Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin.

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin

Mechanical properties of epithelial cells in domes investigated using atomic force microscopy

As epithelial cells in vitro reach a highly confluent state, the cells often form a microscale dome-like architecture that encloses a fluid-filled lumen. The domes are stabilized by mechanical stress and luminal pressure. However, the mechanical properties of cells that form epithelial domes remain poorly characterized at the single-cell level. In this study, we used atomic force microscopy (AFM) to measure the mechanical properties of cells forming epithelial domes. AFM showed that the apparent Young’s modulus of cells in domes was significantly higher when compared with that in the surrounding monolayer. AFM also showed that the stiffness and tension of cells in domes were positively correlated with the apical cell area, depending on the degree of cell stretching. This correlation disappeared when actin filaments were depolymerized or when the ATPase activity of myosin II was inhibited, which often led to a large fluctuation in dome formation. The results indicated that heterogeneous actomyosin structures organized by stretching single cells played a crucial role in stabilizing dome formation. Our findings provide new insights into the mechanical properties of three-dimensional deformable tissue explored using AFM at the single-cell level

Pathogenic Roles of Cardiac Fibroblasts in Pediatric Dilated Cardiomyopathy

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure in children. Despite intensive genetic analyses, pathogenic gene variants have not been identified in most patients with DCM, which suggests that cardiomyocytes are not solely responsible for DCM. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. They have several roles in maintaining cardiac function; however, the pathological role of CFs in DCM remains unknown. METHODS AND RESULTS: Four primary cultured CF cell lines were established from pediatric patients with DCM and compared with 3 CF lines from healthy controls. There were no significant differences in cellular proliferation, adhesion, migration, ap-optosis, or myofibroblast activation between DCM CFs compared with healthy CFs. Atomic force microscopy revealed that cellular stiffness, fluidity, and viscosity were not significantly changed in DCM CFs. However, when DCM CFs were cocultured with healthy cardiomyocytes, they deteriorated the contractile and diastolic functions of cardiomyocytes. RNA sequencing revealed markedly different comprehensive gene expression profiles in DCM CFs compared with healthy CFs. Several hu-moral factors and the extracellular matrix were significantly upregulated or downregulated in DCM CFs. The pathway analysis revealed that extracellular matrix receptor interactions, focal adhesion signaling, Hippo signaling, and transforming growth factor-β signaling pathways were significantly affected in DCM CFs. In contrast, single-cell RNA sequencing revealed that there was no specific subpopulation in the DCM CFs that contributed to the alterations in gene expression. CONCLUSIONS: Although cellular physiological behavior was not altered in DCM CFs, they deteriorated the contractile and diastolic functions of healthy cardiomyocytes through humoral factors and direct cell–cell contact.Tsuru H., Yoshihara C., Suginobe H., et al. Pathogenic Roles of Cardiac Fibroblasts in Pediatric Dilated Cardiomyopathy. Journal of the American Heart Association 12, e029676 (2023); https://doi.org/10.1161/JAHA.123.029676

Mapping power-law rheology of living cells using multi-frequency force modulation atomic force microscopy

We present multi-frequency force modulation atomic force microscopy (AFM) for mapping the complex shear modulus G* of living cells as a function of frequency over the range of 50-500 Hz in the same measurement time as the single-frequency force modulation measurement. The AFM technique enables us to reconstruct image maps of rheological parameters, which exhibit a frequency-dependent power-law behavior with respect to G*. These quantitative rheological measurements reveal a large spatial variation in G* in this frequency range for single cells. Moreover, we find that the reconstructed images of the power-law rheological parameters are much different from those obtained in force-curve or single-frequency force modulation measurements. This indicates that the former provide information about intracellular mechanical structures of the cells that are usually not resolved with the conventional force measurement methods. (C) 2015 AIP Publishing LLC

Calibrating the Young’s modulus of soft materials with surface tilt angle measured by atomic force microscopy

We investigated the apparent Young’s modulus, Em, of soft materials with the surface tilt angle, as measured by colloidal probe atomic force microscopy (AFM). The AFM measurements of soft polymer hydrogels and natural unfertilized eggs showed a clear universal behavior of Em as a function of the tilt angle, θ, of the sample surface at the local contact area. We found that the observed θ dependence of Em was well fitted with a simple modified Hertz contact model, in which the pressure distribution in the contact area follows the conventional Hertz contact model, the vertical component of the net force balances the loading force, and the remaining lateral component of the net force is approximately ignored. We demonstrated how a simple analytical formula derived from the modified Hertz contact model can calibrate the Em values for single cells in a spherical embryo and for single isolated cells and a confluent cell monolayer adhered on flat substrates

Cricket tympanal organ revisited: morphology, development and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane

Vertebrates and insects are phylogenetically separated by millions of years but have commonly developed tympanal membranes for efficiently converting airborne sound to mechanical oscillation in hearing. The tympanal organ of the field cricket Gryllus bimaculatus, spanning 200 mu m, is one of the smallest auditory organs among animals. It indirectly links to two tympana in the prothoracic tibia via tracheal vesicles. The anterior tympanal membrane is smaller and thicker than the posterior tympanal membrane and it is thought to have minor function as a sound receiver. Using differential labeling of sensory neurons/surrounding structures and three-dimensional reconstructions, we revealed that a shell-shaped chitin mass and associated tissues are hidden behind the anterior tympanal membrane. The mass, termed the epithelial core, is progressively enlarged by discharge of cylindrical chitin from epithelial cells that start to aggregate immediately after the final molt and it reaches a plateau in size after 6 days. The core, bridging between the anterior tracheal vesicle and the fluid-filled chamber containing sensory neurons, is supported by a taut membrane, suggesting the possibility that anterior displacements of the anterior tracheal vesicle are converted into fluid motion via a lever action of the core. The epithelial core did not exist in tympanal organ homologs of meso- and metathoracic legs or of nymphal legs. Taken together, the findings suggest that the epithelial core, a potential functional homolog to mammalian ossicles, underlies fine sound frequency discrimination required for adult-specific sound communications

Negative-feedback regulation of ATP release : ATP release from cardiomyocytes is strictly regulated during ischemia

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. lschemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2 hours after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes