A mechanism with severing near barbed ends and annealing explains structure and dynamics of dendritic actin networks.

Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/-35° orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology.

A cryo-TSEM with temperature cycling capability allows deep sublimation of ice to uncover fine structures in thick cells.

The scanning electron microscope (SEM) has been reassembled into a new type of cryo-electron microscope (cryo-TSEM) by installing a new cryo-transfer holder and anti-contamination trap, which allowed simultaneous acquisition of both transmission images (STEM images) and surface images (SEM images) in the frozen state. The ultimate temperatures of the holder and the trap reached - 190 °C and - 210 °C, respectively, by applying a liquid nitrogen slush. The STEM images at 30 kV were comparable to, or superior to, the images acquired with conventional transmission electron microscope (100 kV TEM) in contrast and sharpness. The unroofing method was used to observe membrane cytoskeletons instead of the frozen section and the FIB methods. Deep sublimation of ice surrounding unroofed cells by regulating temperature enabled to emerge intracellular fine structures in thick frozen cells. Hence, fine structures in the vicinity of the cell membrane such as the cytoskeleton, polyribosome chains and endoplasmic reticulum (ER) became visible. The ER was distributed as a wide, flat structure beneath the cell membrane, forming a large spatial network with tubular ER.

Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms.

Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.

LSPR-mediated high axial-resolution fluorescence imaging on a silver nanoparticle sheet.

This paper reports our original technique for visualizing cell-attached nanointerfaces with extremely high axial resolution using homogeneously excited localized surface plasmon resonance (LSPR) on self-assembled silver nanoparticle sheets. The LSPR sheet can confine and enhance the fluorescence at the nanointerface, which provides high signal-to-noise ratio images of focal adhesion at the cell-attached interface. The advantage of this LSPR-assisted technique is its usability, which provides comparable or higher-quality nanointerfacial images than TIRF microscopy, even under epifluorescence microscopy. We also report the cytotoxicity of silver nanoparticles, as determined via morphological analysis of adherent cells on the sheet.

A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM).

The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.

High-resolution imaging of a cell-attached nanointerface using a gold-nanoparticle two-dimensional sheet.

This paper proposes a simple, effective, non-scanning method for the visualization of a cell-attached nanointerface. The method uses localized surface plasmon resonance (LSPR) excited homogeneously on a two-dimensional (2D) self-assembled gold-nanoparticle sheet. The LSPR of the gold-nanoparticle sheet provides high-contrast interfacial images due to the confined light within a region a few tens of nanometers from the particles and the enhancement of fluorescence. Test experiments on rat basophilic leukemia (RBL-2H3) cells with fluorescence-labeled actin filaments revealed high axial and lateral resolution even under a regular epifluorescence microscope, which produced higher quality images than those captured under a total internal reflection fluorescence (TIRF) microscope. This non-scanning-type, high-resolution imaging method will be an effective tool for monitoring interfacial phenomena that exhibit relatively rapid reaction kinetics in various cellular and molecular dynamics.

A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy.

An improved unroofing method consisting of tearing off the cell membrane using an adhesive electron microscopy (EM) grid instead of vitreous ice sectioning (cryo-sectioning) has enabled us to panoramically view the membrane cytoskeleton in its native state with extremely high contrast. Grids pre-treated with Alcian blue were placed on cells, and a portion of the dorsal plasma membrane was transferred onto the grid, which was then floated in buffer solution. These membrane fragments contained sufficient cytoskeleton and were of suitable thickness for observation by cryo-EM. Many actin filaments and microtubules were clearly observed on the cytoplasmic surface of the plasma membrane with extremely high contrast because the soluble components of the cytoplasm flowed out and broke away from the cells. Actin filaments extended in all directions in a smooth contour with little branching. Microtubules spread out as far as 3 µm or more while winding gently in their native state. Upon fixation with 1% glutaraldehyde, however, the microtubules became straight and fragmented. Cryo-EM revealed for the first time a smooth endoplasmic reticulum network beneath the cell membrane in native cells. Clathrin coats and caveolae were also observed on the cytoplasmic surface of the plasma membrane, similar to those seen using freeze-etching replica EM (freeze-etching EM). Unroofing was also useful for immuno-labelling in cryo-EM. Antibody-labelled IQGAP1, one of the effector proteins facilitating the formation of actin filament networks, was localized alongside actin filaments. Freeze-etching EM confirmed the morphological findings of cryo-EM.

An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy.

An improved unroofing method enabled the cantilever of an atomic force microscope (AFM) to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in phosphate-buffered saline (PBS) at a higher resolution than conventional electron microscopy. All of the actin filaments clearly exhibited a short periodicity of approximately 5-6 nm, which was derived from globular actins linked to each other to form filaments, as well as a long helical periodicity. The polarity of the actin filaments appeared to be determined by the shape of the periodic striations. Microtubules were identified based on their thickness. Clathrin coats and caveolae were observed on the cytoplasmic surface of cell membranes. The area containing clathrin molecules and their terminal domains was directly visualized. Characteristic ridge structures located at the surface of the caveolae were observed at high resolution, similar to those observed with electron microscopy (EM). Overall, unroofing allowed intracellular AFM imaging in a liquid environment with a level of quality equivalent or superior to that of EM. Thus, AFMs are anticipated to provide cutting-edge findings in cell biology and histology.

Direct observation of the actin filament by tip-scan atomic force microscopy.

Actin filaments, the actin-myosin complex and the actin-tropomyosin complex were observed by a tip-scan atomic force microscope (AFM), which was recently developed by Olympus as the AFM part of a correlative microscope. This newly developed AFM uses cantilevers of similar size as stage-scan AFMs to improve substantially the spatial and temporal resolution. Such an approach has previously never been possible by a tip-scan system, in which a cantilever moves in the x, y and z directions. We evaluated the performance of this developed tip-scan AFM by observing the molecular structure of actin filaments and the actin-tropomyosin complex. In the image of the actin filament, the molecular interval of the actin subunits (∼5.5 nm) was clearly observed as stripes. From the shape of the stripes, the polarity of the actin filament was directly determined and the results were consistent with the polarity determined by myosin binding. In the image of the actin-tropomyosin complex, each tropomyosin molecule (∼2 nm in diameter) on the actin filament was directly observed without averaging images of different molecules. Each tropomyosin molecule on the actin filament has never been directly observed by AFM or electron microscopy. Thus, our developed tip-scan AFM offers significant potential in observing purified proteins and cellular structures at nanometer resolution. Current results represent an important step in the development of a new correlative microscope to observe nm-order structures at an acceptable frame rate (∼10 s/frame) by AFM at the position indicated by the fluorescent dye observed under a light microscope.

Torque generation and utilization in motor enzyme F0F1-ATP synthase: half-torque F1 with short-sized pushrod helix and reduced ATP Synthesis by half-torque F0F1.

ATP synthase (F(0)F(1)) is made of two motors, a proton-driven motor (F(0)) and an ATP-driven motor (F(1)), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F(1), the central γ subunit rotates inside the α(3)ß(3) ring. Here we report structural features of F(1) responsible for torque generation and the catalytic ability of the low-torque F(0)F(1). (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic ß subunit at the rotor/stator contact region generates mutant F(1)s, termed F(1)(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid "pushrod" to push the rotor γ subunit, but the short helix in F(1)(1/2)s would fail to accomplish this task. (ii) Three different half-torque F(0)F(1)(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F(0)F(1)(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F(0)F(1)(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants.