As sessile organisms, plants must constantly adapt to ever-changing environmental conditions. To survive in their habitats, plants have evolved characteristic cellular features that make the cells rigid yet dynamic. These include the cell wall, large vacuole, and cytoplasmic streaming. The cell wall is an elaborate extracellular matrix that surrounds plant cells and provides both physical strength and protection against external forces. The large vacuole is a membrane-bound organelle absent in animal cells. They can absorb water and expand, thereby exerting a force on the cell wall from within and generating turgor pressure that promotes cell expansion. In the narrow cytoplasmic space between the vacuole and the cell wall, intracellular components circulate via rapid flows, a phenomenon known as cytoplasmic streaming. In this review, we summarize how these three characteristic features of plant cells are organized with the help of cytoskeletal elements. This review article is an extended version of the Japanese article, “Cell Wall,” “Large Vacuole,” & “Cytoplasmic Streaming”: How Do Cytoskeletons Build Plant Cells with Unique Physical Properties?” by Takatsuka et al., published in SEIBUTSU BUTSURI Vol. 64, p. 132–136 (2024).

Plant cells are enclosed by rigid cell walls and contain large vacuoles within. Due to these physical characteristics, the intracellular environment of plant cells is highly crowded. Under such extreme conditions, plant cells generate a directed flow of cytoplasm known as cytoplasmic streaming in order to enable large-scale movement of intracellular structures, thereby maintaining intracellular dynamics. In this review, we highlight how the plant cytoskeletons, composed of actin and microtubules, govern these facets to establish the characteristic properties of plant cells—rigid externally yet dynamic internally.

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Chrimson is cation-conducting channelrhodopsin (CCR) with the most red-shifted absorption spectrum, rendering itself as one of the most promising optogenetic tools. However, the molecular mechanisms underlying its red-shifted absorption have not been completely clarified yet. Here, we found a CCR gene showing high sequence similarity to Chrimson from Lake Hula through freshwater metatranscriptome sampling. Interestingly, despite its high similarity to Chrimson, this CCR—named HulaChrimson—showed significantly blue-shifted action and absorption spectra compared to those of Chrimson. Mutations of amino acid residues, which are prominently different from those in Chrimson, in HulaChrimson did not reproduce the red-shifted absorption of Chrimson, suggesting the color-tuning between these proteins achieved by organizing the entire protein architecture, particularly in the broad hydrogen bonding network around the retinal Schiff base counterion, rather than by the difference in several specific residues. The optical characteristics of HulaChrimson distinct from those of Chrimson provide a basis for understanding the color-tuning mechanisms of channelrhodopsins.

HulaChrimson, a cation channelrhodopsin with high similarity to the red-shifted channelrhodopsin Chrimson, was found to exhibit a considerably blue-shifted absorption spectrum compared to Chrimson, and this spectral difference is attributed to distinct color-tuning mechanisms.

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Predicting the binding affinity between proteins and ligands is a critical task in drug discovery. Although various computational methods have been proposed to estimate ligand target affinity, the method of Yasuda et al. (2022) ranks affinities based on the dynamic behavior obtained from molecular dynamics (MD) simulations without requiring structural similarity among ligand substituents. Thus, its applicability is broader than that of relative binding free energy calculations. However, their approach suffers from high computational costs due to the extensive simulation time and the deep learning computations needed for each ligand pair. Moreover, in the absence of experimental ΔG values (oracle), the sign of the correlation can be misinterpreted. In this study, we present an alternative approach inspired by Yasuda et al.’s method, offering an alternative perspective by replacing the distance metric and reducing computational cost. Our contributions are threefold: (1) By introducing the Jensen–Shannon (JS) divergence, we eliminate the need for deep learning-based similarity estimation, thereby significantly reducing computation time; (2) We demonstrate that production run simulation times can be halved while maintaining comparable accuracy; and (3) We propose a method to predict the sign of the correlation between the first principal component (PC1) and ΔG by using coarse ΔG estimations obtained via AutoDock Vina.

This study proposes a method to estimate protein-ligand binding affinity using molecular dynamics (MD) simulation trajectories. Structural fluctuations of protein-ligand complexes are quantified by computing the Jensen-Shannon (JS) divergence between trajectory-derived distributions. The resulting similarity matrix is subjected to principal component analysis (PCA), where the primary components reflect the diversity in protein responses to different ligands. By projecting each ligand onto this reduced space, we investigate correlations between the PCA axes and experimental binding free energies. This framework enables rapid comparison of ligand-induced protein dynamics and offers a computationally efficient approach to prioritize compounds based on their dynamic impact.

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Quality assessment and characterization of liquid milk by means of electronic sensor remains an intensive area of research. In this work, cylinder-in-cylinder type sample holder is fabricated to measure the bioimpedance of milk as a function of frequency. Experiments on bioimpedance spectroscopy were conducted during adulteration (at control temperature, 23°C) of branded (pasteurized) milk as well as raw milk. Cole equivalent circuit is considered as a characterizing model for milk. Cole parameters were extracted from the experimental data. From Cole parameters, relaxation-time was estimated and state of the milk sample has been expressed in terms of relaxation-time. Analysis of variance was performed on relaxation-time of the samples to gain statistical significance. Our method is capable to discriminate liquid milk from different commercial brands. It was found that the relaxation-time decreases monotonically with the progression of adulteration time for all kinds of milk considered in this study. From the changes in relaxation-time, the adulteration was found to be significant in the first three hours. Hence it is not advisable to consume milk after two hours of adulteration if kept at 23°C. Dominant biochemical pathways responsible for adulteration of milk are also presented.

Cole equivalent circuit is considered for the bioimpedance spectroscopy (BIS) study of milk. All Cole parameters are experimentally estimated to characterize the state of the milk undergoing natural adulteration process. Relative changes in relaxation-time is considered as the monitoring parameter for adulteration. In order to gain statistical relevance, multiple measurement (replica) is conducted and ANOVA is incorporated in the BIS study. A simple, low-cost and cylinder-in-cylinder type sample holder is fabricated to study BIS of liquid sample. As an alternative to traditional pathological test, shelf-life of milk is estimated by electrical test.

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This study aimed to elucidate the impact of polyethylene glycol (PEG) conjugation on protein-protein interactions by investigating the properties of PEG-conjugated actin (PEG-actin). Various PEG molecules were covalently bound to actin monomers by reacting maleimide groups with Cys374 on the actin. The apparent polymerization rate constant (k_on^app) and the critical concentration (Cc) were measured by fluorescence spectroscopy using pyrene-labeled actin, as a function of the portion of PEG-actin and the molecular mass of the conjugated PEG (750 to 10000 Da). The k_on^app gradually decreased as the percentage of PEG-actin increased. At 90% PEG-actin, the k_on^app decreased substantially as the PEG size increased, resulting from a modulation of C-terminus by the conjugated PEGs and their steric hindrance. The Cc was slightly increased by PEG conjugation in the content by up to 50%. Meanwhile, 90% PEG-actin exhibited a substantial increase in Cc. The Cc was almost linearly related to the gyration radius of PEG. These results suggest that the PEG conjugation to actin impedes the association of actin with the filament in a PEG size-dependent manner. Furthermore, the stability of PEG-actin against an extrinsic factor was assessed. PEG-actins >2000 Da were more susceptible to digestion than intact actin when the PEG-actin monomer was subjected to α-chymotrypsin. Thus, conjugation of PEG to Cys374 on actin did not protect actin monomers against their proteolysis by α-chymotrypsin.

The critical concentration of actin increases with the size of polyethylene glycol (PEG) conjugated to actin. The left picture shows a schematic diagram of part of an actin filament copolymerized with PEG-conjugated actin and intact actin. The 5-kDa PEG has a gyration radius of 3 nm, and the distance between neighboring actin components in long-helix of the filament is approximately 6 nm. The graph on the right shows the relationship between the critical concentration and the gyration radius of PEG in the case of 90% content of PEG-conjugated actin.

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The crystal structure of Rubrobacter xylanophilus rhodopsin (RxR) reveals a triangular cluster of three water molecules (W413, W415, and W419) at the extracellular proton-release site, near Glu187 and Glu197. Using a quantum mechanical/molecular mechanical approach, we identified the structural nature of this unique water cluster. The triangular shape is best reproduced when all three water molecules are neutral H₂O with protonated Glu187 and deprotonated Glu197. Attempts to place H₃O⁺ at any of these water molecules result in spontaneous proton transfer to one of the acidic residues and significant distortion from the crystal structure. The plane defined by the triangular water cluster extends into the guanidinium plane of Arg71, with both aligned along the W413...W419 axis. This extended plane lies nearly perpendicular to a five-membered, ring-like H-bond network involving two carboxyl oxygen atoms from Glu187 and one from Glu197. The resulting bipartite planar architecture, defined by the water triangle, Arg71, and the Glu187/Glu197 network may reflect the exceptional thermal stability in RxR.

A compact, triangular water cluster at the proton-release site of Rubrobacter xylanophilus rhodopsin (RxR), stabilized by Glu187, Glu197, and Arg71. Quantum mechanical/molecular mechanical (QM/MM) calculations indicate that three neutral water molecules form this geometry, without involving H₃O⁺. This architecture differs from that of bacteriorhodopsin and may relate to the exceptional thermal stability of RxR.

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The bacterial peptidoglycan layer plays an important role in protecting the bacteria from turgor pressure, viruses, and predators. However, it also acts as a barrier in transmitting forces generated on the cell membrane to adhesion proteins on the surface during gliding locomotion. In this study, peptidoglycan layers were isolated from two species of gliding diderm, i.e., gram-negative bacteria, and their structures were visualized by quick-freeze deep-etch replica electron microscopy. The horizontal bonding of peptidoglycan did not differ obviously among the three species. However, the diameter of pores in the peptidoglycan layer of M. xanthus and the area of surface pores were 51 nm and 14.6%, respectively, which were significantly larger than those of E. coli (32 nm and 5.8%) and F. johnsoniae (29 nm and 7.0%). Based on this, we discussed the mechanism by which diderm bacteria transmit forces across the PG layer to the bacterial surface.

This graphycal abstract shows peptidoglycan sacs isolated from two diderm bacteria. The boxed areas in the left panels are magnified as right panels. Myxococcus xanthus sac showed pore sizes larger than Escherichia coli ones. No directionality of pores against cell axis was detected as shown by FFT analysis shown in the right upper.

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