Characterization of phalloidin-negative nuclear actin filaments in U2OS cells expressing cytoplasmic actin-EGFP.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, including fungi, plants, and animals, and exists not only in the cytoplasm as cytoskeleton but also in the nucleus. Recently, we developed a novel actin probe, ß-actin-EGFP fusion protein, which exhibited similar monomeric to filamentous ratio as that of endogenous actin, in contrast to the widely used EGFP-ß-actin fusion protein that over-assembles in cells. Unexpectedly, this novel probe visualized an interconnected meshwork of slightly curved beam-like bundles of actin filaments in the nucleus of U2OS cells. These structures were not labeled with rhodamine phalloidin, Lifeact-EGFP or anti-actin antibodies. In addition, immunofluorescence staining and expression of cofilin-EGFP revealed that this nuclear actin structures contained cofilin. We named these actin filaments as phalloidin-negative intranuclear (PHANIN) actin filaments. Since PHANIN actin filaments could not be detected by general detection methods for actin filaments, we propose that PHANIN actin filaments are different from previously reported nuclear actin structures.

Microneedle Array-Assisted, Direct Delivery of Genome-Editing Proteins Into Plant Tissue.

Genome editing in plants employing recombinant DNA often results in the incorporation of foreign DNA into the host genome. The direct delivery of genome-editing proteins into plant tissues is desired to prevent undesirable genetic alterations. However, in most currently available methods, the point of entry of the genome-editing proteins cannot be controlled and time-consuming processes are required to select the successfully transferred samples. To overcome these limitations, we considered a novel microneedle array (MNA)-based delivery system, in which the needles are horizontally aligned from the substrate surface, giving it a comb-like configuration. We aimed to deliver genome-editing proteins directly into the inner layers of leaf tissues; palisade, the spongy and subepidermal L2 layers of the shoot apical meristem (SAM) which include cells that can differentiate into germlines. The array with needles 2 µm wide and 60 µm long was effective in inserting into Arabidopsis thaliana leaves and Glycine max (L.) Merr. (soybeans) SAM without the needles buckling or breaking. The setup was initially tested for the delivery of Cre recombinase into the leaves of the reporter plant A. thaliana by quantifying the GUS (ß-glucuronidase) expression that occurred by the recombination of the loxP sites. We observed GUS expression at every insertion. Additionally, direct delivery of Cas9 ribonucleoprotein (RNP) targeting the PDS11/18 gene in soybean SAM showed an 11 bp deletion in the Cas9 RNP target site. Therefore, this method effectively delivered genome-editing proteins into plant tissues with precise control over the point of entry.

Development of a universal method for the measurement of binding affinities of antibody drugs towards a living cell based on AFM force spectroscopy.

A universal method to measure the binding affinities of antibody drugs towards their targets on the surface of living cells was developed based on atomic force microscopy (AFM) analysis. Nivolumab, an antibody drug targeting programmed cell death 1 (PD-1), was mainly used as a model for this evaluation. The surface of a tip-less AFM cantilever was coated with nano-capsules, on which immunoglobulin G-binding ZZ domains of protein A were exposed, and nivolumab molecules were immobilized on the cantilever through binding between the antibody Fc domains and the ZZ domains, which controlled the molecular orientation of the antibodies. Model human T lymphocytes (Jurkat), on which PD-1 molecules were highly expressed, were immobilized on a glass substrate via a lipid bilayer-anchoring reagent. The nivolumab-coated AFM cantilever was moved to approach the T cells, and the rupture forces between nivolumab molecules on the AFM cantilever and PD-1 molecules on the cell surface were measured. The average values of the rupture forces were 0.18 ± 0.10, 0.21 ± 0.18, 0.12 ± 0.07, 0.11 ± 0.06, and 0.12 ± 0.06 nN µm-2 at loading forces of 10, 20, 30, 40, and 50 nN, respectively. Application of significantly higher loading forces decreased the S/N ratio, as confirmed by comparison with control T cells with low PD-1 expression, which suggested that a low loading force of less than 20 nN was sufficient for these measurements. A correlation between the expression levels of PD-1 and the rupture force values was confirmed using immunofluorescence. A similar assay was performed by using an antibody drug targeting epidermal growth factor receptor (EGFR) and a model cancer cell expressing EGFR molecules (A431) to evaluate the universal application of the developed method for various antibody drugs, and the same conclusions as that in nivolumab's case were obtained. This method can be applied to living cells without any chemical treatment, which allows the present method to compare the affinities of various antibody drugs towards the same single cell. These results indicated that the present method is useful for selecting the most effective candidates from various antibody drugs from the point of view of binding forces between antibodies and living cells.