Temperature-Switchable Control of Ligand Display on Adlayers of Mixed Poly(lysine)-g-(PEO) and Poly(lysine)-g-(ligand-modified poly-N-isopropylacrylamide).

Adlayers of poly(lysine)-g-PEG comblike copolymer are extensively used to prepare cell-repellant and protein-repellent surfaces by a straightforward coulomb-driven adsorption that is compatible with diverse substrates (glass, Petri dish, etc.). To endow surfaces with functional properties, namely, controlled ligand-protein binding, comblike poly(lysine) derivatives were used to deposit temperature-responsive poly(NIPAM) macrografts mixed with PEG ones on glass surfaces. Simple surface immersion in mixed solutions of biotin-modified poly(lysine)-g-poly(N-isopropylacrylamide) and poly(lysine)-g-poly(ethylene oxide) yielded robust adlayers whose composition reflected the ratio between the two polymers in solution. We show by fluorescence imaging, and comparison with repellent 100% PEGylated patterns, that specific binding of model avidin/particle conjugates (diameters of ca. 10 or 200 nm) was controlled by temperature switch. The biotin ligand was displayed and accessible at low T, or hidden at T > LCST. Topography and mechanical mapping measurements by AFM confirmed the swelling/collapse status of PNIPAM macrografts in the adlayer at low/high T, respectively. Temperature-responsive comblike PLL derivative that can spontaneously cover anionic interfaces is a promising platform enabling good control on the deposition and accessibility of biofunctional groups on various solid surfaces.

Mixed Copolymer Adlayers Allowing Reversible Thermal Control of Single Cell Aspect Ratio.

Dynamic guidance of living cells is achieved by fine-tuning and spatiotemporal modulation on artificial polymer layers enabling reversible peptide display. Adjustment of surface composition and interactions is obtained by coadsorption of mixed poly(lysine) derivatives, grafted with either repellent PEG, RGD adhesion peptides, or T-responsive poly(N-isopropylacrylamide) strands. Deposition of mixed adlayers provides a straightforward mean to optimize complex substrates, which is here implemented to achieve (1) thermal control of ligand accessibility and (2) adjustment of relative adhesiveness between adjacent micropatterns, while preserving cell attachment during thermal cycles. The reversible polarization of HeLa cells along orthogonal stripes mimics guidance along natural matrices.

Optogenetic dissection of Rac1 and Cdc42 gradient shaping.

During cell migration, Rho GTPases spontaneously form spatial gradients that define the front and back of cells. At the front, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Here we report, using a combination of optogenetics and micropatterning, that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by transport mechanisms. Cdc42 simply follows the distribution of Guanine nucleotide Exchange Factors, whereas Rac1 shaping requires the activity of a GTPase-Activating Protein, ß2-chimaerin, which is sharply localized at the tip of the cell through feedbacks from Cdc42 and Rac1. Functionally, the spatial extent of Rho GTPases gradients governs cell migration, a sharp Cdc42 gradient maximizes directionality while an extended Rac1 gradient controls the speed.