Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows ...centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. Here we show that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, we show that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation.
Primary ciliary dyskinesia (PCD) is a rare genetic ciliopathy in which mucociliary clearance is disturbed by the abnormal motion of cilia or there is a severe reduction in the generation of multiple ...motile cilia. Lung damage ensues due to recurrent airway infections, sometimes even resulting in respiratory failure. So far, no causative treatment is available and treatment efforts are primarily aimed at improving mucociliary clearance and early treatment of bacterial airway infections. Treatment guidelines are largely based on cystic fibrosis (CF) guidelines, as few studies have been performed on PCD. In this review, we give a detailed overview of the clinical studies performed investigating PCD to date, including three trials and several case reports. In addition, we explore precision medicine approaches in PCD, including gene therapy, mRNA transcript and read-through therapy.
Missense mutations of the V600E type constitute the vast majority of tumor-associated somatic alterations in the v-RAF murine sarcoma viral oncogene homolog B1 (
BRAF
) gene. Initially described in ...melanoma, colon and papillary thyroid carcinoma, these alterations have also been observed in primary nervous system tumors albeit at a low frequency. We analyzed exon 15 of
BRAF
spanning the V600 locus by direct sequencing in 1,320 adult and pediatric tumors of the nervous system including various types of glial, embryonal, neuronal and glioneuronal, meningeal, adenohypophyseal/sellar, and peripheral nervous system tumors. A total of 96
BRAF
mutations were detected; 93 of the V600E type and 3 cases with a three base pair insertion between codons 599 and 600. The highest frequencies of
BRAF
V600E
mutations were found in WHO grade II pleomorphic xanthoastrocytomas (42/64; 66%) and pleomorphic xanthoastrocytomas with anaplasia (15/23; 65%), as well as WHO grade I gangliogliomas (14/77; 18%), WHO grade III anaplastic gangliogliomas (3/6) and pilocytic astrocytomas (9/97; 9%). In pilocytic astrocytomas
BRAF
V600E
mutation was strongly associated with extra-cerebellar location (
p
= 0.009) and was most frequent in diencephalic tumors (4/12; 33%). Glioblastomas and other gliomas were characterized by a low frequency or absence of mutations. No mutations were detected in non-glial tumors, including embryonal tumors, meningiomas, nerve sheath tumors and pituitary adenomas. The high mutation frequencies in pleomorphic xanthoastrocytomas, gangliogliomas and extra-cerebellar pilocytic astrocytomas implicate
BRAF
V600E
mutation as a valuable diagnostic marker for these rare tumor entities. Future clinical trials should address whether
BRAF
V600E
mutant brain tumor patients will benefit from
BRAF
V600E
-directed targeted therapies.
It is widely thought that Wnt/Lrp6 signaling proceeds through the cytoplasm and that motile cilia are signaling-inert nanomotors. Contrasting both views, we here show in the mucociliary epidermis of ...X. tropicalis embryos that motile cilia transduce a ciliary Wnt signal that is distinct from canonical β-catenin signaling. Instead, it engages a Wnt-Gsk3-Ppp1r11-Pp1 signaling axis. Mucociliary Wnt signaling is essential for ciliogenesis and it engages Lrp6 co-receptors that localize to cilia via a VxP ciliary targeting sequence. Live-cell imaging using a ciliary Gsk3 biosensor reveals an immediate response of motile cilia to Wnt ligand. Wnt treatment stimulates ciliary beating in X. tropicalis embryos and primary human airway mucociliary epithelia. Moreover, Wnt treatment improves ciliary function in X. tropicalis ciliopathy models of male infertility and primary ciliary dyskinesia (ccdc108, gas2l2). We conclude that X. tropicalis motile cilia are Wnt signaling organelles that transduce a distinct Wnt-Pp1 response.
Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model ...organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport ...(IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.
Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects ...are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located β-HC DNAH11 (defining ODA type 1), and the distally localized β-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the β-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient β-HCs such as that of the unicellular Chlamydomonas reinhardtii.
Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is ...associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.
Primary ciliary dyskinesia (PCD) is a heterogeneous genetic condition. European and North American diagnostic guidelines recommend transmission electron microscopy (TEM) as one of a combination of ...tests to confirm a diagnosis. However, there is no definition of what constitutes a defect or consensus on reporting terminology. The aim of this project was to provide an internationally agreed ultrastructural classification for PCD diagnosis by TEM.A consensus guideline was developed by PCD electron microscopy experts representing 18 centres in 14 countries. An initial meeting and discussion were followed by a Delphi consensus process. The agreed guideline was then tested, modified and retested through exchange of samples and electron micrographs between the 18 diagnostic centres.The final guideline a) provides agreed terminology and a definition of Class 1 defects which are diagnostic for PCD; b) identifies Class 2 defects which can indicate a diagnosis of PCD in combination with other supporting evidence; c) describes features which should be included in a ciliary ultrastructure report to assist multidisciplinary diagnosis of PCD; and d) defines adequacy of a diagnostic sample.This tested and externally validated statement provides a clear guideline for the diagnosis of PCD by TEM which can be used to standardise diagnosis internationally.
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