Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first expansion microscopy method was unable to retain native proteins in the gel and used custom made reagents not widely available. Here, we describe protein retention ExM (proExM), a variant of ExM that anchors proteins to the swellable gel allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validate and demonstrate utility of proExM for multi-color super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.
Fumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2). The main effect of FH mutation is fumarate accumulation. The current paradigm posits that the main consequence of fumarate accumulation is HIF-α stabilization. Paradoxically, FH mutation differs from other HIF-α stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We identified that fumarate can directly up-regulate antioxidant response element (ARE)-controlled genes. We demonstrated that aldo-keto reductase family 1 member B10 (AKR1B10) is an ARE-controlled gene and is up-regulated upon FH knockdown as well as in FH null cell lines. AKR1B10 overexpression is also a prominent feature in both hereditary and sporadic PRCC2. This phenotype better explains the similarities between hereditary and sporadic PRCC2.
During development, neurons send out axonal processes that can reach lengths hundreds of times longer than the diameter of their cell bodies. Recent studies indicate that en masse microtubule translocation is a significant mechanism underlying axonal elongation, but how cellular forces drive this process is unknown. Cytoplasmic dynein generates forces on microtubules in axons to power their movement through 'stop-and-go' transport, but whether these forces influence the bulk translocation of long microtubules embedded in the cytoskeletal meshwork has not been tested. Here, we use both function-blocking antibodies targeted to the dynein intermediate chain and the pharmacological dynein inhibitor ciliobrevin D to ask whether dynein forces contribute to en bloc cytoskeleton translocation. By tracking docked mitochondria as fiducial markers for bulk cytoskeleton movements, we find that translocation is reduced after dynein disruption. We then directly measure net force generation after dynein disruption and find a dramatic increase in axonal tension. Taken together, these data indicate that dynein generates forces that push the cytoskeletal meshwork forward en masse during axonal elongation.
Objective. Multimodal measurements at the neuronal level allow for detailed insight into local circuit function. However, most behavioral studies focus on one or two modalities and are generally limited by the available technology. Approach. Here, we show a combined approach of electrophysiology recordings, chemical sensing, and histological localization of the electrode tips within tissue. The key enabling technology is the underlying use of carbon fiber electrodes, which are small, electrically conductive, and sensitive to dopamine. The carbon fibers were functionalized by coating with Parylene C, a thin insulator with a high dielectric constant, coupled with selective re-exposure of the carbon surface using laser ablation. Main results. We demonstrate the use of this technology by implanting 16 channel arrays in the rat nucleus accumbens. Chronic electrophysiology and dopamine signals were detected 1 month post implant. Additionally, electrodes were left in the tissue, sliced in place during histology, and showed minimal tissue damage. Significance. Our results validate our new technology and methods, which will enable a more comprehensive circuit level understanding of the brain.
Dyneins are a small class of molecular motors that bind to microtubules and walk toward their minus ends. They are essential for the transport and distribution of organelles, signaling complexes and cytoskeletal elements. In addition dyneins generate forces on microtubule arrays that power the beating of cilia and flagella, cell division, migration and growth cone motility. Classical approaches to the study of dynein function in axons involve the depletion of dynein, expression of mutant/truncated forms of the motor, or interference with accessory subunits. By necessity, these approaches require prolonged time periods for the expression or manipulation of cellular dynein levels. With the discovery of the ciliobrevins, a class of cell permeable small molecule inhibitors of dynein, it is now possible to acutely disrupt dynein both globally and locally. In this review, we briefly summarize recent work using ciliobrevins to inhibit dynein and discuss the insights ciliobrevins have provided about dynein function in various cell types with a focus on neurons. We temper this with a discussion of the need for studies that will elucidate the mechanism of action of ciliobrevin and as well as the need for experiments to further analyze the specificity of ciliobreviens for dynein. Although much remains to be learned about ciliobrevins, these small molecules are proving themselves to be valuable novel tools to assess the cellular functions of dynein.
Summary This note describes nTracer, an ImageJ plug-in for user-guided, semi-automated tracing of multispectral fluorescent tissue samples. This approach allows for rapid and accurate reconstruction of whole cell morphology of large neuronal populations in densely labeled brains. Availability and implementation nTracer was written as a plug-in for the open source image processing software ImageJ. The software, instructional documentation, tutorial videos, sample image and sample tracing results are available at https://www.cai-lab.org/ntracer-tutorial. Supplementary information Supplementary data are available at Bioinformatics online.
10Identifying the cellular origins and mapping the dendritic and axonal arbors of neurons have 11 been century old quests to understand the heterogeneity among these brain cells. Classical 12 chemical and genetic methods take advantage of light microscopy and sparse labeling to 13 unambiguously, albeit inefficiently, trace a few neuronal lineages or reconstruct their 14 morphologies in each sampled brain. To improve the analysis throughput, we designed Bitbow, 15 a digital format of Brainbow which exponentially expands the color palette to provide tens of 16 thousands of spectrally resolved unique labels. We generated transgenic Bitbow Drosophila 17 lines, established statistical tools, and streamlined sample preparation, image processing and 18 data analysis pipelines to allow conveniently mapping neural lineages, studying neuronal 19 morphology and revealing neural network patterns with an unprecedented speed, scale and 20 resolution. 21 One way to generate more unique labels for lineage tracing is to localize the same FPs to 48 different subcellular compartments. In strategies such as CLoNe and MAGIC, Brainbow 49 cassettes targeted to cytoplasm, cell membrane, nucleus, and/or mitochondria were co-50 electroporated with transposase for genome integration, which allowed the differentiation of 51 neighboring progenies in chick and mouse embryos with fewer color collisions 26,27 . However, 52 the number of expression cassettes being integrated in each cell is random in these 53 experiments, leading to uncertainty in each color's appearance probability which complicates 54 quantitative analysis. The Raeppli strategy solves this problem by generating a transgenic 55 Drosophila which utilizes 4 FPs to create up to 4 x 4 = 16 membrane and nucleus color 56 combinations 16 . In parallel, strategies such as TIE-DYE and MultiColor FlpOut (MCFO) attempt to 57 generate more color combinations by stochastically removing the expression stops from each 58 3 FP module 15,28 . While inserting 3 different modules into 3 genomic loci allows generating up to 59 2 3 -1=7 unique labels, it is difficult to insert more modules to more genomic loci in a single 60 transgenic animal. 61 Here we present Bitbow, a digital format of Brainbow to greatly expand the unique color 62 pool from a single transgenic cassette. Unlike the original Brainbow, whose FP choices are 63 exclusive in one cassette, Bitbow allows each FP to independently express in an ON or OFF state 64 upon recombination. Color coding by each FP's binary status is similar to the information coding 65 by each bit in computer memory, thus leading to the name Bitbow. In a recent study, we 66 implemented the Bitbow1 design to target 5 spectrally distinct FPs to the nucleus for lineage 67 tracing 33 . Here, we present novel Bitbow1 flies which encode up to 32,767 unique "colors" 68 (Bitbow codes) in a single transgenic animal. This allows reliable lineage tracing without 69 complicated statistical tests 33 . To better enable morphology tracing, we generated Bitbow2, 70 which...
In vitro studies conducted in Aplysia and chick sensory neurons indicate that in addition to microtubule assembly, long microtubules in the C-domain of the growth cone move forward as a coherent bundle during axonal elongation. Nonetheless, whether this mode of microtubule translocation contributes to growth cone motility in vivo is unknown. To address this question, we turned to the model system Drosophila. Using docked mitochondria as fiduciary markers for the translocation of long microtubules, we first examined motion along the axon to test if the pattern of axonal elongation is conserved between Drosophila and other species in vitro. When Drosophila neurons were cultured on Drosophila extracellular matrix proteins collected from the Drosophila Kc167 cell line, docked mitochondria moved in a pattern indicative of bulk microtubule translocation, similar to that observed in chick sensory neurons grown on laminin. To investigate whether the C-domain is stationary or advances in vivo, we tracked the movement of mitochondria during elongation of the aCC motor neuron in stage 16 Drosophila embryos. We found docked mitochondria moved forward along the axon shaft and in the growth cone C-domain. This work confirms that the physical mechanism of growth cone advance is similar between Drosophila and vertebrate neurons and suggests forward translocation of the microtubule meshwork in the axon underlies the advance of the growth cone C-domain in vivo. These results highlight the need for incorporating en masse microtubule translocation, in addition to assembly, into models of axonal elongation.
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