CD44 and Ezrin restrict EGF receptor mobility to generate a novel spatial arrangement of cytoskeletal signaling modules driving bleb-based migration.

Cells under high confinement form highly polarized hydrostatic pressure-driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high-resolution time-lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb.

Large-scale control over collective cell migration using light-controlled epidermal growth factor receptors.

Receptor tyrosine kinases (RTKs) are thought to play key roles in coordinating cell movement at single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggested these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled EGF receptor (OptoEGFR) can be deployed in epithelial cell lines for precise, programmable control of long-range tissue movements.

pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells.

Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy.

Substratum stiffness regulates Erk signaling dynamics through receptor-level control.

The EGFR/Erk pathway is triggered by extracellular ligand stimulation, leading to stimulus-dependent dynamics of pathway activity. Although mechanical properties of the microenvironment also affect Erk activity, their effects on Erk signaling dynamics are poorly understood. Here, we characterize how the stiffness of the underlying substratum affects Erk signaling dynamics in mammary epithelial cells.

Revealing epithelial morphogenetic mechanisms through live imaging.

Epithelial morphogenesis is guided by mechanical forces and biochemical signals that vary spatiotemporally. As many morphogenetic events are driven by rapid cellular processes, understanding morphogenesis requires monitoring development in real time. Here, we discuss how live-imaging approaches can help identify morphogenetic mechanisms otherwise missed in static snapshots of development.

Signaling, Deconstructed: Using Optogenetics to Dissect and Direct Information Flow in Biological Systems.

Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision.

Purification, kinetic characterization, and site-directed mutagenesis of RFAP Synthase Produced in .

Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (HMPT) as a one-carbon carrier instead of tetrahydrofolate. In HMPT biosynthesis, β-ribofuranosylaminobenzene 5'-phosphate (RFAP) synthase catalyzes the production of RFAP, CO, and pyrophosphate from -aminobenzoic acid (ABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from was produced in , and site-directed mutagenesis was used to alter arginine 26 (R26) and aspartic acid 19 (D19), located in a conserved sequence of amino acids resembling the ABA binding site of dihydropteroate synthase.

Light-Activated Proteomic Labeling via Photocaged Bioorthogonal Non-Canonical Amino Acids.

This work introduces light-activated bioorthogonal noncanonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging l-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compound remains stable for many hours in culture, but can be photochemically liberated rapidly and on demand with spatial control.

Photomediated oxime ligation as a bioorthogonal tool for spatiotemporally-controlled hydrogel formation and modification.

Click chemistry has proved a valuable tool in biocompatible hydrogel formation for 3D cell culture, owing to its bioorthogonal nature and high efficiency under physiological conditions. While traditional click reactions can be readily employed to create uniform functional materials about living cells, their spontaneity prohibits spatiotemporal control of material properties, thereby limiting their utility in recapitulating the dynamic heterogeneity characteristic of the in vivo microenvironment. Photopolymerization-based techniques gain this desired level of 4D programmability, but often at the expense of introducing propagating free radicals that are prone to non-specific reactions with biological systems.

A Coating-Free Nonfouling Polymeric Elastomer.

Medical devices face nonspecific biofouling from proteins, cells, and microorganisms, which significantly contributes to complications and device failure. Imparting these devices with nonfouling capabilities remains a major challenge, particularly for those made from elastomeric polymers. Current strategies, including surface coating and copolymerization/physical blending, necessitate compromise among nonfouling properties, durability, and mechanical strength.