Evaluating Young's Modulus of Single Yeast Cells Based on Compression Using an Atomic Force Microscope with a Flat Tip.

In this research, atomic force microscopy (AFM) with a flat tip cantilever is utilized to measure Young's modulus of a whole yeast cell (Saccharomyces cerevisiae BY4741). The results acquired from AFM are similar to those obtained using a microfluidic chip compression system. The mechanical properties of single yeast cells are important parameters which can be examined using AFM. Conventional studies apply AFM with a sharp cantilever tip to indent the cell and measure the force-indentation curve, from which Young's modulus can be calculated. However, sharp tips introduce problems because the shape variation can lead to a different result and cannot represent the stiffness of the whole cell. It can lead to a lack of broader meaning when evaluating Young's modulus of yeast cells. In this report, we confirm the differences in results obtained when measuring the compression of a poly(dimethylsiloxane) bead using a commercial sharp tip versus a unique flat tip. The flat tip effectively avoids tip-derived errors, so we use this method to compress whole yeast cells and generate a force­deformation curve. We believe our proposed method is effective for evaluating Young's modulus of whole yeast cells.

A missense variant in PER2 is associated with delayed sleep-wake phase disorder in a Japanese population.

Delayed sleep-wake phase disorder (DSWPD) is a subtype of circadian rhythm sleep-wake disorders, and is characterized by an inability to fall asleep until late at night and wake up at a socially acceptable time in the morning. The study aim was to identify low-frequency nonsense and missense variants that are associated with DSWPD. Candidate variants in circadian rhythm-related genes were extracted by integration of genetic variation databases and in silico assessment. We narrowed down the candidates to six variants. To examine whether the six variants are associated with DSWPD, we performed an association study in 236 Japanese patients with DSWPD and 1436 controls. A low-frequency missense variant (p.Val1205Met) in PER2 showed a significant association with DSWPD (2.5% in cases and 1.1% in controls, P = 0.026, odds ratio (OR) = 2.32). The variant was also associated with idiopathic hypersomnia known to have a tendency toward phase delay (P = 0.038, OR = 2.07). PER2 forms a heterodimer with CRY, and the heterodimer plays an important role in the regulation of circadian rhythms. Val1205 is located in the CRY-binding domain of PER2 and was hypothesized to interact with CRY. The p.Val1205Met substitution could be a potential genetic marker for DSWPD.

Analysis of Arabidopsis TPK2 and KCO3 reveals structural properties required for K+ channel function.

Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K+ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.