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Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 March 2021) | Viewed by 19850

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Dr. Chikara Sato

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Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Interests: electron microscopy; liquid-phase EM; cryo-EM, molecular complex; biophysics; neural network; nanostructure
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Special Issue Information

Dear Colleagues,

The high-resolution observation of biological specimens and material phenomena in liquid is a fascinating field that has a huge frontier. Recent advances in the manufacture of tough electron-transparent films have realized the stable observation of samples by electron microscopy, delivering significant findings in many research fields. This Special Issue will include results from bio-related fields and material sciences and consider the future direction of the electron microscopy of specimens in liquid. The observation of biological samples, the nano-structures of materials and their dynamics, are of high interest for this Special Issue. We also aim to address experimental and technical challenges that have to be met in order to use an instrument that requires a vacuum environment to image a specimen in liquid. We envisage reports of novel specimen preparation methods and adapted instrumentation, e.g., the development of new specimen holders that include a micro-fluidics chamber and of electro-chemical holders that allow phenomena to be clarified at a molecular level and, of course, the modification of scanning, transmission and scanning transmission electron microscopes (TEM, STEM, and SEM) for their use. The study of metal sedimentation and electrochemical reactions are also of high interest for this Special Issue. Contributions relevant to basic biology, applied biology, and diagnostic research (because they exploit the speed of liquid-phase electron microscopy) are encouraged. Partial clinical research and survey studies are not within the scope of this Special Issue.

Dr. Chikara Sato
Guest Editor

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Keywords

Environmental cell
Immuno-electron microscopy
Cell signaling
Comparative anatomy
Diagnosis
Molecular recognition
Molecular complexes
Electro-chemistry
Liquid phase synthesis
Metal sedimentation

Published Papers (7 papers)

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Research

19 pages, 8892 KiB

Open AccessArticle

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae: 2. Role of Filamentous Extracellular Network and Cell-to-Cell Connections at the Biofilm Surface

by Keiko Sato, Masami Naya, Yuri Hatano, Naoki Kasahata, Yoshio Kondo, Mari Sato, Katsuki Takebe, Mariko Naito and Chikara Sato

Int. J. Mol. Sci. 2021, 22(13), 6911; https://doi.org/10.3390/ijms22136911 - 27 Jun 2021

Cited by 4 | Viewed by 2192

Abstract

Flavobacterium johnsoniae forms a thin spreading colony on nutrient-poor agar using gliding motility. As reported in the first paper, WT cells in the colony were sparsely embedded in self-produced extracellular polymeric matrix (EPM), while sprB cells were densely packed in immature biofilm with less matrix. The colony surface is critical for antibiotic resistance and cell survival. We have now developed the Grid Stamp-Peel method whereby the colony surface is attached to a TEM grid for negative-staining microscopy. The images showed that the top of the spreading convex WT colonies was covered by EPM with few interspersed cells. Cells exposed near the colony edge made head-to-tail and/or side-to-side contact and sometimes connected via thin filaments. Nonspreading sprB and gldG and gldK colonies had a more uniform upper surface covered by different EPMs including vesicles and filaments. The EPM of sprB, gldG, and WT colonies contained filaments ~2 nm and ~5 nm in diameter; gldK colonies did not include the latter. Every cell near the edge of WT colonies had one or two dark spots, while cells inside WT colonies and cells in SprB-, GldG-, or GldK-deficient colonies did not. Together, our results suggest that the colony surface structure depends on the capability to expand biofilm. Full article

(This article belongs to the Special Issue Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences)

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17 pages, 4986 KiB

Open AccessArticle

Ca²⁺-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum

by Jun Nakamura, Yuusuke Maruyama, Genichi Tajima, Yuto Komeiji, Makiko Suwa and Chikara Sato

Int. J. Mol. Sci. 2021, 22(5), 2624; https://doi.org/10.3390/ijms22052624 - 5 Mar 2021

Cited by 5 | Viewed by 2289

Abstract

The Ca²⁺-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca²⁺, i.e., in the presence of ATP and ≤ 0.9 nM Ca²⁺, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca²⁺ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca²⁺ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction. Full article

(This article belongs to the Special Issue Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences)

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15 pages, 3651 KiB

Open AccessArticle

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm

by Keiko Sato, Masami Naya, Yuri Hatano, Yoshio Kondo, Mari Sato, Keiji Nagano, Shicheng Chen, Mariko Naito and Chikara Sato

Int. J. Mol. Sci. 2021, 22(4), 1894; https://doi.org/10.3390/ijms22041894 - 14 Feb 2021

Cited by 4 | Viewed by 2711

Abstract

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms. Full article

(This article belongs to the Special Issue Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences)

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22 pages, 4314 KiB

Open AccessArticle

Supra-Molecular Assemblies of ORAI1 at Rest Precede Local Accumulation into Puncta after Activation

by Diana B. Peckys, Daniel Gaa, Dalia Alansary, Barbara A. Niemeyer and Niels de Jonge

Int. J. Mol. Sci. 2021, 22(2), 799; https://doi.org/10.3390/ijms22020799 - 14 Jan 2021

Cited by 4 | Viewed by 2060

Abstract

The Ca²⁺ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca²⁺ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation. Full article

(This article belongs to the Special Issue Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences)

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19 pages, 15552 KiB

Open AccessArticle

EGFR Expression in HER2-Driven Breast Cancer Cells

by Florian Weinberg, Diana B. Peckys and Niels de Jonge

Int. J. Mol. Sci. 2020, 21(23), 9008; https://doi.org/10.3390/ijms21239008 - 27 Nov 2020

Cited by 15 | Viewed by 4225

Abstract

The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5–5) × 10¹ QDs/µm², whereas labeled HER2 expression was higher ranging from (2–10) × 10² QDs/µm². Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells. Full article

(This article belongs to the Special Issue Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences)

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14 pages, 3140 KiB

Open AccessArticle

Pyrene Excimer-Based Fluorescent Labeling of Cysteines Brought into Close Proximity by Protein Dynamics: ASEM-Induced Thiol-Ene Click Reaction for High Spatial Resolution CLEM

by Masami Naya and Chikara Sato

Int. J. Mol. Sci. 2020, 21(20), 7550; https://doi.org/10.3390/ijms21207550 - 13 Oct 2020

Cited by 9 | Viewed by 2917

Abstract

Fluorescence microscopy (FM) has revealed vital molecular mechanisms of life. Mainly, molecules labeled by fluorescent probes are imaged. However, the diversity of labeling probes and their functions remain limited. We synthesized a pyrene-based fluorescent probe targeting SH groups, which are important for protein folding and oxidative stress sensing in cells. The labeling achieved employs thiol-ene click reactions between the probes and SH groups and is triggered by irradiation by UV light or an electron beam. When two tagged pyrene groups were close enough to be excited as a dimer (excimer), they showed red-shifted fluorescence; theoretically, the proximity of two SH residues within ~30 Å can thus be monitored. Moreover, correlative light/electron microscopy (CLEM) was achieved using our atmospheric scanning electron microscope (ASEM); radicals formed in liquid by the electron beam caused the thiol-ene click reactions, and excimer fluorescence of the labeled proteins in cells and tissues was visualized by FM. Since the fluorescent labeling is induced by a narrow electron beam, high spatial resolution labeling is expected. The method can be widely applied to biological fields, for example, to study protein dynamics with or without cysteine mutagenesis, and to beam-induced micro-fabrication and the precise post-modification of materials. Full article

(This article belongs to the Special Issue Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences)

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10 pages, 2767 KiB

Open AccessArticle

Network of Palladium-Based Nanorings Synthesized by Liquid-Phase Reduction Using DMSO-H2O: In Situ Monitoring of Structure Formation and Drying Deformation by ASEM

by Takuki Komenami, Akihiro Yoshimura, Yasunari Matsuno, Mari Sato and Chikara Sato

Int. J. Mol. Sci. 2020, 21(9), 3271; https://doi.org/10.3390/ijms21093271 - 5 May 2020

Cited by 4 | Viewed by 2691

Abstract

We developed a liquid-phase synthesis method for Pd-based nanostructure, in which Pd dissolved in dimethyl sulfoxide (DMSO) solutions was precipitated using acid aqueous solution. In the development of the method, in situ monitoring using atmospheric scanning electron microscopy (ASEM) revealed that three-dimensional (3D) Pd-based nanonetworks were deformed to micrometer-size particles possibly by the surface tension of the solutions during the drying process. To avoid surface tension, critical point drying was employed to dry the Pd-based precipitates. By combining ASEM monitoring with critical point drying, the synthesis parameters were optimized, resulting in the formation of lacelike delicate nanonetworks using citric acid aqueous solutions. Precipitation using HCl acid aqueous solutions allowed formation of 500-nm diameter nanorings connected by nanowires. The 3D nanostructure formation was controllable and modifiable into various shapes using different concentrations of the Pd and Cl ions as the parameters. Full article

(This article belongs to the Special Issue Liquid-Phase Electron Microscopy in Biological Applications and Material Sciences)

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