(A) GFP-actin expressed in ECs adhering on 20-um wide lines of FN.
ECs do not adhere to PEG-coated areas (black). (B) Phase contrast image
of ECs on FN lines. (C) Rhodamine-FN dots contact-printed onto glass.
(D) Dot array created by stamping hexadecanethiol, blocking with PEG-
alkanethiol, and washing with rhoadmine-FN. (E) ECs growing on a region
printed with rhodamine-FN but not on adjacent non-printed, PEG-coated region.
Substantial research advances have been acheived through our current Seed Program on "Fundamental Studies of Cellular Mechanics Enabled by Focal Adhesion to Nanosctructured Substrates" (see Emerging Areas), led by MRSEC faculty Brian Helmke and Michael Reed. The central goal of this program is the creation of nanoscaled patterned regions with differential adhesion properties for biologic molecules.
Towards this end, one research focus is the use of nanostructured surfaces to understand and control how mechanical stimuli such as stretch or exposure to fluid shear stress causes adherent cells to undergo changes in gene and protein expression profiles, cellular contraction, migration, differentiation, and programmed cell death. Center research has demonstrated spatially focused cytoskeletal deformation in living cells after onset of shear stress. Additionally, green fluorescent protein (GEP) fused to actin (GEP-actin) has revealed initiation of edge ruffling in endothelial cells (ECs) induced by onset of shear stress. Such shear-induced ruffling is not apparent in cells under no-flow conditions or in cells within a confluent layer. Importantly, this edge-ruffling response is significantly altered in ECs adhering on microcontact printed 20-um lines of extracellular matrix (ECM) proteins and is likely to depend on the orientation of the lines relative to the axis of applied shear stress (shown above in Figures A and B). This novel observation has led to a new hypothesis for EC mechanotransduction: flow-induced actin dynamics represent an active response by ECs to stabilize cell contact and prevent increases in vascular permeability in vivo.
In order to investigate mechanisms by which spatial cues on the substrate control cell migration, we have extended this work to nanoscale patterns of ECM proteins that mimic the physiological distribution of adhesion protein clusters at the cell-biomaterial interface. This should enable the first studies of ECM remodeling under living cells in a simple model that excludes adhesive substrate interactions. Using nanocontact printing, we have generated the first surfaces with 150-nm dots of functional rhoadmine-FN. Rhodamine-FN was printed onto glass substrates (shown above in Figure C), or bound to exposed alkane dots (Figure D). ECs plated onto the surface of the latter surface established normal morphology on patterned areas but avoided adhering to the non-patterned areas (Figure E).
