Deep learning enables cross-modality super-resolution in fluorescence microscopy.

We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.

Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale.

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.

Exploring Subcellular Cerebellar Fractions with the Electron Microscope.

Differential ultracentrifugation and subcellular fractionation historically helped to study the components of the cell, to discover new cellular organelles, and to decipher their morphological and molecular properties. In neuroscience, the technique has yielded important results on neuron biochemistry and the mechanisms of synaptic transmission. This Cerebellar Classic is devoted to the pioneering work of Manuel del Cerro, Ray S. Snider, and Mary Lou Oster-Granite, who isolated purified fractions after successive centrifugations of the rat cerebellum from birth to adulthood and studied them under the electron microscope.

Commentary on "Subcellular Fractions of Adult and Developing Rat Cerebellum, by M. del Cerro, R. S. Snider and M. L. Oster, July 1969".

Subcellular fractionation by differential ultracentrifugation has allowed the study of the cell and its organelles from a morphological, physiological, and biochemical perspective. Combined with electron microscopy, and by using animals at different stages of postnatal development, these methods yielded useful results concerning the ontogeny of synaptosomes, mitochondria, and myelin and broadened the possibilities to investigate the molecular underpinnings of cerebellar histogenesis.

Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography.

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focused-ion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, "ridge-like prominences" (RLPs).

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons.

Clostridium botulinum C3 transferase (C3bot) ADP-ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild-type with Vim-/- mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP-ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin-binding RGD motif (C3bot-G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross-talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild-type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim-/- astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.

Subcellular origin of mitochondrial DNA deletions in human skeletal muscle.

OBJECTIVE: In patients with mitochondrial DNA (mtDNA) maintenance disorders and with aging, mtDNA deletions sporadically form and clonally expand within individual muscle fibers, causing respiratory chain deficiency. This study aimed to identify the sub-cellular origin and potential mechanisms underlying this process. METHODS: Serial skeletal muscle cryosections from patients with multiple mtDNA deletions were subjected to subcellular immunofluorescent, histochemical, and genetic analysis. RESULTS: We report respiratory chain-deficient perinuclear foci containing mtDNA deletions, which show local elevations of both mitochondrial mass and mtDNA copy number. These subcellular foci of respiratory chain deficiency are associated with a local increase in mitochondrial biogenesis and unfolded protein response signaling pathways. We also find that the commonly reported segmental pattern of mitochondrial deficiency is consistent with the three-dimensional organization of the human skeletal muscle mitochondrial network. INTERPRETATION: We propose that mtDNA deletions first exceed the biochemical threshold causing biochemical deficiency in focal regions adjacent to the myonuclei, and induce mitochondrial biogenesis before spreading across the muscle fiber. These subcellular resolution data provide new insights into the possible origin of mitochondrial respiratory chain deficiency in mitochondrial myopathy. Ann Neurol 2018;84:289-301.

Subcellular distribution and tolerance of cadmium in Canna indica L.

Canna indica L. is a promising species for heavy metal phytoremediation due to its fast growth rate and large biomass. However, few studies have investigated cadmium (Cd) tolerance mechanisms. In the present study, Canna plants were cultivated under hydroponic conditions with increasing Cd concentrations (0, 5, 10, 15 mg/L). We found that the plants performed well under 5 mg/L Cd2+ stress, but damage was observed under higher Cd exposure, such as leaf chlorosis, growth inhibition, a decreased chlorophyll content, and destruction of the ultrastructure of leaf cells. Additionally, Canna alleviated Cd toxicity to a certain extent. After Canna was exposed to 5, 10 and 15 mg/L Cd2+ for 45 d, the highest Cd concentration was exhibited in roots, which was almost 17-47 times the Cd concentration in leaves and 8-20 times that in stems. At the subcellular level, cellular debris and heat-stable proteins (HSPs) were the main binding sites for Cd, and the proportion of Cd in the two subcellular fractions accounted for 71.4-94.2% of the total Cd. Furthermore, we found that granules could participate in the detoxification process when Cd stress was enhanced. Our results indicated that Canna indica L. can tolerate Cd toxicity by sequestering heavy metals in root tissues, fencing out by cell wall, and binding with biologically detoxified fractions (granules and HSPs).

Mitochondria Maintain Distinct Ca2+ Pools in Cone Photoreceptors.

Ca2+ ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca2+ domains are maintained by Ca2+ uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment. The endoplasmic reticulum surrounds the basal and lateral surfaces of this cluster, but does not reach the apical surface or penetrate into the cluster. Using genetically encoded Ca2+ sensors, we found that mitochondria take up Ca2+ when it accumulates either in the cone cell body or outer segment. Blocking mitochondrial Ca2+ uniporter activity compromises the ability of mitochondria to maintain distinct Ca2+ domains. Together, our findings indicate that mitochondria can modulate subcellular functional specialization in photoreceptors.SIGNIFICANCE STATEMENT Ca2+ homeostasis is essential for the survival and function of retinal photoreceptors. Separate pools of Ca2+ regulate phototransduction in the outer segment, metabolism in the cell body, and neurotransmitter release at the synaptic terminal. We investigated the role of mitochondria in compartmentalization of Ca2+ We found that mitochondria form a dense cluster that acts as a diffusion barrier between the outer segment and cell body. The cluster is surprisingly only partially surrounded by the endoplasmic reticulum, a key mediator of mitochondrial Ca2+ uptake. Blocking the uptake of Ca2+ by mitochondria causes redistribution of Ca2+ throughout the cell. Our results show that mitochondrial Ca2+ uptake in photoreceptors is complex and plays an essential role in normal function.

Metabolomics and Lipidomics Profiling of a Combined Mitochondrial Plus Endoplasmic Reticulum Fraction of Human Fibroblasts: A Robust Tool for Clinical Studies.

Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles' functions, dysfunctions, and interactions. We developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with excellent reproducibility. Seventy-two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12%, among which many were specific to the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and "omics" combination will be useful for investigating the pathophysiology of complex diseases.

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats.

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1 day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3 days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28 days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.

Strategy and software for the statistical spatial analysis of 3D intracellular distributions.

The localization of proteins in specific domains or compartments in the 3D cellular space is essential for many fundamental processes in eukaryotic cells. Deciphering spatial organization principles within cells is a challenging task, in particular because of the large morphological variations between individual cells. We present here an approach for normalizing variations in cell morphology and for statistically analyzing spatial distributions of intracellular compartments from collections of 3D images. The method relies on the processing and analysis of 3D geometrical models that are generated from image stacks and that are used to build representations at progressively increasing levels of integration, ultimately revealing statistical significant traits of spatial distributions. To make this methodology widely available to end-users, we implemented our algorithmic pipeline into a user-friendly, multi-platform, and freely available software. To validate our approach, we generated 3D statistical maps of endomembrane compartments at subcellular resolution within an average epidermal root cell from collections of image stacks. This revealed unsuspected polar distribution patterns of organelles that were not detectable in individual images. By reversing the classical 'measure-then-average' paradigm, one major benefit of the proposed strategy is the production and display of statistical 3D representations of spatial organizations, thus fully preserving the spatial dimension of image data and at the same time allowing their integration over individual observations. The approach and software are generic and should be of general interest for experimental and modeling studies of spatial organizations at multiple scales (subcellular, cellular, tissular) in biological systems.

Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes.

High-throughput precision measurement of subcellular localization in single cells.

To quantify visual and spatial information in single cells with a throughput of thousands of cells per second, we developed Subcellular Localization Assay (SLA). This adaptation of Proximity Ligation Assay expands the capabilities of flow cytometry to include data relating to localization of proteins to and within organelles. We used SLA to detect the nuclear import of transcription factors across cell subsets in complex samples. We further measured intranuclear re-localization of target proteins across the cell cycle and upon DNA damage induction. SLA combines multiple single-cell methods to bring about a new dimension of inquiry and analysis in complex cell populations. © 2017 International Society for Advancement of Cytometry.

Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with µ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT: The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.

GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome.

NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex, GluN1 is combined primarily with GluN2A and GluN2B, which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B, the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components, in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis, revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions.

Dissecting motility signaling through activation of specific Src-effector complexes.

We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed rapamycin-regulated targeted activation of pathways (RapRTAP), was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src-induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.

Dynamical Localization of DivL and PleC in the Asymmetric Division Cycle of Caulobacter crescentus: A Theoretical Investigation of Alternative Models.

Cell-fate asymmetry in the predivisional cell of Caulobacter crescentus requires that the regulatory protein DivL localizes to the new pole of the cell where it up-regulates CckA kinase, resulting in a gradient of CtrA~P across the cell. In the preceding stage of the cell cycle (the "stalked" cell), DivL is localized uniformly along the cell membrane and maintained in an inactive form by DivK~P. It is unclear how DivL overcomes inhibition by DivK~P in the predivisional cell simply by changing its location to the new pole. It has been suggested that co-localization of DivL with PleC phosphatase at the new pole is essential to DivL's activity there. However, there are contrasting views on whether the bifunctional enzyme, PleC, acts as a kinase or phosphatase at the new pole. To explore these ambiguities, we formulated a mathematical model of the spatiotemporal distributions of DivL, PleC and associated proteins (DivJ, DivK, CckA, and CtrA) during the asymmetric division cycle of a Caulobacter cell. By varying localization profiles of DivL and PleC in our model, we show how the physiologically observed spatial distributions of these proteins are essential for the transition from a stalked cell to a predivisional cell. Our simulations suggest that PleC is a kinase in predivisional cells, and that, by sequestering DivK~P, the kinase form of PleC enables DivL to be reactivated at the new pole. Hence, co-localization of PleC kinase and DivL is essential to establishing cellular asymmetry. Our simulations reproduce the experimentally observed spatial distribution and phosphorylation status of CtrA in wild-type and mutant cells. Based on the model, we explore novel combinations of mutant alleles, making predictions that can be tested experimentally.

Preparation of Fluorescent Dye-Doped Biocompatible Nanoparticles for Cell Labeling.

In this paper, we report a series of fluorescent biocompatible nanoparticles (NPs), prepared by a facile reprecipitation-encapsulation method, for cellular labeling. The as-prepared NPs exhibit a narrow size distribution of 70-110 nm, and a core-shell structure comprised of a hybrid core doped with different dyes and a poly-L-lysine (PLL) shell. With coumarin 6, nile red, and meso- tetraphenylporphyrin as the imaging agents, the fluorescent NPs gave green, orange, and red emissions respectively. Due to the positively charged PLL shell, the fluorescent NPs exhibit neglected cytotoxicity and efficient cellular uptake. After incubation with living cells, the results obtained by laser confocal microscope from green, orange, and red channels all clearly show that the fluores- cent NPs are inhomogenously localized inside the cytoplasm without penetrating into the nucleus. Since such PLL-modified NPs can encapsulate other hydrophobic dyes, a wide spectrum of nanoimaging agents is thus expected. Furthermore, the surface amino groups on the PLL shell afford an anchoring site for further bioconjugation, and targeted imaging is also very promising.

Preparation, Characterization and Intracellular Imaging of 2,4-Dichlorophenoxyacetic Acid Conjugated Gold Nanorods.

Visualizing the biodistribution of pesticides inside living cells is great importance for enhancing targeting of pesticides. Here we reported for the first time that gold nanorods (Au NRs) with size of 39.4 nm x 11.3 nm could be used as a fluorescent tracer to examine the distribution of a typical herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in tobacco bright yellow 2 (BY-2) cells. The nanostructures of hybrid materials were analyzed by using Raman spectra and X-ray photoelectron spectroscopy (XPS), including spectra assignments and electronic property. These data revealed 2,4-D has successfully conjugated MP-Au NRs according to Raman and XPS. The biodistribution of the conjugates inside BY-2 cells was directly examined at 12 and 24 h by the two-photon microscopy. The intensity of two-photon luminescence (TPL) inside cells demonstrated that the conjugates could be localized and excluded by BY-2 cells. Thus, this labeling approach opens up new avenues to the facile and efficient labeling of pesticides.