Multidrug Sensitive Yeast Strains, Useful Tools for Chemical Genetics

The budding yeast Saccharomyces cerevisiae is a useful eukaryote model organism for application to chemical biology studies, for example, drug screening, drug evaluation, and target identification. To use yeast for chemical biology research, however, it has been necessary to construct yeast strains suitable for various compounds because of their high drug resistance. Hence, the deletion of all multidrug resistance genes except for those that are important for viability and for genetic experiments/manipulation could increase the drug sensitivity without influencing the transformation, mating, or sporulation efficiency. There are two major factors conferring multidrug resistance in S. cerevisiae: one is the drug efflux system and the other is the permeability barrier. We therefore constructed a strain which shows high sensitivity to multiple drugs by disrupting the drug efflux system using ATP-binding cassette transporters and suppressing the membrane barrier system by introducing an ERG6-inducible system. In this review, we discuss the construction of our multidrug-sensitive yeast strains and their application in chemical biology

Mechanisms of spindle bipolarity establishment in acentrosomal human cells

Centrosomes are not absolutely essential for cell division; acentrosomal bipolar spindles can be established in oocytes and centrosome-eliminated somatic cells. However, the detailed mechanisms describing how spindle bipolarity is established without centrosomes are not completely understood. We have recently demonstrated that in acentrosomal human cells, nuclear mitotic apparatus protein (NuMA) assemblies-mediated microtubule asters and EG5 promote spindle bipolarization in early mitosis

An antioxidant screen identifies ascorbic acid for prevention of light-induced mitotic prolongation in live cell imaging

Abstract Phototoxicity is an important issue in fluorescence live imaging of light-sensitive cellular processes such as mitosis. Among several approaches to reduce phototoxicity, the addition of antioxidants to the media has been used as a simple method. Here, we analyzed the impact of phototoxicity on the mitotic progression in fluorescence live imaging of human cells and performed a screen to identify the most efficient antioxidative agents that reduce it. Quantitative analysis shows that high amounts of light illumination cause various mitotic defects such as prolonged mitosis and delays of chromosome alignment and centrosome separation. Among several antioxidants, our screen reveals that ascorbic acid significantly alleviates these phototoxic effects in mitosis. Furthermore, we demonstrate that adding ascorbic acid to the media enables fluorescence imaging of mitotic events at very high temporal resolution without obvious photodamage. Thus, this study provides an optimal method to effectively reduce the phototoxic effects in fluorescence live cell imaging

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells

Abstract Background Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. Results In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. Conclusions These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells

Inhibition of Microtubule Assembly by a Complex of Actin and Antitumor Macrolide Aplyronine A

Aplyronine A (ApA) is a marine natural product that shows potent antitumor activity. While both ApA and ApC, a derivative of ApA that lacks a trimethylserine ester moiety, inhibit actin polymerization in vitro to the same extent, only ApA shows potent cytotoxicity. Therefore, the molecular targets and mechanisms of action of ApA in cells have remained unclear. We report that ApA inhibits tubulin polymerization in a hitherto unprecedented way. ApA forms a 1:1:1 heterotrimeric complex with actin and tubulin, in association with actin synergistically binding to tubulin, and inhibits tubulin polymerization. Tubulin-targeting agents have been widely used in cancer chemotherapy, but there are no previous descriptions of microtubule inhibitors that also bind to actin and affect microtubule assembly. ApA inhibits spindle formation and mitosis in HeLa S3 cells at 100 pM, a much lower concentration than is needed for the disassembly of the actin cytoskeleton. The results of the present study indicate that ApA represents a rare type of natural product, which binds to two different cytoplasmic proteins to exert highly potent biological activities