vesicles. Development of templated SLBs with controlled curvatures provides a new experimental platform, especially at the nanoscale, at which membrane events such as lipid sorting, phase separation, and protein binding can be studied.
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