Nanometric Protein-Patch Arrays on Glass and Polydimethylsiloxane for Cell Adhesion Studies

Pi, Fuwei; Dillard, Pierre; Limozin, Laurent; Charrier, Anne; Sengupta, Kheya

doi:10.1021/nl401696m

Cited by 17 publications

(29 citation statements)

References 42 publications

(72 reference statements)

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“…A glass coverslide as prepared above was set at an angle and a pre-calibrated volume of the colloidal suspension was allowed to spread on the slide. Slow evaporation at ambient conditions coupled with strategic change of the angle results in uniform and large area coating with a monolayer of colloidal beads (see also Pi et al 39 ).…”

Section: Nano-patterning Of Substratesmentioning

confidence: 99%

“…Colloidal bead lithography has been proposed as an alternative, and has been used either with gold chemistry 38 or with an all-organic approach 39 . The latter metal-free approach is compatible with TIRF and RICM imaging 39,40 , and is the technique of choice here. Our implementation has the additional advantage of offering variable pitch and dot-size.…”

Section: Introductionmentioning

confidence: 99%

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Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization

Dillard

Lellouch

et al. 2016

Integr. Biol.

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We investigate the adhesion and molecular organization of the plasma membrane of T lymphocytes interacting with a surrogate antigen presenting cell comprising glass supported ordered arrays of antibody (α-CD3) nano-dots dispersed in a non-adhesive matrix of polyethylene glycol (PEG). The local membrane adhesion and topography, as well as the distribution of the T cell receptors (TCRs) and the kinase ZAP-70, are influenced by dot-geometry, whereas the cell spreading area is determined by the overall average density of the ligands rather than specific characteristics of the dots. TCR clusters are recruited preferentially to the nano-dots and the TCR cluster size distribution has a weak dot-size dependence. On the patterns, the clusters are larger, more numerous, and more enriched in TCRs, as compared to the homogeneously distributed ligands at comparable concentrations. These observations support the idea that non-ligated TCRs residing in the non-adhered parts of the proximal membrane are able to diffuse and enrich the existing clusters at the ligand dots. However, long distance transport is impaired and cluster centralization in the form of a central supramolecular cluster (cSMAC) is not observed. Time-lapse imaging of early cell-surface contacts indicates that the ZAP-70 microclusters are directly recruited to the site of the antibody dots and this process is concomitant with membrane adhesion. These results together point to a complex interplay of adhesion, molecular organization and activation in response to spatially modulated stimulation.

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Section: Nano-patterning Of Substratesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization

Dillard

Lellouch

et al. 2016

Integr. Biol.

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“…At the same time, it should be pointed out that this is a legitimate approach since anti-CD3 is known to elicit the same signaling pathways as pMHC ligation ( 35 ) and the CD3 domain mediates T-cell mechanotransduction ( 36 ). We use a combination of colloidal bead lithography and metal sputtering to fabricate sub-micron sized ligand clusters on glass ( 37 , 38 ). These clusters are then surrounded by supported lipid bilayers, optionally functionalized with ICAM-1 or B7.2 2 to form substrates that mimic APCs.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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“…There has been also a constant effort to control more and more precisely the physical environment of the ligands in order to mimic one or more properties of cellular membranes and cellular material ( Figure 2A); this approach is often guided by the idea of mimicking as closely as possible the observed properties of APCs (Hivroz and Saitakis 2015). Metal barriers have been used to confine mobile ligands bound to a supported bilayer (Mossman 2005) mimicking corralling effects of the APC's cytoskeleton (Comrie 2015); ligands have been presented on islands of different spacing and size varying from micron (Doh and Irvine 2006), to sub-micron (Pi 2013(Pi , 2015 and nanometer (Delcassian 2013) scales. This helps to assess the role of different length scales on receptor behaviour and to reproduce various spatial organisations of receptors on the APC ( Figure 2B).…”

Section: Reductionist and Bio-mimetic Engineering Approachesmentioning

confidence: 99%

Membrane Organization and Physical Regulation of Lymphocyte Antigen Receptors: A Biophysicist’s Perspective

Limozin

Puech

2019

J Membrane Biol

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Receptors at the membrane of immune cells are the central players of innate and adaptative immunity, providing effective defence mechanisms against pathogens or cancer cells. Their function is intimately linked to their position at and within the membrane which provides accessibility, mobility as well as membrane proximal cytoskeleton anchoring, all of these elements playing important roles in the final function and links to cellular actions. Understanding how immune cells integrate the specific signals received at their membrane to take a decision remains an immense challenge and a very active field of fundamental and applied research. Recent progress in imaging and micromanipulation techniques have led to an unprecedented refinement in the description of molecular structures and supramolecular assemblies at the immune cell membrane, and provided a glimpse into their dynamics and regulation by force. Several key elements have been scrutinized such as the roles of geometry (relative sizes of molecules), lateral organisation, motion in the membrane of the receptors, but also physical cues such as forces, mediated by cellular substrates of different rigidities or applied by the cell itself, in conjunction with its partner cell. We review here these recent discoveries associated with a description of the biophysical methods used. While a conclusive picture integrating all of these components is still lacking, mainly due to the implication of diverse and different mechanisms and spatiotemporal scales involved, the amount of quantitative data available opens the way for physical modelling and numerical simulations and new avenues for experimental research.

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Nanometric Protein-Patch Arrays on Glass and Polydimethylsiloxane for Cell Adhesion Studies

Cited by 17 publications

References 42 publications

Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization

Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization

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

Membrane Organization and Physical Regulation of Lymphocyte Antigen Receptors: A Biophysicist’s Perspective

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