Laboratoire P.A.S.T.E.U.R

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

Biomacromolecules2016 May 9;17(5):1727-36

 

Adlayers of poly(lysine)-g -PEG comblike copolymer are extensively used to prepare cell-repellant and proteinrepellent 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-modifi ed poly(lysine)-g -poly(N -isopropylacrylamide) and poly(lysine)-g -poly(ethylene oxide) yielded robust adlayers whose composition refl ected the ratio between the two polymers in solution. We show by fluorescence imaging, and comparison with repellent 100% PEGylated patterns, that specifi c 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 confi rmed 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

Cellular heterogeneity mediates inherent sensitivity– specificity tradeoff in cancer targeting by synthetic circuits

Proc. Natl. Acad. Sci. USA2016

 

Synthetic gene circuits are emerging as a versatile means to target cancer with enhanced specificity by combinatorial integration of multiple expression markers. Such circuits must also be tuned to be highly sensitive because escape of even a few cells might be detrimental. However, the error rates of decision-making circuits in light of cellular variability in gene expression have so far remained unexplored. Here, we measure the single-cell response function of a tunable logicANDgate actingon twopromoters in heterogeneous cell populations. Our analysis reveals an inherent tradeoff between specificity and sensitivity that is controlled by the AND gate amplification gain and activation threshold. We implement a tumor-mimicking cellculture model of cancer cells emerging in a background of normal ones, and show that molecular parameters of the synthetic circuits control specificity and sensitivity in a killing assay. This suggests that, beyond the inherent tradeoff, synthetic circuits operating in a heterogeneous environment could be optimized to efficiently target malignant state with minimal loss of specificity.

On-Chip Quantitative Measurement of Mechanical Stresses During Cell Migration with Emulsion Droplets

    

Long-term in vivo single-cell lineage tracing of deep structures using three-photon activation

Light: Science & Applications (2016) 5, e16084

 

Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.

 

Dynamical Disorder in the DNA Hydration Shell

J. Am. Chem. Soc.2016

 

The reorientation and hydrogen-bond dynamics of water molecules within the hydration shell of a B-DNA dodecamer, which are of interest for many of its biochemical functions, are investigated via molecular dynamics simulations and an analytic jump model, which provide valuable new molecular level insights into these dynamics. Different sources of heterogeneity in the hydration shell dynamics are determined. First, a pronounced spatial heterogeneity is found at the DNA interface and explained via the jump model by the diversity in local DNA interfacial topographies and DNA− water H-bond interactions. While most of the hydration shell is moderately retarded with respect to the bulk, some water molecules confined in the narrow minor groove exhibit very slow dynamics. An additional source of heterogeneity is found to be caused by the DNA conformational fluctuations, which modulate the water dynamics. The groove widening aids the approach of, and the jump to, a new water H-bond partner. This temporal heterogeneity is especially strong in the minor groove, where groove width fluctuations occur on the same time scale as the water H-bond rearrangements, leading to a strong dynamical disorder. The usual simplifying assumption that hydration shell dynamics is much faster than DNA dynamics is thus not valid; our results show that biomolecular conformational fluctuations are essential to facilitate the water motions and accelerate the hydration dynamics in confined groove sites.