Electron Microscopy Analysis of Membraneless Condensates in Plant Cells.

Biomolecular condensates are triggered by multivalent interactions conferred by the intrinsically disordered regions and/or interacting domains within the constituents. While light microscopy has provided powerful tools to study the dynamics of intracellular condensates, electron microscopy (EM) gives more detailed insights into their ultrastructure and spatial connectivity with membrane system. In this chapter, we describe the methods for detecting the membraneless condensates in plant cells by high-pressure freezing -based EM coupled with immuno-gold labeling and correlative light electron microscopy techniques, which may benefit researchers in future studies.

How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological question.

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.

GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABAA receptors.

Striatal dopamine axons co-release dopamine and gamma-aminobutyric acid (GABA), using GABA provided by uptake via GABA transporter-1 (GAT1). Functions of GABA co-release are poorly understood. We asked whether co-released GABA autoinhibits dopamine release via axonal GABA type A receptors (GABAARs), complementing established inhibition by dopamine acting at axonal D2 autoreceptors. We show that dopamine axons express α3-GABAAR subunits in mouse striatum. Enhanced dopamine release evoked by single-pulse optical stimulation in striatal slices with GABAAR antagonism confirms that an endogenous GABA tone limits dopamine release. Strikingly, an additional inhibitory component is seen when multiple pulses are used to mimic phasic axonal activity, revealing the role of GABAAR-mediated autoinhibition of dopamine release. This autoregulation is lost in conditional GAT1-knockout mice lacking GABA co-release. Given the faster kinetics of ionotropic GABAARs than G-protein-coupled D2 autoreceptors, our data reveal a mechanism whereby co-released GABA acts as a first responder to dampen phasic-to-tonic dopamine signaling.

Quantitative proteomics of tau and Aß in detergent fractions from Alzheimer's disease brains.

The two hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aß drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aß mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aß species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aß42 at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT181 (~22%) and pT217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non-filamentous form (N-terminal intact tau) co-localized with Aß in the sarkosyl-insoluble pellets along with tau filaments (N-truncated MTBR tau). Our results suggest a model that Aß and tau interact forming globular aggregates, from which filamentous tau and Aß emerge. These characterizations contribute towards unravelling the sequence of events which lead to end-stage AD changes.

An Improved Method for Assessing Antigen Presentation on the Surface of Plasmodium falciparum-Infected Erythrocytes by Immuno-Electron Microscopy.

Immuno-electron microscopy can detect and localize antigens in cells or tissues at a resolution of several nanometers. In the case of P. falciparum-infected erythrocytes, immuno-EM studies are frequently hampered by the electron-dense nature of the hemoglobin and access of antibodies to antigenic sites, particularly if the targeted protein is presented on the host cell surface or lies in proximity to the host cell cytoskeleton. Here, we describe an improved immuno-EM protocol that overcomes these problems. The improved signal to noise ratio and the enhanced access to antigenic sites now allows one to obtain information regarding target density and distribution and, hence, additional insights into the architecture and function of parasite-induced, or -affected, structures.

Large-Area Fluorescence and Electron Microscopic Correlative Imaging With Multibeam Scanning Electron Microscopy.

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.

The invasive cell coat at the microsporidian Trachipleistophora hominis-host cell interface contains secreted hexokinases.

Microsporidia are obligate intracellular parasites causing significant disease in humans and economically important animals. In parallel to their extreme genetic reduction, Microsporidia have evolved novel mechanisms for exploiting host metabolism. A number of microsporidians confer secretion of otherwise cytosolic proteins by coding for signal peptides that direct entry into the endoplasmic reticulum. The human pathogen Trachipleistophora hominis encodes for four hexokinases, three of which have signal peptides at the N-terminus. Here, we localized hexokinase 2 and hexokinase 3 through developmental stages of T. hominis using light and electron microscopy. Both proteins were concentrated in an extracellular coat previously termed the plaque matrix (PQM). The PQM (containing hexokinases) was morphologically dynamic, infiltrating the host cytoplasm predominantly during replicative stages. Throughout development the PQM interacted closely with endoplasmic reticulum that was demonstrated to be active in membrane protein biosynthesis and export. The impact of hexokinase on the host metabolism was probed using the fluorescent analog of glucose, 2-NBDG, which displayed spatially restricted increases in signal intensity at the parasite/vacuole surface, coincident with hexokinase/PQM distribution. Gross metabolic aberrations, measured using metabolic profiling with the Seahorse XF Analyzer, were not detectable in mixed stage cocultures. Overall, these results highlight a role for the extended cell coat of T. hominis in host-parasite interactions, within which secreted hexokinases may work as part of a metabolic machine to increase glycolytic capacity or ATP generation close to the parasite surface.

Immuno-TEM/STEM in Retinal Research.

Immuno-EM is a method that can determine the localization of a protein in a tissue at the ultrastructural level. Generally, membrane structures in immuno-EM specimens have very low contrast because fixation is performed without osmium. Here, by using high-angle annular dark field (HAADF)-scanning transmission electron microscopy (STEM) instead of transmission electron microscopy (TEM) for observation of immuno-EM samples, we demonstrate that photoreceptor disk membranes are clearly visible and that the procedures described in this article can be extended to visualize other membrane structures.

Electron Tomography and Correlative Approaches in Platelet Studies.

Blood platelets play a central role in the arrest of bleeding and the development of thrombosis. Unraveling the complex processes of platelet biogenesis from megakaryocytes, platelet adhesion, aggregation, and secretory responses are important topics in the field of hemostasis and thrombosis. Analysis of the ultrastructural changes that occur during these processes is essential for understanding the rapid membrane dynamics and has contributed substantially to our present knowledge of platelet formation and functioning. Recent developments in real-time imaging, correlative light and electron microscopy imaging (CLEM), and 3D (cryo) electron microscopy and tomography offer exciting opportunities to improve studies of the platelet adhesive responses and secretion at the ultrastructural level in a close to native environment. In this chapter we discuss and illustrate cryo preparation techniques (high-pressure freezing, vitrification), correlative LM and EM workflows, and 3D cryo-electron tomography that we apply in our current research projects.

Using Microscopy Tools to Visualize Autophagosomal Structures in Plant Cells.

Macroautophagy (hereafter as autophagy), is a metabolic process for sequestration of cytoplasmic cargos into a double membrane structure named as autophagosome. In plants, autophagy is required for nutrition mobilization/recycling and clearance of protein aggregates or damaged organelles during starvation or other unfavorable conditions, as well as for plant immunity during pathogen infection. Multiple experimental approaches have been developed to elucidate the autophagic activity. To facilitate further investigations on the potential involvement of autophagy in protein secretion process in plant cells, here we describe detailed protocols to measure the autophagic activity in model plant Arabidopsis. Using the autophagosome marker ATG8 and a novel autophagic regulator SH3P2 as examples, we illustrate the major cell biology tools and methods using microscopy to analyze the autophagosomal structures in plant cells, including BTH-induced autophagic response, transient expression and colocalization analysis, as well as immuno-EM labeling.

Scanning EM of non-heavy metal stained biosamples: Large-field of view, high contrast and highly efficient immunolabeling.

Scanning electron microscopy (SEM) is increasing its application in life sciences for electron density measurements of ultrathin sections. These are traditionally analyzed with transmission electron microscopy (TEM); by most labs, SEM analysis still is associated with surface imaging only. Here we report several advantages of SEM for thin sections over TEM, both for structural inspection, as well as analyzing immuno-targeted labels such as quantum dots (QDs) and gold, where we find that QD-labeling is ten times more efficient than gold-labeling. Furthermore, we find that omitting post-staining with uranyl and lead leads to QDs readily detectable over the ultrastructure, but under these conditions ultrastructural contrast was even almost invisible in TEM examination. Importantly, imaging in SEM with STEM detection leads to both outstanding QDs and ultrastructural contrast. STEM imaging is superior over back-scattered electron imaging of these non-contrasted samples, whereas secondary electron detection cannot be used at all. We conclude that examination of ultrathin sections by SEM, which may be immunolabeled with QDs, will allow rapid and straightforward analysis of large fields with more efficient labeling than can be achieved with immunogold. The large fields of view routinely achieved with SEM, but not with TEM, allows straightforward raw data sharing using virtual microscopy, also known as nanotomy when this concerns EM data in the life sciences.

Native immunogold labeling of cell surface proteins and viral glycoproteins for cryo-electron microscopy and cryo-electron tomography applications.

Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.

Immuno correlative light and electron microscopy on Tokuyasu cryosections.

Finding a rare structure by electron microscopy is the equivalent of finding a "needle in a haystack." Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.

Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM.

Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future.

Anna S. Tikhonenko: Electron microscopist extraordinary.

Anna Sergeyevna Tikhonenko (1925-2010) is to be remembered for the excellency of her electron microscopical work, particularly with bacteriophages. She published 113 articles and one book, Ultrastructure of Bacterial Viruses (Izdadelstvo Nauka, Moscow 1968; Plenum Press, New York, 1972). It included 134 micrographs and a complete overview of the 316 phages then examined by electron microscopy. Most micrographs were of exceptional quality. This book, a rarity in those days of strict separation of Soviet and Western research, was the first bacteriophage atlas in the literature and presented a morphological classification of phages into five categories of family level, similar to a scheme presented in 1965 by D.E. Bradley (J Royal Microsc Soc 84:257-316). Her book remains one of the fundamentals of phage research.

Insights into cell entry and intracellular trafficking of peptide and protein drugs provided by electron microscopy.

For widening the arsenal of protein and peptide therapeutics that act within cells, their cell-entry mechanisms, intracellular trafficking and distribution need to be characterized in detail. Immunofluorescence microscopy has been a prevalent tool for these studies. However, due to the limited resolution, it is often complemented with other methods. This article focuses on the perspectives of electron microscopy in tracking the intracellular delivery and trafficking of proteins, peptides and their carriers. This review introduces the electron microscopy techniques and labeling methods currently used for studying the cellular whereabouts of peptides and proteins with a focus on their intracellular trafficking. Since cell-penetrating peptides have widely been harnessed as carriers for proteins and peptides, and their usage is rapidly expanding, a particular emphasis has been placed on their applications and cell-entry mechanisms.