Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.

High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Immuno EM-OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM).

In the atmospheric scanning electron microscope (ASEM), an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized the cytoskeletons formed by actin and tubulin, the chaperone PDI that catalyses native disulfide bond formation of proteins in the endoplasmic reticulum (ER) and the calcium sensor STIM1 that is integrated in ER membranes, using established cell lines. In particular, a dynamic string-like gathering of STIM1 was observed on the ER in Jurkat T cells in response to Ca(2+) store depletion. We have also visualized filamentous actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca(2+) signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework. Based on this work, ASEM correlative microscopy promises to allow the dynamics of various protein complexes to be investigated in the near future.

Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy.

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called "crystallization bottleneck". Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iß in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few µm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.

Inhibitory synaptic modulation of renshaw cell activity in the lumbar spinal cord of neonatal mice.

In the mammalian spinal cord, Renshaw cells (RCs) are excited by axon collaterals of motoneurons (MNs), and in turn, provide recurrent inhibition of MNs. They are considered an important element in controlling the motor output. However, how RCs are modulated by spinal circuits during motor behaviors remains unclear. In this study, the physiological nature of inhibitory synaptic inputs to RCs in the lumbar segment during spontaneous motoneuronal activity was examined in the isolated spinal cord taken from glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse neonates. Whole cell recordings of RCs in current-clamp mode showed that they receive phasic inhibition that could modulate the RC firing evoked by excitation of MNs. In voltage-clamp recording, we observed a barrage of spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by glycine and/or GABA. These sIPSCs persisted in the presence of mecamylamine, a nicotinic receptor antagonist, indicating that excitation of other RCs by MN axon collaterals may not be essential for these inhibitory actions. Simultaneous recording of RC and the ventral root in the same segment showed that the RCs received inhibitory inputs when spontaneous MN firing occurred. Paired recordings of a RC and a MN showed that during the bursting activity in the ventral root, the magnitude of the RC sIPSCs and the magnitude of the excitatory inputs that MNs receive are highly correlated. These results indicate that RCs are modulated by inhibition that matches the MN excitation in timing and amplitude during motor behaviors.

New Alzheimer's disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid ß oligomers.

Oligomers of intracellular amyloid ß protein (Aß) are strongly cytotoxic and play crucial roles in synaptic transmission and cognitive function in Alzheimer's disease (AD). However, there is currently no AD model mouse in which to specifically analyze the function of Aß oligomers only. We have now developed a novel AD model mouse, an Aß-GFP transgenic mouse (Aß-GFP Tg), that expresses the GFP-fused human Aß1-42 protein, which forms only Aß oligomers within neurons throughout their life. The fusion proteins are expressed mainly in the hippocampal CA1-CA2 region and cerebral cortex, and are not secreted extracellularly. The Aß-GFP Tg mice exhibit increased tau phosphorylation, altered spine morphology, decreased expressions of the GluN2B receptor and neuroligin in synaptic regions, attenuated hippocampal long-term potentiation, and impaired object recognition memory compared with non-Tg littermates. Interestingly, these dysfunctions have already appeared in 2-3-months-old animals. The Aß-GFP fusion protein is bioactive and highly toxic, and induces the similar synaptic dysfunctions as the naturally generated Aß oligomer derived from postmortem AD patient brains and synthetic Aß oligomers. Thus, Aß-GFP Tg mouse is a new tool specialized to analyze the function of Aß oligomers in vivo and to find subtle changes in synapses in early symptoms of AD.

Primary cultured neuronal networks and type 2 diabetes model mouse fatty liver tissues in aqueous liquid observed by atmospheric SEM (ASEM): Staining preferences of metal solutions.

Neural networking, including axon targeting and synapse formation, is the basis of various brain functions, including memory and learning. Diabetes-mellitus affects peripheral nerves and is known to cause fatty liver disease. Electron microscopy (EM) provides the resolution required to observe changes in fine subcellular structures caused by such physiological and pathological processes, but samples are observed in vacuum. Environmental capsule EM can directly observe cells in a more natural aqueous environment, but the size-limited capsules restrict cell culturability. The recently developed atmospheric scanning electron microscope (ASEM) has an open, 35 mm sample dish, allowing the culture of primary cells, including neurons, on the electron-transparent film window fabricated in its base. The system's inverted scanning electron microscope observes aldehyde-fixed cells or tissues from below through the silicon nitride film; the optical microscope located above allows direct correlation of fluorescence labeling. To observe fixed biological samples, damage due to low dose electron radiation is minimized in three ways. First, knock on damage that pushes out atoms is decreased by the low accelerating voltage of 10-30 kV. Second, increased radical generation due to the decreased acceleration voltage is countered by the addition of a radical scavenger, glucose or ascorbic acid, to the sample solution. Third, the large volume (max. 2 ml) of aqueous buffer surrounding the sample has a high specific heat capacity, minimizing the temperature increase caused by irradiation. Using ASEM, we have developed protocols for heavy metal staining in solution to selectively visualize intracellular structures. Various EM staining methods served as a starting point. Uranyl acetate preferably stains proteins and nucleic acid, and prior tannic acid treatment enhances membranes. Osmium tetroxide is suggested to enhance lipids, especially oil droplets. Imaging primary-culture neurons stained with platinum blue or uranyl acetate revealed growth cones, synapses, and 50-500 nm spines, together with neurite backbones and their associated structures. Correlative microscopy with immuno-fluorescence labeling suggested that these were mainly microtubule associated objects; some showed signs of a fission process and were, thus, possibly mitochondria. Liver tissue excised from the ob/ob type 2 diabetes model mouse, was stained with osmium tetroxide and observed using ASEM. Swollen bright balls occupied a large area of the cytoplasm and could be distinguished from vacuoles, suggesting that they are oil droplets. In some of the images, oil-like droplets were pressing surrounding structures, including sinusoids, significant for blood circulation in the liver. Based on these studies, ASEM combined with metal staining methods promises to allow the study of various mesoscopic-scale phenomena of cells and tissues immersed in natural aqueous environment in the near future. The quick nature of ASEM could facilitate not only the precise imaging for neuroscience but also the diagnosis of fatty liver disease and related diseases.

Cre complementation with variable dimerizers for inducible expression in neurons.

Cre complementation is a process of reconstitution of the activity of DNA recombinase by noncovalent association of multiple segments of Cre recombinase, which are enzymatically inactive by themselves. Cre complementation is potentially useful in restriction of Cre activity in a specific subset of cells, with temporal regulation, by limiting overlap in expression of Cre fragments. We analyzed the efficiency of Cre complementation using three different dimerizing modules in the context of non-neuronal cells and found differential Cre complementation efficiency. We further tested the efficiency of Cre complementation in primary hippocampal neurons derived from transgenic mice harboring a reporter gene flanked by loxP sites and confirmed differential activity of dimerization modules in Cre-dependent recombination of the transgene. These results suggest possible application of dimerizer-based Cre complementation in inducible expression/inactivation of target genes in a specific subset of neurons in the complex environment of nervous tissue in vivo.

Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels.

Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca2+ from intracellular stores and a sustained influx of external Ca2+ in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca2+ from intracellular stores and a sustained influx of Ca2+ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca2+ pools by irreversibly inhibiting the Ca2+ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca2+ stores, and that these stores are a subset of intracellular stores depleted by the Ca2+ ionophore ionomycin. Intracellular Ca2+ stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca2+, but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca2+ levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca2+ into internal stores and a delayed decrease that correlates with the inhibition of a Mn2+-permeable Ca2+ channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca2+ influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca2+ influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn2+, and (iii) identical rates of Ca2+ influx are observed when Ca2+ is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca2+. Taken together, these data suggest that the sustained influx of extracellular Ca2+ observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn2+-permeable, Ca2+ store-operated Ca2+ channel.

Development of new fusion proteins for visualizing amyloid-ß oligomers in vivo.

The intracellular accumulation of amyloid-ß (Aß) oligomers critically contributes to disease progression in Alzheimer's disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aß oligomers in vivo is key for drug discovery research, however, it has been challenging because Aß aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aß1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of Aß-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between Aß and GFP, we generated two fusion proteins with "a long-linker" and "a short-linker", and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with Aß-GFP plasmids and Aß-GFP transgenic C. elegans. We found that Aß-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel Aß-GFP fusion proteins could be utilized for studying the physiological functions of Aß oligomers in living cells and animals, and for drug screening by analyzing Aß toxicity.

Synchronized formation and remodeling of postsynaptic densities: long-term visualization of hippocampal neurons expressing postsynaptic density proteins tagged with green fluorescent protein.

To explore mechanisms governing the formation and remodeling of postsynaptic density (PSD), we used dissociated cultures of hippocampal neurons isolated from transgenic embryos expressing green fluorescent protein (GFP)-tagged PSD proteins PSD-Zip45 (Homer 1c) and PSD-95. Expression of GFP-tagged PSD molecules was stable, and the remodeling process of PSDs could be followed for >1 week. A higher expression level of GFP-PSD-Zip45 enabled us to quantitatively analyze the amount of PSD-Zip45 clusters during development. Repetitive imaging of the same cell populations between 11 and 17 d in culture revealed an increase of the average PSD-Zip45 cluster density from 0.32 to 0.73/microm. Newly generated dendrites rapidly acquired GFP-PSD-Zip45 clusters, and their density reached the level of parental dendrites within a few days. Temporal profiles of GFP-PSD-Zip45 cluster density showed a variety of patterns. Some dendrites showed a monotonous increase of clusters, whereas others showed complex patterns, including short decremental stages. Analysis of long-term remodeling of PSD-95-GFP clusters confirmed that the decremental stages were not specific to the PSD-Zip45 clusters. Comparison of the temporal profiles of the cluster density among neurons indicated synchronization of both GFP-PSD-Zip45 and PSD-95 clustering within individual cells. Furthermore, activation of cAMP-dependent protein kinase suppressed the decremental stages of cluster remodeling. These observations suggest the presence of signaling mechanisms that can induce synchronized addition or elimination of PSD proteins throughout dendritic arborization of a single neuron.

Observation of tissues in open aqueous solution by atmospheric scanning electron microscopy: applicability to intraoperative cancer diagnosis.

In the atmospheric scanning electron microscope (ASEM), a 2- to 3-µm layer of the sample resting on a silicon nitride-film window in the base of an open sample dish is imaged, in liquid, at atmospheric pressure, from below by an inverted SEM. Thus, the time-consuming pretreatments generally required for biological samples to withstand the vacuum of a standard electron microscope are avoided. In the present study, various mouse tissues (brain, spinal cord, muscle, heart, lung, liver, kidney, spleen and stomach) were fixed, stained with heavy metals, and visualized in radical scavenger D-glucose solution using the ASEM. While some stains made the nuclei of cells very prominent (platinum-blue, phosphotungstic acid), others also emphasized cell organelles and membranous structures (uranium acetate or the NCMIR method). Notably, symbiotic bacteria were sometimes observed on stomach mucosa. Furthermore, kidney tissue could be stained and successfully imaged in <30 min. Lung and spinal cord tissue from normal mice and mice metastasized with breast cancer cells was also examined. Cancer cells present in lung alveoli and in parts of the spine tissue clearly had larger nuclei than normal cells. The results indicate that the ASEM has the potential to accelerate intraoperative cancer diagnosis, the diagnosis of kidney diseases and pathogen detection. Importantly, in the course of the present study it was possible to increase the observable tissue area by using a new multi-windowed ASEM sample dish and sliding the tissue across its eight windows.

DHP-insensitive L-type-like Ca channel of ascidian acquires sensitivity to DHP with single amino acid change in domain III P-region.

TuCa1, an ascidian homolog of L-type Ca channel alpha(1)-subunit, has many critical sites required for binding 1,4-dihydropyridines (DHPs), but is insensitive to DHPs and methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL-64176). We have substituted Ser for Ala(1016) at the P-region of domain III in TuCa1 (TuCa1/A1016S) and functionally expressed the channel in Xenopus oocyte along with rabbit alpha(2)/delta and beta(2b). TuCa1/A1016S has gained DHP sensitivity as high as that of a mammalian neuronal L-type Ca channel (rbCII), but remained resistant to FPL-64176. These results reinforce the view that Ser(1016) in TuCa1/A1016S participates in DHP binding, but there exist other novel sites that fully acquire sensitivity to FPL-64176.

Coexpression of a Ca(v)1.2 protein lacking an N-terminus and the first domain specifically suppresses L-type calcium channel activity.

L-type Ca(2) channels play a critical role in many types of cells, including nerve, muscle and endocrine cells. The most popular and effective tools for analyzing the roles of L-type calcium channels (L-channels) are specific antagonists such as dihydropyrigines. With these drugs however, it is difficult to target specific cells. One solution is to develop a genetically targetable inhibitor coded by DNA. As a candidate for such an inhibitor, a dominant negative mutant of Ca(v)1.2 was designed by mimicking an ascidian 3-domain-type alpha1 subunit (that inhibits the full-length subunit's current). The 3-domain-type Ca(v)1.2 subunit significantly inhibited wild-type Ca(v)1.2 current, but not other ionic currents such as Ca(v)2.1 and Na(v) channels in Xenopus oocyte expression systems. Western blot analysis showed that the expression of the wild-type protein into the plasma membrane was significantly suppressed on coexpression with the truncated protein. These findings support that an N-terminus-truncated mutant could serve as a specific genetically encoded inhibitor for L-channels.

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.

Simultaneous observation of stably associated presynaptic varicosities and postsynaptic spines: morphological alterations of CA3-CA1 synapses in hippocampal slice cultures.

Dendritic spines are highly motile structures, but the extent and mode of coordination in motility between spines and presynaptic varicosities with synaptic contacts is not clear. To analyze movements of dendritic spines and axonal varicosities simultaneously, we labeled CA1 pyramidal cells with green fluorescent protein and CA3 pyramidal cells with rhodamine-dextran in hippocampal slice cultures. Varicosities and spines were visualized using two-photon microscopy to detect close association of two components. Time-lapse imaging revealed that they performed rapid morphological changes without losing their contacts. The extent of overall structural changes between varicosities and spines was correlated, while the direction of short-term volume changes was regulated independently. Furthermore, alterations of dendritic morphology induced by strong electrical stimulation had little effects on their association. These results indicate the presence of local regulatory mechanisms to coordinate presynaptic and postsynaptic motility.

The ascidian dihydropyridine-resistant calcium channel as the prototype of chordate L-type calcium channel.

This review describes recent findings on voltage-gated Ca channel (Cav channel) cloned from ascidians, the most primitive chordates. Ascidian L-type like Cav channel has several unusual features: (1). it is closely related to the prototype of chordate L-type Cav channels by sequence alignment; (2). it is resistant to dihydropyridine due to single amino acid change in the pore region, and (3). maternally provided RNA putatively encodes a truncated protein which has remarkable suppressive effect on Cav channel expression during development. Ascidian Cav channel will provide a useful molecular clue in the future to understand Ca(2+)-regulated cell differentiation and physiology with the background of recently defined ascidian genome and molecular biological tools.