Mechanical detection of interactions between proteins related to intermediate filament and transcriptional regulation in living cells.

Intermediate filaments (IF) bind to various proteins and regulate cell function in the cytoplasm. Recently, IFs were found to regulate gene expression by acting as capture scaffolds for transcription-related proteins and preventing their translocation into the nucleus. To reveal such transcriptional regulatory mechanisms controlled by IFs, a method to analyze the interaction between IFs and transcription-related proteins is necessary. Although there are many methods to observe interactions in living cells, it is still challenging to measure protein-protein interactions in living cells in their unmodified and native state. In this study, we utilized a nanoneedle that can access the cytosol by insertion into the cell. Modification of antibody recognizing transcription-related proteins allows the needle to detect mechanical force required to unbind the interaction between antibody and target proteins interacting with IFs during retraction of the needle from the cell. We focused on IF vimentin, a marker of epithelial-mesenchymal transition, to mechanically detect transcription-related proteins trapped by vimentin filaments. Prohibitin 2 (PHB2), a transcription-related factor, was selected as the candidate vimentin-binding protein. We conducted mechanical detection of PHB2 using atomic force microscopy and anti-PHB2 antibody-modified nanoneedles in vimentin-expressing mouse breast cancer and vimentin-knockout (VKO) cells. Significantly larger unbinding forces were detected in the vimentin-expressing cells than in the VKO cells. The results demonstrate that this method is useful for in-cell mechanical detection of IF-binding proteins.

Exploring New Functional Aspects of HTLV-1 RNA-Binding Protein Rex: How Does Rex Control Viral Replication?

After integration to the human genome as a provirus, human T-cell leukemia virus type 1 (HTLV-1) utilizes host T cell gene expression machinery for viral replication. The viral RNA-binding protein, Rex, is known to transport unspliced/incompletely spliced viral mRNAs encoding viral structural proteins out of the nucleus to enhance virus particle formation. However, the detailed mechanism of how Rex avoids extra splicing of unspliced/incompletely spliced viral mRNAs and stabilizes them for effective translation is still unclear. To elucidate the underlying molecular mechanism of Rex function, we comprehensively analyzed the changes in gene expression and splicing patterns in Rex-overexpressing T cells. In addition, we identified 81 human proteins interacting with Rex, involved in transcription, splicing, translation, and mRNA quality control. In particular, Rex interacts with NONO and SFPQ, which play important roles in the regulation of transcription and splicing. Accordingly, expression profiles and splicing patterns of a wide variety of genes are significantly changed in Rex-expressing T cells. Especially, the level of vPD-L1 mRNA that lacks the part of exon 4, thus encodes soluble PD-L1 was significantly increased in Rex-expressing cells. Overall, by integrated analysis of these three datasets, we showed for the first time that Rex intervenes the host gene expression machinery throughout the pathway, probably to escort viral unstable mRNAs from transcription (start) to translation (end). Upon exerting its function, Rex may alter the expression level and splicing patterns of various genes, thus influencing the phenotype of the host cell.

Cell culture and genetic transfection methods for the Japanese scallop, Patinopecten yessoensis.

Cell cultures can simplify assays of biological phenomena; therefore, cell culture systems have been established for many species, even invertebrates. However, there are few primary culture systems from marine invertebrates that can be maintained long term. The Japanese scallop, Patinopecten yessoensis, is a marine bivalve. Cell culture systems for the scallop have only been established for a few organ-derived cell types and for embryonic cells. We developed a primary culture system for cells from male and female scallop gonads, hepatopancreas, and adductor muscle by utilizing culture conditions closer to those in nature, with regard to temperature, osmolarity, and nutrition. Primary cultured female gonadal cells were maintained for more than 1 month and had potential for proliferation. Furthermore, a genetic transfection system was attempted using a scallop-derived promoter and a lipofection reagent. GFP-positive cells were detected in the attempt. These technical developments would promote our understanding of biochemical mechanisms in scallops as well as providing clues for establishment of immortalized molluscan cell lines.