μManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, μManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced μManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging.

Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton1: protrusion of the leading edge requires assembly of a network of actin filaments2, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly3, it is not clear how activity of these locally acting proteins could be coordinated over the whole-cell distance scale. Here we show that nonmuscle myosin II plays a direct role in actin network disassembly in crawling cells. In moving fish keratocytes, myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actomyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.

Hydrogen bonds play major roles in biological structure and function. Nonetheless, hydrogen-bonded protons are not typically observed by X-ray crystallography, and most structural studies provide limited insight into the conformational plasticity of individual hydrogen bonds or the dynamical coupling present within hydrogen bond networks. We report the NMR detection of the hydrogen-bonded protons donated by Tyr-42 and Glu-46 to the chromophore oxygen in the active site of the bacterial photoreceptor, photoactive yellow protein (PYP). We have used the NMR resonances for these hydrogen bonds to probe their conformational properties and ability to rearrange in response to nearby electronic perturbation. The detection of geometric isotope effects transmitted between the Tyr-42 and Glu-46 hydrogen bonds provides strong evidence for robust coupling of their equilibrium conformations. Incorporation of a modified chromophore containing an electron-withdrawing cyano group to delocalize negative charge from the chromophore oxygen, analogous to the electronic rearrangement detected upon photon absorption, results in a lengthening of the Tyr-42 and Glu-46 hydrogen bonds and an attenuated hydrogen bond coupling. The results herein elucidate fundamental properties of hydrogen bonds within the complex environment of a protein interior. Furthermore, the robust conformational coupling and plasticity of hydrogen bonds observed in the PYP active site may facilitate the larger-scale dynamical coupling and signal transduction inherent to the biological function that PYP has evolved to carry out and may provide a model for other coupled dynamic systems.charge delocalization ͉ hydrogen bond coupling ͉ protein structure ͉ signal transduction

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