Multiwell Combinatorial Hydrogel Array for High-Throughput Analysis of Cell-ECM Interactions.

Biophysical cues in the extracellular matrix (ECM) regulate cell behavior in a complex, nonlinear, and interdependent manner. To quantify these important regulatory relationships and gain a comprehensive understanding of mechanotransduction, there is a need for high-throughput matrix platforms that enable parallel culture and analysis of cells in various matrix conditions. Here we describe a multiwell hyaluronic acid (HA) platform in which cells are cultured on combinatorial arrays of hydrogels spanning a range of elasticities and adhesivities. Our strategy utilizes orthogonal photopatterning of stiffness and adhesivity gradients, with the stiffness gradient implemented by a programmable light illumination system. The resulting platform allows individual treatment and analysis of each matrix environment while eliminating contributions of haptotaxis and durotaxis. In human mesenchymal stem cells, our platform recapitulates expected relationships between matrix stiffness, adhesivity, and cell mechanosensing. We further applied the platform to show that as integrin ligand density falls, cell adhesion and migration depend more strongly on CD44-mediated interactions with the HA backbone. We anticipate that our system could bear great value for mechanistic discovery and screening where matrix mechanics and adhesivity are expected to influence phenotype.

Getting the big picture of cell-matrix interactions: High-throughput biomaterial platforms and systems-level measurements.

Living cells interact with the extracellular matrix (ECM) in a complex and reciprocal manner. Much has been learned over the past few decades about cell-ECM interactions from targeted studies in which a specific matrix parameter (e.g. stiffness, adhesivity) has been varied across a few discrete values, or in which the level or activity of a protein is controlled in an isolated fashion. As the field moves forward, there is growing interest in addressing cell-matrix interactions from a systems perspective, which has spurred a new generation of matrix platforms capable of interrogating multiple ECM inputs in a combinatorial and parallelized fashion. Efforts are also actively underway to integrate specialized, synthetic ECM platforms with global measures of cell behaviors, including at the transcriptomic, proteomic and epigenomic levels. Here we review recent advances in both areas. We describe how new combinatorial ECM technologies are revealing unexpected crosstalk and nonlinearity in the relationship between cell phenotype and matrix properties. Similarly, efforts to integrate "omics" measurements with synthetic ECM platforms are illuminating how ECM properties can control cell biology in surprising and functionally important ways. We expect that advances in both areas will deepen the field's understanding of cell-ECM interactions and offer valuable insight into the design of biomaterials for specific biomedical applications.

Structural Regulation of a Neurofilament-Inspired Intrinsically Disordered Protein Brush by Multisite Phosphorylation.

Intrinsically disordered proteins (IDPs) play central roles in numerous cellular processes. While IDP structure and function are often regulated by multisite phosphorylation, the biophysical mechanisms linking these post-translational modifications to IDP structure remain elusive. For example, the intrinsically disordered C-terminal sidearm domain of the neurofilament heavy subunit (NFH-SA) forms a dense brush along axonal NF backbones and is subject to extensive serine phosphorylation. Yet, biophysical insight into the relationship between phosphorylation and structure has been limited by the lack of paradigms in which NF brush conformational responses can be measured in the setting of controlled phosphorylation. Here, we approach this question by immobilizing a recombinant NFH-SA (rNFH-SA) as IDP brushes onto glass, and controllably phosphorylating the sequence in situ with mitogen-activated protein kinase 1 (ERK2) preactivated by mitogen-activated protein kinase kinase (MKK). We then monitor brush height changes using atomic force microscopy, which shows that phosphorylation induces significant brush swelling to an extent that strongly depends upon pH and ionic strength, consistent with a mechanism in which phosphorylation regulates brush structure through local electrostatic interactions. Further consistent with this mechanism, the phosphorylated rNFH-SA brush may be dramatically condensed with micromolar concentrations of divalent cations. Phosphorylation-induced height changes are qualitatively reversible via alkaline phosphatase-mediated dephosphorylation. Our study demonstrates that multisite phosphorylation controls NFH-SA structure through modulation of chain electrostatics and points to a general strategy for engineering IDP-based interfaces that can be reversibly and dynamically modulated by enzymes.

Mechanistic Investigation and Multiplexing of Liposome-Assisted Metabolic Glycan Labeling.

Metabolic labeling of glycans with bioorthogonal reporters has been widely used for glycan imaging and glycoproteomic profiling. One of the intrinsic limitations of metabolic glycan labeling is the lack of cell-type selectivity. The recently developed liposome-assisted bioorthogonal reporter (LABOR) strategy provides a promising means to overcome this limitation, but the mechanism of LABOR has not been investigated in detail. In this work, we performed a mechanistic study on LABOR and explored its multiplexing capability. Our studies support an endocytosis-salvage mechanism. The ligand-targeted liposomes encapsulating azidosugars are internalized into the endosome via the receptor-mediated endocytosis. Unlike the conventional drug delivery, LABOR does not rely on the endosomal escape pathways. Rather, the liposomes are allowed to enter the lysosome, inside which the azidosugars are released from the liposomes. The released azidosugars then intercept the salvage pathways of monosaccharides and get transported into the cytosol by lysosomal sugar transporters. Based on this mechanism, we expanded the scope of LABOR by evaluating a series of ligand-receptor pairs for targeting sialoglycans in various cell types. Different ligand types including small molecules, antibodies, aptamers, and peptides could be easily implemented into LABOR. Finally, we demonstrated that the sialoglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters by performing a multiplexed LABOR labeling.

Targeted imaging and proteomic analysis of tumor-associated glycans in living animals.

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor-associated glycans by using ligand-targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer-cell-specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue-specific manner. Subsequently, a copper-free click reaction was performed in vivo to chemoselectively conjugate the azides with a near-infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.