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2432 Janelia Publications
Showing 1661-1670 of 2432 resultsCryo-electron microscopy (cryo-EM) of single-particle specimens is used to determine the structure of proteins and macromolecular complexes without the need for crystals. Recent advances in detector technology and software algorithms now allow images of unprecedented quality to be recorded and structures to be determined at near-atomic resolution. However, compared with X-ray crystallography, cryo-EM is a young technique with distinct challenges. This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners.
The brain receives information about the direction of object motion from several types of retinal ganglion cells (RGCs). On-Off direction-selective (DS) RGCs respond preferentially to stimuli moving quickly in one of four directions and provide a significant (but difficult to quantify) fraction of RGC input to the SC. On DS RGCs, in comparison, respond preferentially to stimuli moving slowly in one of three directions and are thought to only target retinorecipient nuclei comprising the accessory optic system, e.g., the medial terminal nucleus (MTN). To determine the fraction of SC-projecting RGCs that exhibit direction selectivity, and the specificity with which On-Off and On DS RGCs target retinorecipient areas, we performed optical and electrophysiological recordings from RGCs retrogradely labeled from the mouse SC and MTN. We found, surprisingly, that both On-Off and On DS RGCs innervate the SC; collectively they constitute nearly 40% of SC-projecting RGCs. In comparison, only On DS RGCs project to the MTN. Subsequent experiments revealed that individual On DS RGCs innervate either the SC or MTN and exhibit robust projection-specific differences in somatodendritic morphology, cellular excitability, and light-evoked activity; several projection-specific differences in the output of On DS RGCs correspond closely to differences in excitatory synaptic input the cells receive. Our results reveal a robust projection of On DS RGCs to the SC, projection-specific differences in the response properties of On DS RGCs, and biophysical and synaptic mechanisms that underlie these functional differences.
Comprehensive measurement of neural activity remains challenging due to the large numbers of neurons in each brain area. We used volumetric two-photon imaging in mice expressing GCaMP6s and nuclear red fluorescent proteins to sample activity in 75% of superficial barrel cortex neurons across the relevant cortical columns, approximately 12,000 neurons per animal, during performance of a single whisker object localization task. Task-related activity peaked during object palpation. An encoding model related activity to behavioral variables. In the column corresponding to the spared whisker, 300 layer (L) 2/3 pyramidal neurons (17%) each encoded touch and whisker movements. Touch representation declined by half in surrounding columns; whisker movement representation was unchanged. Following the emergence of stereotyped task-related movement, sensory representations showed no measurable plasticity. Touch direction was topographically organized, with distinct organization for passive and active touch. Our work reveals sparse and spatially intermingled representations of multiple tactile features.
Natural events present multiple types of sensory cues, each detected by a specialized sensory modality. Combining information from several modalities is essential for the selection of appropriate actions. Key to understanding multimodal computations is determining the structural patterns of multimodal convergence and how these patterns contribute to behaviour. Modalities could converge early, late or at multiple levels in the sensory processing hierarchy. Here we show that combining mechanosensory and nociceptive cues synergistically enhances the selection of the fastest mode of escape locomotion in Drosophila larvae. In an electron microscopy volume that spans the entire insect nervous system, we reconstructed the multisensory circuit supporting the synergy, spanning multiple levels of the sensory processing hierarchy. The wiring diagram revealed a complex multilevel multimodal convergence architecture. Using behavioural and physiological studies, we identified functionally connected circuit nodes that trigger the fastest locomotor mode, and others that facilitate it, and we provide evidence that multiple levels of multimodal integration contribute to escape mode selection. We propose that the multilevel multimodal convergence architecture may be a general feature of multisensory circuits enabling complex input–output functions and selective tuning to ecologically relevant combinations of cues.
Modern high-throughput bioimaging techniques pose the unprecedented challenge of exploring and analyzing the produced Terabyte-scale volumetric images directly in their 3D space. Without expensive virtual reality devices and/or parallel computing infrastructures, this becomes even more demanding and calls for new, more scalable tools that help exploring these very large 3D data also on common laptops and graphic hardware. To this end, we developed a plugin for the open-source, cross-platform Vaa3D system to extend its powerful 3D visualization and analysis capabilities to images of potentially unlimited size. When used with large volumetric images up to 2.5 Terabyte in size, Vaa3D-TeraFly exhibited real-time (subsecond) performance that scaled constantly on image size. The tool has been implemented in C++ with Qt and OpenGL and it is freely and publicly available both as open-source and as binary package along with the main Vaa3D distribution.
In the field of biomedical imaging analysis on single-cell level, reliable and fast segmentation of the cell nuclei from the background on three-dimensional images is highly needed for the further analysis. In this work we propose an interactive cell segmentation toolkit that first establishes a set of exemplar regions from user input through an easy and intuitive interface in both 2D and 3D in real-time, then
extracts the shape and intensity features from those exemplars. Based on a local contrast-constrained region growing scheme, each connected component in the whole image would be filtered by the features from exemplars, forming an “exemplar-matching” group which passed the filtering and would be part of the final segmentation result, and a “non-exemplar-matching” group in which components
would be further segmented by the gradient vector field based algorithm. The results of the filtering process are visualized back to the user in near real-time, thus enhancing the experience in exemplar selecting and parameter tuning. The toolkit is distributed as a plugin within the open source Vaa3D system (http://vaa3d.org).
Neural computations are implemented by activity in spatially distributed neural circuits. Cellular imaging fills a unique niche in linking activity of specific types of neurons to behavior, over spatial scales spanning single neurons to entire brain regions, and temporal scales from milliseconds to months. Imaging may soon make it possible to track activity of all neurons in a brain region, such as a cortical column. We review recent methodological advances that facilitate optical imaging of neuronal populations in vivo, with an emphasis on calcium imaging using protein indicators in mice. We point out areas that are particularly ripe for future developments.
The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.
Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In experiments reported here, we have used spinning disk confocal and lattice light sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2-EGFP and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium, as well as at the approximate boundary between the lamellipodium and lamella, and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary-coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.
The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.