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Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell–cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

Two very polarized views exist for understanding the cellular plasma membrane (PM). For some, it is the simple fluid described by the original Singer–Nicolson fluid mosaic model. For others, due to the presence of thousands of molecular species that extensively interact with each other, the PM forms various clusters and domains that are constantly changing and therefore, no simple rules exist that can explain the structure and molecular dynamics of the PM. In this article, we propose that viewing the PM from its two predominant components, cholesterol and actin filaments, provides an excellent and transparent perspective of PM organization, dynamics, and mechanisms for its functions. We focus on the actin-induced membrane compartmentalization and lipid raft domains coexisting in the PM and how they interact with each other to perform PM functions. This view provides an important update of the fluid mosaic model.

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized. They revealed the dynamic nanoorganization of the focal adhesion (FA), leading to the compartmentalized archipelago FA model, consisting of FA-protein islands with broad diversities in size (13–100 nm; mean island diameter ≈30 nm), protein copy numbers, compositions, and stoichiometries, which dot the partitioned fluid membrane (74-nm compartments in the FA vs. 109-nm compartments outside the FA). Integrins are recruited to these islands by hop diffusion. The FA-protein islands form loose ≈320 nm clusters and function as units for recruiting FA proteins.

In Japan, small-scale forestry, known as 'self-logging (Zibatsu)', has attracted the attention of young people in recent years. In 2014 the Association for the Promotion of Self-logging type Forestry (Zibatsu-gata) was established as a non-profit organization (NPO). The NPO promotes small-scale forestry by providing training in chainsaw thinning and disaster-resistant spur roads, as well as management advice. However, it is unclear which demographic groups participate in the NPO's activities and what their expectations are of the small-scale forestry. Therefore, a questionnaire by google forms was conducted with 1,040 registered the direct mailing list of the NPOs to examine the attributes of the members and their expectations on Zibatsu forestry. The number of valid responses was 184 (17.7%). More than half of the respondents were under 50 years of age. Respondents could be divided into three categories: those who planned to engage in self-logging forestry (Candidates: 57%), those already working in self-logging forestry (Practitioners: 30%) and those who were supporters of the NPO activities (Supporters: 13%). The prospective workers were seeking to acquire skills in self-logging forestry and were particularly interested in learning spur roads for disaster resilient forestry. In particular, a high percentage of the candidates did not own forest land, indicating the importance of matching aging forest owners with candidates. It was also clear that few of the practitioners and candidates planned to make a living solely from income from Zibatsu, and many planned to combine it with other self-employment businesses.