In order to respond to external stimuli and their own internal states, cells recognize these information through intracellular signal transduction. Although the molecular players forming the signaling network have been elucidated, it has recently attracted attention that information of external stimuli and internal states is encoded in the temporal dynamics of cell signaling. Live cell imaging is a very powerful tool to measure the dynamics of signal transduction, and the development of various biosensors allows us to understand the mechanisms underlying complex biological phenomena. In addition, the advent of optogenetics has made it possible to apply reversible manipulation of signal transduction with observing the change in cell signaling concomitantly. In this review, we introduce the state-of-the-art live cell imaging by genetically encoding biosensors and signaling manipulation by optogenetics.

Rho and Ras family small GTPases are activated by extracellular stimuli and regulate numerous cellular processes, including cell motility, proliferation, and differentiation. Although biosensors have revealed the distribution of their intracellular activity, it is necessary to manipulate their activity and observe cellular responses to understand their functions accurately. Optogenetics is a technology that uses light-responsive proteins to manipulate the activity of signaling molecules by light irradiation. Optogenetics has made it possible to manipulate small GTPases in living cells with high spatiotemporal resolution. In this article, we review the manipulation of small GTPases by optogenetics and observation of their intracellular functions using biosensors, focusing on our recent study in which we constructed optogenetic tools to control six members of small GTPases and analyzed the crosstalk between small GTPases and intracellular calcium signaling.

It is known that activation of Rac1, a small GTPase molecular switch, is essential for various actin-dependent cell motilities including cell migration, macropinocytosis, and phagocytosis. However, the significant role of its deactivation has not received much attention. Using the optogenetics of photoactivatable (PA)-Rac1, we can reversibly turn on and off the Rac1 switch by irradiating a live cell with blue light. Taking advantage of the features of PA-Rac1, we have analyzed the roles of Rac1 ON-OFF in the macropinocytosis and phagocytosis processes. In this paper, we described the spatiotemporal control of macropinosome and phagosome formation by Rac1 ON-OFF switching, focusing on our recent research using Rac1 optogenetics and live-cell microscopy.

Plasma membrane is associated with the underlying actin filaments. When the intracellular pressure increased or the actin filaments are locally disrupted, the membrane protrudes. This spherical membrane protrusion is called membrane blebs. Membrane blebs are often observed during cytokinesis and cell migration. Blebs regress when the actin cytoskeleton re-accumulates under the plasma membrane. However, it remains unclear how bleb expansion and retraction are regulated. In our previous study, we addressed this issue by using live imaging and demonstrated that the interplay between two small GTPases, Rnd3 and RhoA, is important for the establishment of bleb cycle. Recently, we report that the cytoplasmic fluidity is regulated in the blebbing cells; the cytoplasm of rapidly expanding membrane blebs is more disordered than the cytoplasm of retracting blebs. The increase of cytoplasmic fluidity in the expanding bleb is caused by a sharp rise in the calcium concentration. The STIM1-Orai1 pathway regulates this rapid and restricted increase of calcium in the expanding blebs. In this review, we focus on the molecular mechanism controlling cytoplasmic fluidity that enables dynamic morphological changes during membrane blebbing.

The development of a new generation of monochromators improves the energy resolution of electron energy loss spectroscopy (EELS) and makes it possible to analyze local electronic states with high spatial resolution by combining it with a spherical aberration-corrected scanning transmission electron microscope (STEM). In order to maximize the features of the STEM-EELS method, it is essential to measure high-quality spectra. In this paper, we first introduce a new method to dramatically improve the signal-to-noise ratio of spectra by accurately removing the dark noise of the spectrum detector (CCD). Then three recent works on the analysis of energy-loss near-edge fine structures are described; the atomic resolution hole mapping of the high-Tc superconductor, the extraction of local structure of the oxygen octahedron in the transition metal oxide and the analysis of carbon K-edge spectra of organic thin films. Finally, the study of surface plasmon polaritons of a branched silver nanorode is also shown. All the spectra shown here were measured by the monochromated STEM-EELS.

Negative staining method has played a key role in electron microscopic observation of biological samples, for more than half a century. This method does not require any special and expensive equipment, such as cryo-electron microscope or rapid freezing robot. It is a powerful and useful method that can acquire images with high contrast and good resolution, though the sample preparation technique itself is extremely simple and rapid. The target of negative staining method range from huge viruses to small protein complexes with a molecular weight of 100 k or less. By combining with single particle analysis, the 3D-structure of the macromolecular complex composed of proteins, nucleic acids, can be quickly obtained with a resolution of around 2 nm. This paper outlines the principle of the negative staining method, the actual sample preparation protocol, as well as image recording procedure of the electron microscopic image of macromolecular complex. Furthermore, we will introduce briefly the three-dimensional structure analysis of such complex by single particle analysis.

Nano-diffraction imaging (NDI) is a novel imaging technique based on scanning transmission electron microscopy (STEM). It uses a nanometer-size electron beam to scan across a specimen, and the electron diffraction (ED) pattern at each position is recorded onto a two-dimensional (2D) pixelated detector. Here, we used NDI to image the nanoscale spatial distribution and orientation of lamellar crystals of polyethylene (PE) without any pretreatment (e.g., RuO4 staining). 2D ED patterns were obtained for two PE samples featuring significantly different crystal orientations, i.e., non-oriented and oriented samples. Spot-like diffractions, corresponding to orthorhombic PE 110 and 200 peaks, were observed. The detailed analysis of the diffractions provides the spatial distribution and orientation of lamellar crystals at nanometer resolutions. Though no distinct morphologies were observed under conventional STEM, we identified a substantial difference in lamellar orientation between non-oriented and oriented samples. Such local structural information is key to understanding higher-level hierarchical elements, e.g., spherulite, but cannot be obtained by conventional diffraction/scattering methods.