Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss.
ABSTRACT: DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C-terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N-terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient-derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR1204-1207) at the DAD C-terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID-DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG-tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID-DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1.
Project description:Dia1, which belongs to the diaphanous-related formin family, influences a variety of cellular processes through straight actin elongation activity. Recently, novel DIA1 mutants such as p.R1213X (p.R1204X) and p.A265S, have been reported to cause an autosomal dominant sensorineural hearing loss (DFNA1). Additionally, active DIA1 mutants induce progressive hearing loss in a gain-of-function manner. However, the subcellular localization and pathological function of DIA1(R1213X/R1204X) remains unknown. In the present study, we demonstrated the localization of endogenous Dia1 and the constitutively active DIA1 mutant in the cochlea, using transgenic mice expressing FLAG-tagged DIA1(R1204X) (DIA1-TG). Endogenous Dia1 and the DIA1 mutant were regionally expressed at the organ of Corti and the spiral ganglion from early life; alongside cochlear maturation, they became localized at the apical junctional complexes (AJCs) between hair cells (HCs) and supporting cells (SCs). To investigate HC vulnerability in the DIA1-TG mice, we exposed 4-week-old mice to moderate noise, which induced temporary threshold shifts with cochlear synaptopathy and ultrastructural changes in stereocilia 4 weeks post noise exposure. Furthermore, we established a knock-in (KI) mouse line expressing AcGFP-tagged DIA1(R1213X) (DIA1-KI) and confirmed mutant localization at AJCs and the tips of stereocilia in HCs. In MDCKAcGFP-DIA1(R1213X) cells with stable expression of AcGFP-DIA1(R1213X), AcGFP-DIA1(R1213X) revealed marked localization at microvilli on the apical surface of cells and decreased localization at cell-cell junctions. The DIA1-TG mice demonstrated hazy and ruffled circumferential actin belts at AJCs and abnormal stereocilia accompanied with HC loss at 5 months of age. In conclusion, Dia1 plays a pivotal role in the development and maintenance of AJCs and stereocilia, ensuring cochlear and HC integrity. Subclinical/latent vulnerability of HCs may be the cause of progressive hearing loss in DFNA1 patients, thus suggesting new therapeutic targets for preventing HC degeneration and progressive hearing loss associated with DFNA1.
Project description:The Diaphanous-related formin Dia1 nucleates actin polymerization, thereby regulating cell shape and motility. Mechanisms that control the cellular location of Dia1 to spatially define actin polymerization are largely unknown. In this study, we identify the cytoskeletal scaffold protein IQGAP1 as a Dia1-binding protein that is necessary for its subcellular location. IQGAP1 interacts with Dia1 through a region within the Diaphanous inhibitory domain after the RhoA-mediated release of Dia1 autoinhibition. Both proteins colocalize at the front of migrating cells but also at the actin-rich phagocytic cup in macrophages. We show that IQGAP1 interaction with Dia1 is required for phagocytosis and phagocytic cup formation. Thus, we identify IQGAP1 as a novel component involved in the regulation of phagocytosis by mediating the localization of the actin filament nucleator Dia1.
Project description:Early-onset hearing loss is mostly of genetic origin. The complexity of the hearing process is reflected by its extensive genetic heterogeneity, with probably many causative genes remaining to be identified. Here, we aimed at identifying the genetic basis for autosomal dominant non-syndromic hearing loss (ADNSHL) in a large German family.A panel of 66 known deafness genes was analyzed for mutations by next-generation sequencing (NGS) in the index patient. We then conducted genome-wide linkage analysis, and whole-exome sequencing was carried out with samples of two patients. Expression of Osbpl2 in the mouse cochlea was determined by immunohistochemistry. Because Osbpl2 has been proposed as a target of miR-96, we investigated homozygous Mir96 mutant mice for its upregulation.Onset of hearing loss in the investigated ADNSHL family is in childhood, initially affecting the high frequencies and progressing to profound deafness in adulthood. However, there is considerable intrafamilial variability. We mapped a novel ADNSHL locus, DFNA67, to chromosome 20q13.2-q13.33, and subsequently identified a co-segregating heterozygous frameshift mutation, c.141_142delTG (p.Arg50Alafs*103), in OSBPL2, encoding a protein known to interact with the DFNA1 protein, DIAPH1. In mice, Osbpl2 was prominently expressed in stereocilia of cochlear outer and inner hair cells. We found no significant Osbpl2 upregulation at the mRNA level in homozygous Mir96 mutant mice.The function of OSBPL2 in the hearing process remains to be determined. Our study and the recent description of another frameshift mutation in a Chinese ADNSHL family identify OSBPL2 as a novel gene for progressive deafness.
Project description:Signal-peptide-mediated ER localization of mRNAs encoding for membrane and secreted proteins, and RNA-zipcode-mediated intracellular targeting of mRNAs encoding for cytosolic proteins are two well-known mechanisms for mRNA localization. Here, we report a previously unidentified mechanism by which mRNA encoding for Dia1, a cytosolic protein without the signal peptide, is localized to the perinuclear ER in an RNA-zipcode-independent manner in fibroblasts. Dia1 mRNA localization is also independent of the actin and microtubule cytoskeleton but requires translation and the association of Dia1 nascent peptide with the ribosome-mRNA complex. Sequence mapping suggests that interactions of the GTPase binding domain of Dia1 peptide with active Rho are important for Dia1 mRNA localization. This mechanism can override the ?-actin RNA zipcode and redirect ?-actin mRNA to the perinuclear region, providing a new way to manipulate intracellular mRNA localization.
Project description:Many kinases are still 'orphans,' which means knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family, is shown to be present in the endoplasmic reticulum and to influence trafficking via the secretory pathway. Subsequently, DIA1 is subjected to phosphoproteomics analysis to cast light on its signalling pathways. A liquid chromatography-tandem mass spectrometry proteomic approach with phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK293T cells, and phosphosites dependent on the presence of DIA1 are elucidated. Most of these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, the proteomics of proteins immunoprecipitated with DIA1 reported its probable interactors. This pilot study provides the basis for deeper studies of DIA1 signalling.
Project description:Developing tissues change shape and tumors initiate spreading through collective cell motility. Conserved mechanisms by which tissues initiate motility into their surroundings are not known. We investigated cytoskeletal regulators during collective invasion by mouse tumor organoids and epithelial Madin-Darby canine kidney (MDCK) acini undergoing branching morphogenesis in collagen. Use of the broad-spectrum formin inhibitor SMIFH2 prevented the formation of migrating cell fronts in both cell types. Focusing on the role of the formin Dia1 in branching morphogenesis, we found that its depletion in MDCK cells does not alter planar cell motility either within the acinus or in two-dimensional scattering assays. However, Dia1 was required to stabilize protrusions extending into the collagen matrix. Live imaging of actin, myosin, and collagen in control acini revealed adhesions that deformed individual collagen fibrils and generated large traction forces, whereas Dia1-depleted acini exhibited unstable adhesions with minimal collagen deformation and lower force generation. This work identifies Dia1 as an essential regulator of tissue shape changes through its role in stabilizing focal adhesions.
Project description:It is clear that RhoA activates the DRF (diaphanous-related formin) mDia2 by disrupting the molecular interaction between the DAD (diaphanous autoregulatory domain) and the DID (diaphanous inhibitory domain). Previous studies indicate that a basic motif within the DAD contributes to mDia2 auto-inhibition, and results shown in the present study suggest these residues bind a conserved acidic region within the DID. Furthermore, we demonstrate that mDia2 is phosphorylated by ROCK (Rho-kinase) at two conserved residues (Thr(1061) and Ser(1070)) just C-terminal to the DAD basic region. Phosphomimetic mutations to these residues in the context of the full-length molecule enhanced mDia2 activity as measured by increased actin polymerization, SRF (serum response factor)-dependent smooth muscle-specific gene transcription, and nuclear localization of myocardin-related transcription factor B. Biochemical and functional data indicate that the T1061E/S1070E mutation significantly inhibited the ability of DAD to interact with DID and enhanced mDia2 activation by RhoA. Taken together, the results of the present study indicate that ROCK-dependent phosphorylation of the mDia2 DAD is an important determinant of mDia2 activity and that this signalling mechanism affects actin polymerization and smooth muscle cell-specific gene expression.
Project description:Cancer bioinformatics has been used to screen possible key cancer genes and pathways. Here, through bioinformatics analysis, we found that high expression of diaphanous related formin 1 (DIAPH1) was associated with poor overall survival in head and neck squamous cell carcinoma and laryngeal squamous cell carcinoma (LSCC). The effect of DIAPH1 in LSCC has not been previously investigated. Therefore, we evaluated the expression, function, and molecular mechanisms of DIAPH1 in LSCC. Immunohistochemistry and western blot analysis confirmed the significant upregulation of DIAPH1 in LSCC. We used DIAPH1 RNA interference to construct two DIAPH1-knockdown LSCC cell lines, AMC-HN-8 and FD-LSC-1, and validated the knockdown efficiency. Flow cytometry data showed that DIAPH1 inhibited apoptosis. Further, western blot analysis revealed that DIAPH1 knockdown increased the protein levels of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9. Thus, DIAPH1 is upregulated in LSCC and may act as an oncogene by inhibiting apoptosis through the ATR/p53/caspase-3 pathway in LSCC cells.
Project description:Exome sequencing identified homozygous loss-of-function variants in DIAPH1 (c.2769delT; p.F923fs and c.3145C>T; p.R1049X) in four affected individuals from two unrelated consanguineous families. The affected individuals in our report were diagnosed with postnatal microcephaly, early-onset epilepsy, severe vision impairment, and pulmonary symptoms including bronchiectasis and recurrent respiratory infections. A heterozygous DIAPH1 mutation was originally reported in one family with autosomal dominant deafness. Recently, however, a homozygous nonsense DIAPH1 mutation (c.2332C4T; p.Q778X) was reported in five siblings in a single family affected by microcephaly, blindness, early onset seizures, developmental delay, and bronchiectasis. The role of DIAPH1 was supported using parametric linkage analysis, RNA and protein studies in their patients' cell lines and further studies in human neural progenitors cells and a diap1 knockout mouse. In this report, the proband was initially brought to medical attention for profound metopic synostosis. Additional concerns arose when his head circumference did not increase after surgical release at 5 months of age and he was diagnosed with microcephaly and epilepsy at 6 months of age. Clinical exome analysis identified a homozygous DIAPH1 mutation. Another homozygous DIAPH1 mutation was identified in the research exome analysis of a second family with three siblings presenting with a similar phenotype. Importantly, no hearing impairment is reported in the homozygous affected individuals or in the heterozygous carrier parents in any of the families demonstrating the autosomal recessive microcephaly phenotype. These additional families provide further evidence of the likely causal relationship between DIAPH1 mutations and a neurodevelopmental disorder.
Project description:Drosophila homologue of Diaphanous 1 (DIAPH1) regulates actin polymerization and microtubule (MT) stabilization upon stimulation with lysophosphatidic acid (LPA). Recently, we showed strongly reduced lung metastasis of DIAPH1-depleted colon cancer cells but we found accumulations of DIAPH1-depleted cells in bone marrow. Here, we analyzed possible organ- or tissue-specific metastasis of DIAPH1-depleted HCT-116 cells. Our data confirmed that depletion of DIAPH1 strongly inhibited lung metastasis and revealed that, in contrast to control cells, DIAPH1-depleted cells did not form metastases in further organs. Detailed mechanistic analysis on cells that were not stimulated with LPA to activate the cytoskeleton-modulating activity of DIAPH1, revealed that even under basal conditions DIAPH1 was essential for cellular adhesion to collagen. In non-stimulated cells DIAPH1 did not control actin dynamics but, interestingly, was essential for stabilization of microtubules (MTs). Additionally, DIAPH1 controlled directed vesicle trafficking and with this, local clustering of the adhesion protein integrin-?1 at the plasma membrane. Therefore, we conclude that under non-stimulating conditions DIAPH1 controls cellular adhesion by stabilizing MTs required for local clustering of integrin-?1 at the plasma membrane. Thus, blockade of DIAPH1-tubulin interaction may be a promising approach to inhibit one of the earliest steps in the metastatic cascade of colon cancer.