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2496 Janelia Publications
Showing 2441-2450 of 2496 resultsThe transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.
Glutamate is the principal excitatory neurotransmitter, and occasionally subserves inhibitory roles, in the vertebrate nervous system. Glutamatergic synapses are dense in the vertebrate brain, at \textasciitilde1/μm3. Glutamate is released from and onto diverse components of the nervous system, including neurons, glia, and other cells. Methods for glutamate detection are critically important for understanding the function of synapses and neural circuits in normal physiology, development, and disease. Here we describe the development, optimization, and deployment of genetically encoded fluorescent glutamate indicators. We review the theoretical considerations governing glutamate sensor properties from first principles of synapse biology, microscopy, and protein structure-function relationships. We provide case studies of the state-of-the-art iGluSnFR glutamate sensor, encompassing design and optimization, mechanism of action, in vivo imaging, data analysis, and future directions. We include detailed protocols for iGluSnFR imaging in common preparations (bacteria, cell culture, and brain slices) and model organisms (worm, fly, fish, rodent).
Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the polymer gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors, for example using iterative expansion, but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures to carry out. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick tissue sections and cells enabling high-resolution subcellular imaging in a single expansion step. We show that applying TREx on antibody-stained samples can be combined with off-the-shelf small molecule stains for both total protein and membranes to provide ultrastructural context to subcellular protein localization.
During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.
In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.
View Publication PageOur ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.
Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional nanofilm that facilitates subcellular visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈3.2 µm (full width at half maximum) and are observed with a mean spatial frequency of 1 hotspot per ≈7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots colocalized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.
The importance of mechanical force in biology is evident across diverse length scales, ranging from tissue morphogenesis during embryo development to mechanotransduction across single adhesion proteins at the cell surface. Consequently, many force measurement techniques rely on optical microscopy to measure forces being applied by cells on their environment, to visualize specimen deformations due to external forces, or even to directly apply a physical perturbation to the sample via photoablation or optogenetic tools. Recent developments in advanced microscopy offer improved approaches to enhance spatiotemporal resolution, imaging depth, and sample viability. These advances can be coupled with already existing force measurement methods to improve sensitivity, duration and speed, amongst other parameters. However, gaining access to advanced microscopy instrumentation and the expertise necessary to extract meaningful insights from these techniques is an unavoidable hurdle. In this Live Cell Imaging special issue Review, we survey common microscopy-based force measurement techniques and examine how they can be bolstered by emerging microscopy methods. We further explore challenges related to the accompanying data analysis in biomechanical studies and discuss the various resources available to tackle the global issue of technology dissemination, an important avenue for biologists to gain access to pre-commercial instruments that can be leveraged for biomechanical studies.
The assembly of sequence-specific enhancer-binding transcription factors (TFs) at cis-regulatory elements in the genome has long been regarded as the fundamental mechanism driving cell type-specific gene expression. However, despite extensive biochemical, genetic, and genomic studies in the past three decades, our understanding of molecular mechanisms underlying enhancer-mediated gene regulation remains incomplete. Recent advances in imaging technologies now enable direct visualization of TF-driven regulatory events and transcriptional activities at the single-cell, single-molecule level. The ability to observe the remarkably dynamic behavior of individual TFs in live cells at high spatiotemporal resolution has begun to provide novel mechanistic insights and promises new advances in deciphering causal-functional relationships of TF targeting, genome organization, and gene activation. In this review, we review current transcription imaging techniques and summarize converging results from various lines of research that may instigate a revision of models to describe key features of eukaryotic gene regulation.
The nature of nervous system function and development is inherently global, since all components eventually influence one another. Networks communicate through dense synaptic, electric, and modulatory connections and develop through concurrent growth and interlinking of their neurons, processes, glia, and blood vessels. These factors drive the development of techniques capable of imaging neural signaling, anatomy, and developmental processes at ever-larger scales. Here, we discuss the nature of questions benefitting from large-scale imaging techniques and introduce recent applications. We focus on emerging light-sheet microscopy approaches, which are well suited for live imaging of large systems with high spatiotemporal resolution and over long periods of time. We also discuss computational methods suitable for extracting biological information from the resulting system-level image data sets. Together with new tools for reporting and manipulating neuronal activity and gene expression, these techniques promise new insights into the large-scale function and development of neural systems.