Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

Two clichés of science journalism have now played out around the ENCODE project. ENCODE’s publicity first presented a misleading "all the textbooks are wrong" narrative about noncoding human DNA. Now several critiques of ENCODE’s narrative have been published, and one was so vitriolic that it fueled "undignified academic squabble" stories that focused on tone more than substance. Neither story line does justice to our actual understanding of genomes, to ENCODE’s results, or to the role of big science in biology.

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

Object detection and classification are key tasks in computer vision that can facilitate high-throughput image analysis of microscopy data. We present a set of local image descriptors for three-dimensional (3D) microscopy datasets inspired by the well-known Haar wavelet framework. We add orientation, illumination and scale information by assuming that the neighborhood surrounding points of interests in the image can be described with ellipsoids, and we increase discriminative power by incorporating edge and shape information into the features. The calculation of the local image descriptors is implemented in a Graphics Processing Unit (GPU) in order to reduce computation time to 1 millisecond per object of interest. We present results for cell division detection in 3D time-lapse fluorescence microscopy with 97.6% accuracy.

Understanding the development of complex multicellular organisms as a function of the underlying cell behavior is one of the most fundamental goals of developmental biology. The ability to quantitatively follow cell dynamics in entire developing embryos is an indispensable step towards such a system-level understanding. In recent years, light-sheet fluorescence microscopy has emerged as a particularly promising strategy for recording the in vivo data required to realize this goal. Using light-sheet fluorescence microscopy, entire complex organisms can be rapidly imaged in three dimensions at sub-cellular resolution, achieving high temporal sampling and excellent signal-to-noise ratio without damaging the living specimen or bleaching fluorescent markers. The resulting datasets allow following individual cells in vertebrate and higher invertebrate embryos over up to several days of development. However, the complexity and size of these multi-terabyte recordings typically preclude comprehensive manual analyses. Thus, new computational approaches are required to automatically segment cell morphologies, accurately track cell identities and systematically analyze cell behavior throughout embryonic development. We review current efforts in light-sheet microscopy and bioimage informatics towards this goal, and argue that comprehensive cell lineage reconstructions are finally within reach for many key model organisms, including fruit fly, zebrafish and mouse.

Objective: While the contribution of α-Synuclein to neurodegeneration in Parkinson’s disease is well accepted, the putative impact of its close homologue, β-Synuclein, is enigmatic. β-Synuclein is widely expressed throughout the central nervous system as is α-Synuclein, but the physiological functions of both proteins remain unknown. Recent findings supported the view that β-Synuclein can act as an ameliorating regulator of α-Synuclein-induced neurotoxicity, having neuroprotective rather than neurodegenerative capabilities, and being non-aggregating due to absence of most part of the aggregation-promoting NAC domain. However, a mutation of β-Synuclein linked to dementia with Lewy bodies rendered the protein neurotoxic in transgenic mice and fibrillation of β-Synuclein has been demonstrated in vitro. Methods / Results: Supporting the hypothesis that β-Synuclein can act as a neurodegeneration-inducing factor we now demonstrate that wild-type β-Synuclein is neurotoxic for cultured primary neurons. Furthermore, β-Synuclein formed proteinase K resistant aggregates in dopaminergic neurons in vivo, leading to pronounced and progressive neurodegeneration in rats. Expression of β-Synuclein caused mitochondrial fragmentation, but this fragmentation did not render mitochondria non-functional in terms of ion handling and respiration even in late stages of neurodegeneration. A comparison of the neurodegenerative effects induced by α-, β-, and γ-Synuclein revealed that β-Synuclein was eventually as neurotoxic as α-Synuclein for nigral dopaminergic neurons, while γ-Synuclein proved to be non-toxic and had very low aggregation propensity. Interpretation: Our results suggest that the role of β-Synuclein as a putative modulator of neuropathology in aggregopathies like Parkinson’s disease and dementia with Lewy bodies needs to be revisited. ANN NEUROL 2013. © 2013 American Neurological Association.

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

Developmental signals such as Wnts are often presented to cells in an oriented manner. To examine the consequences of local Wnt signaling, we immobilized Wnt proteins on beads and introduced them to embryonic stem cells in culture. At the single-cell level, the Wnt-bead induced asymmetric distribution of Wnt-β-catenin signaling components, oriented the plane of mitotic division, and directed asymmetric inheritance of centrosomes. Before cytokinesis was completed, the Wnt-proximal daughter cell expressed high levels of nuclear β-catenin and pluripotency genes, whereas the distal daughter cell acquired hallmarks of differentiation. We suggest that a spatially restricted Wnt signal induces an oriented cell division that generates distinct cell fates at predictable positions relative to the Wnt source.

Automatic 3D digital reconstruction (tracing) of neurons embedded in noisy microscopic images is challenging, especially when the cell morphology is complex.

Neural processes that direct an animal’s actions toward environmental goals are critical elements for understanding behavior. The hypothalamus is closely associated with motivated behaviors required for survival and reproduction. Intense feeding, drinking, aggressive, and sexual behaviors can be produced by a simple neuronal stimulus applied to discrete hypothalamic regions. What can these "evoked behaviors" teach us about the neural processes that determine behavioral intent and intensity? Small populations of neurons sufficient to evoke a complex motivated behavior may be used as entry points to identify circuits that energize and direct behavior to specific goals. Here, I review recent applications of molecular genetic, optogenetic, and pharmacogenetic approaches that overcome previous limitations for analyzing anatomically complex hypothalamic circuits and their interactions with the rest of the brain. These new tools have the potential to bridge the gaps between neurobiological and psychological thinking about the mechanisms of complex motivated behavior.