Ryo Suzuki

Traditional amide I spectral decomposition requires prior specification of component number and initial parameters from previous studies. Here, we apply Bayesian free-energy model selection to compare Gaussian models with different numbers of components for a coherent anti-Stokes Raman scattering (CARS)-derived spontaneous-Raman-equivalent Im[χ(3)] spectrum. When applied to the amide I spectrum of a drug-treated binucleated human mammary epithelial cell (HMEC), the four-component model showed the lowest Bayesian free energy and the highest posterior model probability of 72.4% among the tested candidate models. In a separate dataset from an untreated mononucleated HMEC, Bayesian free energy exhibited internal minima at K = 3 for the amide I region and K = 6 for the CH stretching region, whereas RSS decreased monotonically with increasing model complexity in both cases. This workflow reduces user-dependent bias and improves the reproducibility of spectral decomposition in single-cell CARS analysis when the likelihood, prior ranges, candidate models, and preprocessing procedures are explicitly specified.

The “eat me” signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is activated by caspase-mediated cleavage and binding of the XRCC4 fragment. Here, we show that extracellular calcium is an additional factor needed to activate Xkr4. The constitutively active mutant of Xkr4 is found to induce phospholipid scrambling in an extracellular, but not intracellular, calcium-dependent manner. Importantly, other Xkr family members also require extracellular calcium for activation. Alanine scanning shows that D123 and D127 of TM1 and E310 of TM3 coordinate calcium binding. Moreover, lysine scanning demonstrates that the E310K mutation-mediated salt bridge between TM1 and TM3 bypasses the requirement of calcium. Cysteine scanning proves that disulfide bond formation between TM1 and TM3 also activates phospholipid scrambling without calcium. Collectively, this study shows that extracellular calcium functions as a molecular glue for TM1 and TM3 of Xkr proteins for activation, thus demonstrating a regulatory mechanism for multi-transmembrane region-containing proteins.

Using three-dimensional scanning force microscopy (3D-SFM) combined with frequency modulation detection, we detected the cadmium ion-specific nanoscale compaction of poly(acrylic acid) brushes modified with cysteine.

Dynamic spatiotemporal patterns that arise from out-of-equilibrium biochemical reactions generate forces in living cells. Despite considerable recent efforts, rational design of spatiotemporal patterns in artificial molecular systems remains at an early stage of development. Here, we describe force generation by a propagating wave of supramolecular nanofibers. Inspired by actin dynamics, a reaction network is designed to control the formation and degradation of nanofibers by two chemically orthogonal stimuli. Real-time fluorescent imaging successfully visualizes the propagating wave based on spatiotemporally coupled generation and collapse of nanofibers. Numerical simulation indicates that the concentration gradient of degradation stimulus and the smaller diffusion coefficient of the nanofiber are critical for wave emergence. Moreover, the force (0.005 pN) generated by chemophoresis and/or depletion force of this propagating wave can move nanobeads along the wave direction.

Biological cells in tissues adapt their shapes, positions, and orientations in response to dynamic changes in their physical microenvironments. Here, we investigated the dynamic response of myoblast cells by fabricating substrates displaying microwrinkles that can reversibly change the direction within 60 s by axial compression and relaxation. To quantitatively assess the collective order of cells, we introduced the nematic order parameter of cells that takes not only the distribution of cell-wrinkle angles but also the degree of cell elongation into account. On the sub-cellular level, we also calculated the nematic order parameter of actin cytoskeletons that takes the rearrangement of actin filaments into consideration. The results obtained on substrates with different wrinkle wavelengths implied the presence of a characteristic wavelength beyond which the order parameters of both cells and actin cytoskeletons level off. Immunofluorescence labeling of vinculin showed that the focal adhesions were all concentrated on the peaks of wrinkles when the wavelength is below the characteristic value. On the other hand, we found the focal adhesions on both the peaks and the troughs of wrinkles when the wavelength exceeds the characteristic level. The emergence of collective ordering of cytoskeletons as well as the adaptation of cell shapes and orientations were monitored by live cell imaging after the seeding of cells from suspensions. After the cells had reached the steady state, the orientation of wrinkles was abruptly changed by 90 °. The dynamic response of myoblasts to the drastic change in surface topography was monitored, demonstrating the coordination of the shape and orientation of cells and the nematic ordering of actin cytoskeletons. The “dynamic” substrates established in this study can be used as a powerful tool in mechanobiology that helps us understand how cytoskeletons, cells, and cell ensembles respond to the dynamic contact guidance cues.

A balance between motion and cooperation: In active matter systems, the infusion of energy and motion drives ordering processes. Huber et al. present a combination of experiments and numerical simulations on an active matter system consisting of actin filaments propelled across a surface by surface-attached myosin motor proteins. Adding a depletion agent—polymer chains that weakened interactions between the actin filaments—drove the system between ferromagnetic (polar) and nematic (liquid crystal) ordering. Science , this issue p. 255

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca²⁺ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca²⁺ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

Most living systems, ranging from animal flocks, self-motile microorganisms to the cytoskeleton rely on self-organization processes to perform their own specific function. Despite its importance, the general understanding of how individual active constituents initiate the intriguing pattern formation phenomena on all these different length scales still remains elusive. Here, using a high density actomyosin motility assay system, we show that the observed collective motion arises from a seeding process driven by enhanced acute angle collisions. Once a critical size is reached, the clusters coarsen into high and low density phases each with fixed filament concentrations. The steady state is defined by a balance of collision induced randomization and alignment effects of the filaments by multi-filament collisions within ordered clusters.

Regeneration of a tissue fragment of freshwater polyp Hydra is accompanied by significant morphological fluctuations, suggesting the generation of active forces. In this study, we utilized a two fingered micro-robotic hand to gain insights into the mechanics of regenerating tissues. Taking advantage of a high force sensitivity (∼1 nN) of our micro-hand, we non-invasively acquired the bulk elastic modulus of tissues by keeping the strain levels low (ε < 0.15). Moreover, by keeping the strain at a constant level, we monitored the stress relaxation of the Hydra tissue and determined both viscous modulus and elastic modulus simultaneously, following a simple Maxwell model. We further investigated the correlation between the frequency of force fluctuation and that of morphological fluctuation by monitoring one “tweezed” tissue and the other “intact” tissue at the same time. The obtained results clearly indicated that the magnitude and periodicity of the changes in force and shape are directly correlated, confirming that our two fingered micro-hand can precisely quantify the mechanics of soft, dynamic tissue during the regeneration and development in a non-invasive manner.

Significance: Biological active systems like swimming bacteria or molecular motors in the cell cytoskeleton consume energy locally to create movements or stresses. Although the surrounding fluid gives rise to thermal fluctuations of the system’s dynamic variables, the corresponding statistics can deviate significantly from a thermal Boltzmann distribution. To quantify these differences, we compare the statistics and dynamics of actin filaments driven by molecular motors with Brownian filaments. In both cases, energy injection is local and random; however, for active filaments, motors can generate sudden jump-like changes in the system’s dynamic variables, leading to nonequilibrium energy dissipation. Motor activity results in a nonthermal distribution of filament curvatures and a faster relaxation of filament bends than in the thermal case.