Laurent Limozin

Nanometric protein-patch arrays on glass and polydimethylsiloxane for cell adhesion studies.

We present a simple cost-effective benchtop protocol to functionalize glass and polydimethylsiloxane (PDMS) with nanometric protein patches for cell adhesion studies. Evaporation masks, covering macroscopic areas on glass, were made using improved strategies for self-assembly of colloidal microbeads which then served as templates for creating the protein patch arrays via the intermediate steps of organo-aminosilane deposition and polyethylene-glycol grafting. The diameter of the patches could be varied down to about 80 nm. The glass substrates were used for advanced optical imaging of T-lymphocytes to explore adhesion by reflection interference contrast microscopy and the possible colocalization of T-cell receptor microclusters and the activating protein patches by total internal reflection fluorescence microscopy. The selectively functionalized glass could also serve as template for transferring the protein nanopatches to the surface of a soft elastomer. We demonstrated successful reverse contact printing onto the surface of thin layers of PDMS with stiffness ranging from 30 KPa to 3 MPa.

Coupling artificial actin cortices to biofunctionalized lipid monolayers.

We report the assembly of protein supramolecular structures at an air-water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin-biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.

Size-Tunable Organic Nanodot Arrays: A Versatile Platform for Manipulating and Imaging Cells

Arrays of protein nanodots with dot-size tuned independently of spacing (e.g., ∼100 to 600 nm diameter for 900 nm spacing) are fabricated. The mechanism of size control is demonstrated, by numerical simulations, to arise from shadow effects during deposition of a sacrificial metal mask. We functionalize the nanodots with antibodies and embed them in a polymer-cushion or in lipid-bilayers or transfer them to soft elastomers. Their ability to influence cell architecture and local membrane organization is demonstrated in T-lymphocytes, using reflection interference contrast and total internal reflection fluorescence microscopy.

Printing Functional Protein Nanodots on Soft Elastomers: From Transfer Mechanism to Cell Mechanosensing

Living cells sense the physical and chemical nature of their micro/nano environment with exquisite sensitivity. In this context, there is a growing need to functionalize soft materials with micro/nanoscale biochemical patterns for applications in mechanobiology. This, however, is still an engineering challenge. Here a new method is proposed, where submicronic protein-patterns are first formed on glass and are then printed on to an elastomer. The degree of transfer is shown to be governed mainly by hydrophobic interactions and to be influenced by grafting an appropriate fluorophore onto the core protein of interest. The transfer mechanism is probed by measuring the forces of adhesion/cohesion using atomic force microscopy. The transfer of functional arrays of dots with size down to about 400 nm, on elastomers with stiffness ranging from 3 kPa to 7 MPa, is demonstrated. Pilot studies on adhesion of T lymphocytes on such soft patterned substrates are reported.

T Cells on Engineered Substrates: The Impact of TCR Clustering Is Enhanced by LFA-1 Engagement

We created APC-mimetic synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit antibodies directed against the TCR-complex in the form of a patterned array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. We show that for T cell adhesion mediated by T cell receptor (TCR) alone, in the patterned, but not in the corresponding homogeneous controls, the TCR, ZAP-70 and actin are present in the form of clusters or patches that co-localize with the ligand-dots. However, global cell scale parameters like cell area and actin distribution are only weakly impacted by ligand clustering. In presence of ICAM-1 - the ligand of the T cell integrin LFA-1 - on the SLB, the TCR is still clustered due to the patterning of its ligands, but now global parameters are also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, whereas the cell area increases significantly. These observations taken together point to a possible pivotal role for LFA-1 in amplifying the effect of TCR-clustering. No such effect is evident for co-engagement of CD28, affected via its ligand B7.2. Unlike on ICAM-1, on B7.2 cell spreading and actin organization are similar for homogeneous and patterned substrates. However, TCR and ZAP-70 clusters are still formed in the patterned case. These results indicate complementary role for LFA-1 and CD28 in the regulation and putative coupling of TCR micro-clusters to actin. The engineered substrates presented here clearly have the potential to act as platform for fundamental research in immune cell biology, as well as translational analyses in immunotherapy, for example to screen molecules for their role in T cell adhesion/activation.