Biology subjects

Iwashita, S.

Publications and source records attributed to Iwashita, S..

3 recordsLinked to original sources

bioRxiv2025

Macromolecular Crowding-Affected Mobility of Bcnt/Cfdp1, a Disordered Component of the Srcap Complex, from the Nucleus to Cytosol in Subcellular Fractionation

Metazoan Bucentaur/Craniofacial Development Protein 1 (Bcnt/Cfdp1) is crucial for embryonic development, and its yeast homolog protein, SWC5, functions as part of the SWR1 complex in histone exchange reactions. Bcnt/Cfdp1, an intrinsically disordered protein (IDP) subject to various post-translational modifications, contains a putative nuclear localization sequence. Nevertheless, its subcellular localization remains ambiguous. We investigated its intracellular trafficking using diverse approaches. In C2C12 myoblasts, fluorescent protein-tagged Bcnt/Cfdp1 was predominantly localized within the nucleus, preferentially in regions of low DAPI density in myoblasts, and the localization persisted in differentiated multinucleated myotubes. Conversely, detergent-based biochemical fractionation of C2C12 and HEK293T cells indicated a substantial presence of Bcnt/Cfdp1 in cytoplasmic fractions. Quantitative proteomic analysis of fractionated proteins revealed that six IDPs, including a PEST-containing nuclear protein, demonstrated similar distribution patterns to Bcnt/Cfdp1; despite being annotated as nuclear proteins, they were detected in cytoplasmic fractions. Direct treatment of both cell lines with digitonin enabled the collection of released proteins in three steps to accurately isolate cytoplasmic proteins. Bcnt/Cfdp1 exhibited distinct extraction patterns compared to cytoplasmic soluble protein extracts in Ficoll/sucrose solution, while no differences were noted when cells underwent digitonin treatment in an isotonic solution. Quantitative proteomic analysis of these extracts highlighted the mobility or integrity of each protein within the cytoplasm. Consequently, although Bcnt/Cfdp1 is essential within the nucleus, our findings suggest its additional presence in the cytoplasm. We hypothesize that the elastic properties of Bcnt/Cfdp1 facilitate its translocation from the nucleus during subcellular fractionation, which is affected by macromolecular crowding.

biochemistry↗

bioRxiv2019

Assessment of a western blot signal for the Bcnt/Cfdp1, a tentative component of Srcap chromatin remodeling complex; trial to overcome off-target problems

The BCNT (Bucentaur) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in fungi, fly, and chicken. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the Srcap (SNF2-Related CBP Activator Protein) chromatin remodeling complex, but little is known about its properties, partly because there are few suitable antibodies to detect the endogenous protein. We used multiple anti-Bcnt/Cfdp1 antibodies against unrelated immunogens derived from BCNT-C domain and mouse-specific N-terminal peptide. To assign western blot signals and evaluate these antibodies, we utilized a stem cell line from mutant embryos of mouse Bcnt/Cfdp1, whose mRNA expression levels were reduced to 75% of the parental cells. In western blotting of these mutant and parental cell extracts with the anti-Bcnt/Cfdp1 antibodies, mouse Bcnt/Cfdp1 was detected as a doublet of approximately 45 kDa. LC-MS/MS analysis of the corresponding doublet for the Flag-tagged mouse Bcnt/Cfdp1 constitutively expressed in T-REx 293 cell (a HEK293 derivative) exhibited that the upper band was much more phosphorylated than the lower band and that there was preferential Ser phosphorylation in the WESF motif in the BCNT-C domain. Western blot with these validated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development in mouse and rat, which is consistent with the expression of Bcnt/Cfdp1 mRNA. This article describes the evaluation of anti-Bcnt/Cfdp1 antibodies, including a scheme to prepare a potential negative control for western blot, and discusses immune-cross reactions with off-target proteins, particularly immunoreaction probabilities.

biochemistry↗

bioRxiv2018

N1 component of event-related potential evoked by simple composite figures in the lateral occipital cortex

Since Sugawara and Morotomi (1991) first reported that illusory contour (IC) figures, or Kanizsa square inducers (KSI), evoked a significant N1 component, the first visually evoked negative component of event-related potentials (ERPs), in the lateral occipital cortex (LO), many ERP studies have confirmed their finding, and these studies showed that Kanizsa-type inducers alone evoked a relatively large N1 in the LO, even when the inducers are oriented to not form an IC (see Murray and Herrmann, 2013). In the present study, we used non-IC simple composite figures composed of several elementary shapes (e.g., triplet squares or quadruplet circles) as visual stimuli, and found that the composite figures evoked a significant N1 comparable to that evoked by KSI in the anterior LO, whereas solitary elementary shapes did not. Thus, it appears that the LO is activated by not only an IC but also the compositeness of figures, which implies that the LO might analyze the geometrical relationships among multiple 2D shapes that compose a single composite figure.

neuroscience↗