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In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear.

ABSTRACT: Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.

SUBMITTER: Chiereghin C

PROVIDER: S-EPMC9870134 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear.

Chiereghin Chiara C Robusto Michela M Lewis Morag A MA Caetano Susana S Massa Valentina V Castorina Pierangela P Ambrosetti Umberto U Steel Karen P KP Duga Stefano S Asselta Rosanna R Soldà Giulia G

PloS one 20230123 1

Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserv ...[more]

PMID: 36689403

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Project description:BackgroundDiaphanous-related formins govern actin-based processes involved in many cellular functions, such as cell movement and invasion. Possible connections to developmental processes and cellular changes associated with malignant phenotype make them interesting study targets. In spite of this, very little is known of the tissue distribution and cellular location of any mammalian formin. Here we have carried out a comprehensive analysis of the formin family member formin -like 2 (FMNL2) in human tissues.ResultsAn FMNL2 antibody was raised and characterized. The affinity-purified FMNL2 antibody was validated by Western blotting, Northern blotting, a peptide competition assay and siRNA experiments. Bioinformatics-based mRNA profiling indicated that FMNL2 is widely expressed in human tissues. The highest mRNA levels were seen in central and peripheral nervous systems. Immunohistochemical analysis of 26 different human tissues showed that FMNL2 is widely expressed, in agreement with the mRNA profile. The widest expression was detected in the central nervous system, since both neurons and glial cells expressed FMNL2. Strong expression was also seen in many epithelia. However, the expression in different cell types was not ubiquitous. Many mesenchymal cell types showed weak immunoreactivity and cells lacking expression were seen in many tissues. The subcellular location of FMNL2 was cytoplasmic, and in some tissues a strong perinuclear dot was detected. In cultured cells FMNL2 showed mostly a cytoplasmic localization with perinuclear accumulation consistent with the Golgi apparatus. Furthermore, FMNL2 co-localized with F-actin to the tips of cellular protrusions in WM164 human melanoma cells. This finding is in line with FMNL2's proposed function in the formation of actin filaments in cellular protrusions, during amoeboid cellular migration.ConclusionFMNL2 is expressed in multiple human tissues, not only in the central nervous system. The expression is especially strong in gastrointestinal and mammary epithelia, lymphatic tissues, placenta, and in the reproductive tract. In cultured melanoma cells, FMNL2 co-localizes with F-actin dots at the tips of cellular protrusions.

Dataset Information

In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear.

Publications

In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear.

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