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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. We begin with a summary of live-imaging strategies, including recent advances that push the limits of spatiotemporal resolution and specimen size. We then describe recent efforts that employ live imaging to uncover morphogenetic mechanisms. We conclude by discussing how information collected from live imaging can be enhanced by genetically-encoded biosensors and spatiotemporal perturbation techniques to determine the dynamics of patterning of developmental signals and their importance for guiding morphogenesis.

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. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology.

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. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags opens the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs.HighlightspYtags report on signaling of user-defined RTKs in living cellsEGFR signaling dynamics depend on ligand identity and dimer strengthrthogonal pYtags enable reporter multiplexingpYtags can report on signaling of endogenously expressed RTKs

This work introduces light-activated bioorthogonal non-canonical 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. Upon directed light exposure, the uncaged amino acid is available for local translation, enabling downstream proteomic interrogation via bioorthogonal conjugation. Exploiting the reactive azide moiety present on Aha’s amino acid side chain, we demonstrate that newly synthesized proteins can be purified for quantitative proteomics or visualized in synthetic tissues with a new level of spatiotemporal control. Shedding light on when and where proteins are translated within living samples, we anticipate that laBONCAT will aid in understanding the progression of complex protein-related disorders.

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. Here we present a strategy for bioorthogonal hydrogel formation and modification that does not rely on propagating free radicals, proceeding through oxime ligation moderated by a photocaged alkoxyamine. Upon mild near UV light exposure, the photocage is cleaved, liberating the alkoxyamine and permitting localized condensation with an aldehyde. Multi-arm crosslinkers, functionalized with either benzaldehydes or photocaged alkoxyamines, formed oxime-based hydrogels within minutes of light exposure in the presence of live cells. Polymerization rates and final mechanical properties of these gels could be systematically tuned by varying crosslinker concentrations, light intensity, aniline catalyst equivalents, and pH. Moreover, hydrogel geometry and final mechanical properties were controlled by the location and extent of UV exposure, respectively. Photomediated oxime ligation was then translated to the biochemical modification of hydrogels, where full-length proteins containing photocaged alkoxyamines were immobilized in user-defined regions exposed to UV light. The programmability afforded by photomediated oxime ligation can recapitulate dynamically anisotropic mechanical and biochemical aspects of the native extracellular matrix. Consequently, photopolymerized oxime-based hydrogels are expected to enable an enhanced understanding of cell-matrix interactions by serving as improved 4D cell culture platforms.

SUMMARY 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. We find that soft microenvironments attenuate Erk signaling, both at steady state and in response to epidermal growth factor (EGF) stimulation. Optogenetic manipulation at multiple signaling nodes reveals that intracellular signal transmission is largely unaffected by substratum stiffness. Instead, we find that soft microenvironments decrease EGF receptor (EGFR) expression and alter the amount and spatial distribution of EGF binding at cell membranes. Our data demonstrate that the mechanical microenvironment tunes Erk signaling dynamics via receptor-ligand interactions, underscoring how multiple microenvironmental signals are jointly processed through a highly conserved pathway that regulates tissue development, homeostasis, and disease progression., Graphical Abstract, In brief Farahani et al. investigate how soft microenvironments limit signal transmission through the EGFR/Erk pathway. Optogenetic stimulation reveals that the intracellular portion of the EGFR/Erk pathway remains signaling competent regardless of substratum stiffness. Rather, soft microenvironments limit Erk signaling by decreasing interactions between EGFR and its cognate ligand.

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. Here, a new strategy is reported to achieve both high bulk mechanical strength and excellent surface nonfouling properties, which are typically contradictory, in one material. This is realized through a nonfouling polymeric elastomer based on zwitterionic polycarboxybetaine derivatives. By hiding both charged moieties of the zwitterionic compounds with hydrocarbon ester and tertiary amine groups, the bulk polymer itself is elastomeric and hydrophobic while its superhydrophilic surface properties are restored upon hydrolysis. This coating-free nonfouling elastomer is a highly promising biomaterial for biomedical and engineering applications.