SummaryThe arrangement of β cells within islets of Langerhans is critical for insulin release through the generation of rhythmic activity. A privileged role for individual β cells in orchestrating these responses has long been suspected, but not directly demonstrated. We show here that the β cell population in situ is operationally heterogeneous. Mapping of islet functional architecture revealed the presence of hub cells with pacemaker properties, which remain stable over recording periods of 2 to 3 hr. Using a dual optogenetic/photopharmacological strategy, silencing of hubs abolished coordinated islet responses to glucose, whereas specific stimulation restored communication patterns. Hubs were metabolically adapted and targeted by both pro-inflammatory and glucolipotoxic insults to induce widespread β cell dysfunction. Thus, the islet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.

Synthetic photoswitches have been known for many years, but their usefulness in biology, pharmacology, and medicine has only recently been systematically explored. Over the past decade photopharmacology has grown into a vibrant field. As the photophysical, pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biological targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), soluble proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, we provide an overview of photopharmacology using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. We discuss the scope and limitations of this approach to study biological function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.

Light is a fascinating phenomenon that ties together physics, chemistry, and biology. It is unmatched in its ability to confer information with temporal and spatial precision and has been used to map objects on the scale of tens of nanometers (10(-8) m) to light years (10(16) m). This information, gathered through super-resolution microscopes or space-based telescopes, is ultimately funneled through the human visual system, which is a miracle in itself. It allows us to see the Andromeda galaxy at night, an object that is 2.5 million light years away and very dim, and ski the next day in bright sunlight at an intensity that is 12 orders of magnitude higher. Human vision is only one of many photoreceptive systems that have evolved on earth and are found in all kingdoms of life. These systems rely on molecular photoswitches, such as retinal or tetrapyrrols, which undergo transient bond isomerizations or bond formations upon irradiation. The set of chromophores that have been employed in Nature for this purpose is surprisingly small. Nevertheless, they control a wide variety of biological functions, which have recently been significantly increased through the rapid development of optogenetics. Optogenetics originated as an effort to control neural function with genetically encoded photoreceptors that use abundant chromophores, in particular retinal. It now covers a variety of cellular functions other than excitability and has revolutionized the control of biological pathways in neuroscience and beyond. Chemistry has provided a large repertoire of synthetic photoswitches with highly tunable properties. Like their natural counterparts, these chromophores can be attached to proteins to effectively put them under optical control. This approach has enabled a new type of synthetic photobiology that has gone under various names to distinguish it from optogenetics. We now call it photopharmacology. Here we trace our involvement in this field, starting with the first light-sensitive potassium channel (SPARK) and concluding with our most recent work on photoswitchable fatty acids. Instead of simply providing a historical account of our efforts, we discuss the design criteria that guided our choice of molecules and receptors. As such, we hope to provide a roadmap to success in photopharmacology and make a case as to why synthetic photoswitches, properly designed and made available through well-planned and efficient syntheses, should have a bright future in biology and medicine.

Neurons have ion channels that are directly gated by voltage, ligands and temperature but not by light. Using structure-based design, we have developed a new chemical gate that confers light sensitivity to an ion channel. The gate includes a functional group for selective conjugation to an engineered K + channel, a pore blocker and a photoisomerizable azobenzene. Long-wavelength light drives the azobenzene moiety into its extended trans configuration, allowing the blocker to reach the pore. Short-wavelength light generates the shorter cis configuration, retracting the blocker and allowing conduction. Exogenous expression of these channels in rat hippocampal neurons, followed by chemical modification with the photoswitchable gate, enables different wavelengths of light to switch action potential firing on and off. These synthetic photoisomerizable azobenzene-regulated K + (SPARK) channels allow rapid, precise and reversible control over neuronal firing, with potential applications for dissecting neural circuits and controlling activity downstream from sites of neural damage or degeneration.Natural photoreceptive proteins, such as rhodopsin, have a covalently attached chromophore that directly activates the protein on exposure to light. Several strategies have been used to enable light regulation of proteins that are not intrinsically light sensitive. Light can be used indirectly to activate receptor proteins, for example, by making a ligand available from a caged precursor 1 . Light can also be used to directly photoisomerize a synthetic molecule that is covalently attached to a protein, thereby imposing conformational changes 2,3 . We have combined these ideas by synthesizing a photoisomerizable tether that attaches a specific ligand on a protein near its normal binding site. Photoswitching of the tethered ligand rapidly regulates its ability to reach its binding site, thereby switching the protein on and off without causing unnatural conformational changes. Photoswitchable tethered ligands can be agonists (for example for nicotinic acetylcholine receptors 4 ), antagonists, or other regulators of protein function. We have applied this general design principle to an ion channel, where the chosen ligand is a blocker of the channel's pore. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diffusion kinetics. Recently, the exogenous expression of genes encoding ion channels has been used to influence electrical activity in specific neurons 7-9 , but the onset and reversal of gene expression is slow. A modification of this technique, in which a receptor from one species is introduced into another that lacks a natural ligand, dramatically improves temporal control 10,11 , but this approach still relies on a diffusible ligand whose time course of removal limits reversibility. Finally, photic regulation has been conferred on neurons by introducing a rhodopsin-based signal transduction cascade 12 . This technique requires coordinated exogenous expression of three different genes and produ...

The precise regulation of protein activity is fundamental to life. The allosteric control of an active site by a remote regulatory binding site is a mechanism of regulation found across protein classes, from enzymes to motors to signaling proteins. We describe a general approach for manipulating allosteric control using synthetic optical switches. Our strategy is exemplified by a ligand-gated ion channel of central importance in neuroscience, the ionotropic glutamate receptor (iGluR). Using structure-based design, we have modified its ubiquitous clamshell-type ligand-binding domain to develop a light-activated channel, which we call LiGluR. An agonist is covalently tethered to the protein through an azobenzene moiety, which functions as the optical switch. The agonist is reversibly presented to the binding site upon photoisomerization, initiating clamshell domain closure and concomitant channel gating. Photoswitching occurs on a millisecond timescale, with channel conductances that reflect the photostationary state of the azobenzene at a given wavelength. Our device has potential uses not only in biology but also in bioelectronics and nanotechnology.Many proteins function like molecular machines that undergo mechanical movements in response to input signals. These signals can consist of changes in voltage, membrane tension, temperature or, most commonly, ligand concentration. Ligands provide information about events in the external world or about the energetic or biosynthetic state of the cell. They can be as small as a proton or as large as a whole protein. In allostery, ligand binding induces a structural change of a sensor domain, which propagates to a functional domain of the protein and alters its behavior. Such conformational control can operate over long distances, crossing a membrane or passing from one protein to another in a complex.Reengineering of nanoscopic protein machines to contain artificial control elements would be a major benefit for biology and technology. Optical switches would be especially powerful, as they could be activated remotely with precise temporal and spatial control 1,2 . A simple design Correspondence should be addressed to E.I. (ehud@berkeley.edu) and D.T. (trauner@berkeley.edu).. 5 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Chemical Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript strategy would be to modify a protein by attaching a synthetic ligand whose binding ability could be altered by light. These ligands tethered via an optical switch could function in two ways. They could conditionally block the active site of an enzyme or the pore of a channel without inducing major conformational changes in the protein, or they could reversibly present an agonist to an allosteric binding site and conditionally trigger the normal conformational changes of ac...

We have observed reversible light-induced mechanical switching for individual organic molecules bound to a metal surface. Scanning tunneling microscopy (STM) was used to image the features of individual azobenzene molecules on Au(111) before and after reversibly cycling their mechanical structure between trans and cis states using light. Azobenzene molecules were engineered to increase their surface photomechanical activity by attaching varying numbers of tert-butyl (TB) ligands ("legs") to the azobenzene phenyl rings. STM images show that increasing the number of TB legs "lifts" the azobenzene molecules from the substrate, thereby increasing molecular photomechanical activity by decreasing molecule-surface coupling.

Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements1. We have identified a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics2 in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr (KA) cell3, which extends cilia into the central cerebrospinal fluid containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments4. Genetically silencing KA cells reduced the frequency of spontaneous free swimming, indicating that KA cell activity provides necessary tone for spontaneous forward swimming. KA cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in low vertebrates these cells provide a positive drive to the spinal CPG for spontaneous locomotion.