Kakihara, Y. | Search bioRxiv

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↗

bioRxiv2023

Coordinated multiple cellular processes in tongue development

Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested migration of other mesoderm-derived cells and CNCC. Some aberrant CNCC abnormally differentiated into osteoblasts due to the lack of Hh signal, which migrated into tongue to form ectopic bone. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations in CNCC to disturb cellular processes which were disrupted in mutant mice. Thus, tongue development requires coordinated multiple cellular processes (cell-cell contact, migration and differentiation). Our results provide hints for possible future treatment in ciliopathies.

developmental biology↗