bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2020‒11‒15
nine papers selected by
Céline Gagnieux
École Polytechnique Fédérale de Lausanne (EPFL)

  1. Am J Physiol Renal Physiol. 2020 Nov 09.
      Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the presence of numerous fluid-filled cysts, extensive fibrosis, and the progressive decline in kidney function. Transforming growth factor β1 (TGF-β1), an important mediator for renal fibrosis and chronic kidney disease, is overexpressed by cystic cells compared to normal kidney cells; however, its role in PKD pathogenesis remains undefined. To investigate the effect of TGF-β1 on cyst growth, fibrosis, and disease progression, we overexpressed active TGF-β1 specifically in collecting duct (CD)s of phenotypic normal (Pkd1RC/+) and Pkd1RC/RC mice. In normal mice, CD-specific TGF-β1 overexpression caused tubule dilations by 5 weeks of age that were accompanied by increased levels of phosphorylated SMAD3, α-smooth muscle actin, vimentin, and periostin; however, it did not induce overt cyst formation by 20 weeks. In Pkd1RC/RCmice, CD overexpression of TGF-β1 increased cyst epithelial cell proliferation. However, extensive fibrosis limited cyst enlargement and caused contraction of the kidneys, leading to a loss of renal function and a shortened lifespan of the mice. These data demonstrate that TGF-β1-induced fibrosis constrains cyst growth and kidney enlargement and accelerates the decline of renal function, supporting the hypothesis that a combined therapy that inhibits renal cyst growth and fibrosis will be required to effectively treat ADPKD.
    Keywords:  ADPKD; ALK5; Autosomal dominant polycystic kidney disease; Collecting duct; Epithelial-mesenchymal-transition
  2. Elife. 2020 Nov 09. pii: e60684. [Epub ahead of print]9
      Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here, we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.
    Keywords:  cell biology; molecular biophysics; none; structural biology
  3. Rep Biochem Mol Biol. 2020 Jul;9(2): 193-198
      Background: Autosomal dominant polycystic kidney disease (ADPKD), a multisystem disorder, is the most prevalent type of hereditary kidney disease. Here, we aimed to evaluate methylation of the PKD1 gene (PKD1) promoter and its correlation with PKD1 expression in peripheral blood.Methods: In this case-control study methylation of the PKD1 promoter was evaluated using methylation-sensitive high-resolution melt (MS-HRM) analysis. PKD1 expression was assessed by quantitative real-time PCR. The correlation was evaluated using the Pearson correlation test.
    Results: Twenty subjects from both the patient and control groups (n= 40 for each) were methylated at the PKD1 promoter to various levels (18.9% in patients and 62.5% in controls). This difference was statistically significant (p< 0.0001). PKD1 expression in blood samples was significantly greater in ADPKD patients than in controls (p= 0.0081). Significant correlation was seen between PKD1 expression and its promoter methylation status in peripheral blood (r case= -0.5300, p= 0.0162, and r control = -0.6265, p= 0.0031).
    Conclusion: Methylation of the PKD1 promoter in ADPKD patients was inversely correlated with PKD1 expression.
    Keywords:  Autosomal Dominant Polycystic Kidney Disease (ADPKD); Epigenetic; Methylation; PKD1; methylation-sensitive high-resolution melt (MS-HRM) analysis
  4. Mol Biol Cell. 2020 Nov 11. mbcE20080556
      Cilia sense and transduce extracellular signals via specific receptors. The intraflagellar transport (IFT) machinery mediates not only bidirectional protein trafficking within cilia but also the import/export of ciliary proteins across the ciliary gate. The IFT machinery is known to comprise two multisubunit complexes, namely, IFT-A and IFT-B; however, little is known about how the two complexes cooperate to mediate ciliary protein trafficking. We here show that IFT144-IFT122 from IFT-A and IFT88-IFT52 from IFT-B make major contributions to the interface between the two complexes. Exogenous expression of the IFT88(Δα) mutant, which has decreased binding to IFT-A, partially restores the ciliogenesis defect of IFT88-knockout (KO) cells. However, IFT88(Δα)-expressing IFT88-KO cells demonstrate a defect in IFT-A entry into cilia, aberrant accumulation of IFT-B proteins at the bulged ciliary tips, and impaired import of ciliary GPCRs. Furthermore, overaccumulated IFT proteins at the bulged tips appeared to be released as extracellular vesicles. These phenotypes of IFT88(Δα)-expressing IFT88-KO cells resembled those of IFT144-KO cells. These observations together indicate that the IFT-A complex cooperates with the IFT-B complex to mediate the ciliary entry of GPCRs as well as retrograde trafficking of the IFT machinery from the ciliary tip. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
  5. Sci Rep. 2020 Nov 09. 10(1): 19301
      Ciliopathies are clinical disorders of the primary cilium with widely recognised phenotypic and genetic heterogeneity. Here, we found impaired ciliogenesis in fibroblasts derived from individuals with fetal akinesia deformation sequence (FADS), a broad spectrum of neuromuscular disorders arising from compromised foetal movement. We show that cells derived from FADS individuals have shorter and less primary cilia (PC), in association with alterations in post-translational modifications in α-tubulin. Similarly, siRNA-mediated depletion of two known FADS proteins, the scaffold protein rapsyn and the nucleoporin NUP88, resulted in defective PC formation. Consistent with a role in ciliogenesis, rapsyn and NUP88 localised to centrosomes and PC. Furthermore, proximity-ligation assays confirm the respective vicinity of rapsyn and NUP88 to γ-tubulin. Proximity-ligation assays moreover show that rapsyn and NUP88 are adjacent to each other and that the rapsyn-NUP88 interface is perturbed in the examined FADS cells. We suggest that the perturbed rapsyn-NUP88 interface leads to defects in PC formation and that defective ciliogenesis contributes to the pleiotropic defects seen in FADS.
  6. Curr Opin Cell Biol. 2020 Nov 10. pii: S0955-0674(20)30137-X. [Epub ahead of print]68 98-104
      While microtubule dynamics and organization have been extensively studied invitro, both biochemically and in cultured cells, recent work has begun to extend this into tissues ex vivo and organisms in vivo. Advances in genetic tools and imaging technology have allowed studies on the dynamics, function, and organization of microtubules in the stratified epithelia of the epidermis. Here, we discuss recent work that highlights the varied roles that microtubules play in supporting epidermal function. These findings demonstrate that studying microtubules in tissues has revealed not only novel aspects of epidermal biology but also new principles of microtubule regulation.
    Keywords:  Centrosome; Cilia; Desmosomes; Differentiation; Epidermis; Gammatubulin; Microtubule; Skin
  7. Open Biol. 2020 Nov;10(11): 200221
      The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled-coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2-Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.
    Keywords:  Ana2; Sas6; centrioles; centrosome; hydrogen–deuterium exchange mass spectrometry; protein interaction
  8. Am J Physiol Cell Physiol. 2020 Nov 11.
      Spag6 encodes an axoneme central apparatus protein that is required for normal flagellar and cilia motility. Recent findings suggest that Spag6 also plays a role in ciliogenesis, orientation of cilia basal feet, and planar polarity. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity. They represent a distinctive form of cellular polarity, known as planar cell polarity (PCP). However, a role for Spag6 in the inner ear has not yet been explored. In the present study, the function of Spag6 in the inner ear was examined using Spag6-deficient mice. Our results demonstrate hearing loss in the Spag6 mutants, associated with abnormalities in cellular patterning, cell shape, stereocilia bundles and basal bodies, as well as abnormally distributed Frizzled class receptor 6 (FZD6), suggesting that Spag6 participates in PCP regulation. Moreover, we found that the sub-apical microtubule meshwork was disrupted. Our observations suggest new functions for Spag6 in hearing and PCP in the inner ear.
    Keywords:  Hair cell; Hearing loss; Inner ear; Sperm-associated antigen 6; polarity
  9. Biophys Physicobiol. 2020 ;17 71-85
      Cilia or flagella of eukaryotes are small micro-hair like structures that are indispensable to single-cell motility and play an important role in mammalian biological processes. Cilia or flagella are composed of nine doublet microtubules surrounding a pair of singlet microtubules called the central pair (CP). Together, this arrangement forms a canonical and highly conserved 9+2 axonemal structure. The CP, which is a unique structure exclusive to motile cilia, is a pair of structurally dimorphic singlet microtubules decorated with numerous associated proteins. Mutations of CP-associated proteins cause several different physical symptoms termed as ciliopathies. Thus, it is crucial to understand the architecture of the CP. However, the protein composition of the CP was poorly understood. This was because the traditional method of identification of CP proteins was mostly limited by available Chlamydomonas mutants of CP proteins. Recently, more CP protein candidates were presented based on mass spectrometry results, but most of these proteins were not validated. In this study, we re-evaluated the CP proteins by conducting a similar comprehensive CP proteome analysis comparing the mass spectrometry results of the axoneme sample prepared from Chlamydomonas strains with and without CP complex. We identified a similar set of CP protein candidates and additional new 11 CP protein candidates. Furthermore, by using Chlamydomonas strains lacking specific CP sub-structures, we present a more complete model of localization for these CP proteins. This work has established a new foundation for understanding the function of the CP complex in future studies.
    Keywords:  Central Pair; Cilia; Electron Microscopy; Flagella; Mass Spectrometry