Quantifying postsynaptic receptor dynamics: insights into synaptic function.

The molecular composition of presynaptic and postsynaptic neuronal terminals is dynamic, and yet long-term stabilizations in postsynaptic responses are necessary for synaptic development and long-term plasticity. The need to reconcile these concepts is further complicated by learning- and memory-related plastic changes in the molecular make-up of synapses. Advances in single-particle tracking mean that we can now quantify the number and diffusive properties of specific synaptic molecules, while statistical thermodynamics provides a framework to analyse these molecular fluctuations. In this Review, we discuss the use of these approaches to gain quantitative descriptions of the processes underlying the turnover, long-term stability and plasticity of postsynaptic receptors and show how these can help us to understand the balance between local molecular turnover and synaptic structural identity and integrity.

Identification of a stereotypic molecular arrangement of endogenous glycine receptors at spinal cord synapses.

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far, the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy. We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs µm-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains, and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

Advancing cell instructive biomaterials through increased understanding of cell receptor spacing and material surface functionalization.

Regenerative medicine is aimed at restoring normal tissue function and can benefit from the application of tissue engineering and nano-therapeutics. In order for regenerative therapies to be effective, the spatiotemporal integration of tissue engineered scaffolds by the native tissue, and the binding/release of therapeutic payloads by nano-materials, must be tightly controlled at the nanoscale in order to direct cell fate. However, due to a lack of insight regarding cell-material interactions at the nanoscale and subsequent downstream signaling, the clinical translation of many regenerative therapies is limited due to poor material integration, rapid clearance and complications such as graft-versus-host disease. This review paper is intended to outline our current understanding of cell-material interactions with the aim of highlighting potential areas for knowledge advancement or application in the field of regenerative medicine. This is achieved by reviewing the nanoscale organization of key cell surface receptors, the current techniques used to control the presentation of cell-interactive molecules on material surfaces, as well as the most advanced techniques for characterizing the interactions that occur between cell surface receptors and materials intended for use in regenerative medicine.

IL-1ß mediated nanoscale surface clustering of integrin α5ß1 regulates the adhesion of mesenchymal stem cells.

Clinical use of human mesenchymal stem cells (hMSCs) is limited due to their rapid clearance, reducing their therapeutic efficacy. The inflammatory cytokine IL-1ß activates hMSCs and is known to enhance their engraftment. Consequently, understanding the molecular mechanism of this inflammation-triggered adhesion is of great clinical interest to improving hMSC retention at sites of tissue damage. Integrins are cell-matrix adhesion receptors, and clustering of integrins at the nanoscale underlies cell adhesion. Here, we found that IL-1ß enhances adhesion of hMSCs via increased focal adhesion contacts in an α5ß1 integrin-specific manner. Further, through quantitative super-resolution imaging we elucidated that IL-1ß specifically increases nanoscale integrin α5ß1 availability and clustering at the plasma membrane, whilst conserving cluster area. Taken together, these results demonstrate that hMSC adhesion via IL-1ß stimulation is partly regulated through integrin α5ß1 spatial organization at the cell surface. These results provide new insight into integrin clustering in inflammation and provide a rational basis for design of therapies directed at improving hMSC engraftment.

Nanoscale Molecular Quantification of Stem Cell-Hydrogel Interactions.

A common approach to tailoring synthetic hydrogels for regenerative medicine applications involves incorporating RGD cell adhesion peptides, yet assessing the cellular response to engineered microenvironments at the nanoscale remains challenging. To date, no study has demonstrated how RGD concentration in hydrogels affects the presentation of individual cell surface receptors. Here we studied the interaction between human mesenchymal stem cells (hMSCs) and RGD-functionalized poly(ethylene glycol) hydrogels, by correlating macro- and nanoscale single-cell interfacial quantification techniques. We quantified RGD unbinding forces on a synthetic hydrogel using single cell atomic force spectroscopy, revealing that short-term binding of hMSCs was sensitive to RGD concentration. We also performed direct stochastic optical reconstruction microscopy (dSTORM) to quantify the molecular interactions between integrin α5ß1 and a biomaterial, unexpectedly revealing that increased integrin clustering at the hydrogel-cell interface correlated with fewer available RGD binding sites. Our complementary, quantitative approach uncovered mechanistic insights into specific stem cell-hydrogel interactions, where dSTORM provides nanoscale sensitivity to RGD-dependent differences in cell surface localization of integrin α5ß1. Our findings reveal that it is possible to precisely determine how peptide-functionalized hydrogels interact with cells at the molecular scale, thus providing a basis to fine-tune the spatial presentation of bioactive ligands.

Spatiotemporal quantification of acoustic cell patterning using Voronoï tessellation.

Acoustic patterning using ultrasound standing waves has recently emerged as a potent biotechnology enabling the remote generation of ordered cell systems. This capability has opened up exciting opportunities, for example, in guiding the development of organoid cultures or the organization of complex tissues. The success of these studies is often contingent on the formation of tightly-packed and uniform cell arrays; however, a number of factors can act to disrupt or prevent acoustic patterning. Yet, to the best of our knowledge, there has been no comprehensive assessment of the quality of acoustically-patterned cell populations. In this report we use a mathematical approach, known as Voronoï tessellation, to generate a series of metrics that can be used to measure the effect of cell concentration, pressure amplitude, ultrasound frequency and biomaterial viscosity upon the quality of acoustically-patterned cell systems. Moreover, we extend this approach towards the characterization of spatiotemporal processes, namely, the acoustic patterning of cell suspensions and the migration of patterned, adherent cell clusters. This strategy is simple, unbiased and highly informative, and we anticipate that the methods described here will provide a systematic framework for all stages of acoustic patterning, including the robust quality control of devices, statistical comparison of patterning conditions, the quantitative exploration of parameter limits and the ability to track patterned tissue formation over time.

Inhibitory Receptor Diffusion Dynamics.

The dynamic modulation of receptor diffusion-trapping at inhibitory synapses is crucial to synaptic transmission, stability, and plasticity. In this review article, we will outline the progression of understanding of receptor diffusion dynamics at the plasma membrane. We will discuss how regulation of reversible trapping of receptor-scaffold interactions in combination with theoretical modeling approaches can be used to quantify these chemical interactions at the postsynapse of living cells.

Engineering Extracellular Vesicles with the Tools of Enzyme Prodrug Therapy.

Extracellular vesicles (EVs) have recently gained significant attention as important mediators of intercellular communication, potential drug carriers, and disease biomarkers. These natural cell-derived nanoparticles are postulated to be biocompatible, stable under physiological conditions, and to show reduced immunogenicity as compared to other synthetic nanoparticles. Although initial clinical trials are ongoing, the use of EVs for therapeutic applications may be limited due to undesired off-target activity and potential "dilution effects" upon systemic administration which may affect their ability to reach their target tissues. To fully exploit their therapeutic potential, EVs are embedded into implantable biomaterials designed to achieve local delivery of therapeutics taking advantage of enzyme prodrug therapy (EPT). In this first application of EVs for an EPT approach, EVs are used as smart carriers for stabilizing enzymes in a hydrogel for local controlled conversion of benign prodrugs to active antiinflammatory compounds. It is shown that the natural EVs' antiinflammatory potential is comparable or superior to synthetic carriers, in particular upon repeated long-term incubations and in different macrophage models of inflammation. Moreover, density-dependent color scanning electron microscopy imaging of EVs in a hydrogel is presented herein, an impactful tool for further understanding EVs in biological settings.

Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate.

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1-4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

Self-Assembled 2D Free-Standing Janus Nanosheets with Single-Layer Thickness.

We report the thermodynamically controlled growth of solution-processable and free-standing nanosheets via peptide assembly in two dimensions. By taking advantage of self-sorting between peptide ß-strands and hydrocarbon chains, we have demonstrated the formation of Janus 2D structures with single-layer thickness, which enable a predetermined surface heterofunctionalization. A controlled 2D-to-1D morphological transition was achieved by subtly adjusting the intermolecular forces. These nanosheets provide an ideal substrate for the engineering of guest components (e.g., proteins and nanoparticles), where enhanced enzyme activity was observed. We anticipate that sequence-specific programmed peptides will offer promise as design elements for 2D assemblies with face-selective functionalization.

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information.

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin ß1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the ß1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor ß1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Quantitative volumetric Raman imaging of three dimensional cell cultures.

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

Controlled Sub-Nanometer Epitope Spacing in a Three-Dimensional Self-Assembled Peptide Hydrogel.

Cells in the body use a variety of mechanisms to ensure the specificity and efficacy of signal transduction. One way that this is achieved is through tight spatial control over the position of different proteins, signaling sequences, and biomolecules within and around cells. For instance, the extracellular matrix protein fibronectin presents RGDS and PHSRN sequences that synergistically bind the α5ß1 integrin when separated by 3.2 nm but are unable to bind when this distance is >5.5 nm.1 Building biomaterials to controllably space different epitopes with subnanometer accuracy in a three-dimensional (3D) hydrogel is challenging. Here, we synthesized peptides that self-assemble into nanofiber hydrogels utilizing the ß-sheet motif, which has a known regular spacing along the peptide backbone. By modifying specific locations along the peptide, we are able to controllably space different epitopes with subnanometer accuracy at distances from 0.7 nm to over 6 nm, which is within the size range of many protein clusters. Endothelial cells encapsulated within hydrogels displaying RGDS and PHSRN in the native 3.2 nm spacing showed a significant upregulation in the expression of the alpha 5 integrin subunit compared to those in hydrogels with a 6.2 nm spacing, demonstrating the physiological relevance of the spacing. Furthermore, after 24 h the cells in hydrogels with the 3.2 nm spacing appeared to be more spread with increased staining for the α5ß1 integrin. This self-assembling peptide system can controllably space multiple epitopes with subnanometer accuracy, demonstrating an exciting platform to study the effects of ligand density and location on cells within a synthetic 3D environment.