Tatsuhiko Ebihara

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.

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.

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.

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 Ca2+-regulated cell differentiation and physiology with the background of recently defined ascidian genome and molecular biological tools.

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 cells 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.

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-Iβ 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.

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.

Atmospheric Electron Microscope: Limits of Observable Depth

The new Atmospheric Scanning Electron Microscope (ASEM) is able to directly observe cells fixed in a culture medium under atmospheric pressure [1]. In this system, an electron-permeable window made of pressure-resistant film allows an electron beam to be projected from underneath the sample. The electrons backscattered from the samples are captured by a detector positioned below. In the process, since the electron beam is scattered first by the thin film and further by the sample, the depth of observation is restricted. In this study, we theoretically and experimentally investigate the ASEMs depth limits.

Coexpression of a Cav1.2 protein lacking an N-terminus and the first domain specifically suppresses L-type calcium channel activity

L‐type Ca2 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 Cav1.2 was designed by mimicking an ascidian 3‐domain‐type α1 subunit (that inhibits the full‐length subunits current). The 3‐domain‐type Cav1.2 subunit significantly inhibited wild‐type Cav1.2 current, but not other ionic currents such as Cav2.1 and Nav 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.

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 α1‐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 Ala1016 at the P‐region of domain III in TuCa1 (TuCa1/A1016S) and functionally expressed the channel in Xenopus oocyte along with rabbit α2/δ and β2b. 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 Ser1016 in TuCa1/A1016S participates in DHP binding, but there exist other novel sites that fully acquire sensitivity to FPL‐64176.