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ABSTRACT: Since the discovery that proteins mutated in different forms of polycystic kidney disease (PKD) are tightly associated with primary cilia, strong efforts have been made to define the role of this organelle in the pathogenesis of cyst formation. Cilia are filiform microtubular structures, anchored in the basal body and extending from the apical membrane into the tubular lumen. Early work established that cilia act as flow sensors, eliciting calcium transients in response to bending, which involve the two proteins mutated in autosomal dominant PKD (ADPKD), polycystin-1 and -2. Loss of cilia alone is insufficient to cause cyst formation. Nevertheless, a large body of evidence links flow sensing by cilia to aspects relevant for cyst formation such as cell polarity, Stat6- and mammalian target of rapamycin signalling. This review summarizes the current literature on cilia and flow sensing with respect to PKD and discusses how these findings intercalate with different aspects of cyst formation.
Nephrology Dialysis Transplantation 01/2013; · 3.40 Impact Factor
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ABSTRACT: The microtubular motor Kinesin-2 and its subunit Kif3a are essential for the formation of primary cilia, an organelle implicated in a wide spectrum of developmental abnormalities. Outside cilia, Kinesin-2 mediated transport has been implicated in vesicle and N-cadherin transport, but it is unknown if and how extraciliary Kif3a affects basic cellular functions such as migration or the formation of multicellular structures. Here we show that tetracycline inducible depletion of Kif3a in MDCK cells slows epithelial cell migration. Microtubules at the leading edge of Kif3a depleted cells failed to grow perpendicularly into the leading edge and microtubular dynamics were dampened in Kif3a depleted cells. Loss of Kif3a retarded lateral membrane specification and completely prevented the formation of three-dimensional spheres in collagen. These data uncover that Kif3a regulates the microtubular cytoskeleton in the cell periphery and imply that extra-ciliary Kif3a has an unexpected function in morphogenesis.
PLoS ONE 01/2013; 8(5):e62165. · 4.09 Impact Factor
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Soeren Lienkamp,
Athina Ganner, Christopher Boehlke,
Thorsten Schmidt,
Sebastian J Arnold,
Tobias Schäfer,
Daniel Romaker,
Julia Schuler,
Sylvia Hoff,
Christian Powelske, [......],
Corinna Krönig,
Axel Bullerkotte,
Roland Nitschke,
E Wolfgang Kuehn,
Emily Kim,
Hans Burkhardt,
Thomas Brox,
Olaf Ronneberger,
Joachim Gloy,
Gerd Walz
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ABSTRACT: Mutations of inversin cause type II nephronophthisis, an infantile autosomal recessive disease characterized by cystic kidney disease and developmental defects. Inversin regulates Wnt signaling and is required for convergent extension movements during early embryogenesis. We now show that Inversin is essential for Xenopus pronephros formation, involving two distinct and opposing forms of cell movements. Knockdown of Inversin abrogated both proximal pronephros extension and distal tubule differentiation, phenotypes similar to that of Xenopus deficient in Frizzled-8. Exogenous Inversin rescued the pronephric defects caused by lack of Frizzled-8, indicating that Inversin acts downstream of Frizzled-8 in pronephros morphogenesis. Depletion of Inversin prevents the recruitment of Dishevelled in response to Frizzled-8 and impeded the accumulation of Dishevelled at the apical membrane of tubular epithelial cells in vivo. Thus, defective tubule morphogenesis seems to contribute to the renal pathology observed in patients with nephronophthisis type II.
Proceedings of the National Academy of Sciences 11/2010; 107(47):20388-93. · 9.68 Impact Factor
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Christopher Boehlke,
Fruzsina Kotsis,
Vishal Patel,
Simone Braeg,
Henriette Voelker,
Saskia Bredt,
Theresa Beyer,
Heike Janusch,
Christoph Hamann,
Markus Gödel,
Klaus Müller,
Martin Herbst,
Miriam Hornung,
Mara Doerken,
Michael Köttgen,
Roland Nitschke,
Peter Igarashi,
Gerd Walz,
E Wolfgang Kuehn
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ABSTRACT: The mTOR pathway is the central regulator of cell size. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD), where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins. We therefore investigated if cilia have a role in mTOR regulation. Here, we show that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. Our results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling.
Nature Cell Biology 10/2010; 12(11):1115-22. · 19.49 Impact Factor
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ABSTRACT: The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.
Journal of Cell Science 04/2010; 123(Pt 9):1460-7. · 6.11 Impact Factor
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Hongyu Gao,
Yan Wang,
Tomasz Wegierski,
Kassiani Skouloudaki,
Michael Pütz,
Xiao Fu,
Christina Engel, Christopher Boehlke,
Hongquan Peng,
E Wolfgang Kuehn,
Emily Kim,
Albrecht Kramer-Zucker,
Gerd Walz
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ABSTRACT: Autosomal dominant polycystic liver disease (PCLD) is caused by mutations of either PRKCSH or Sec63, two proteins associated with the endoplasmic reticulum (ER). Both proteins are involved in carbohydrate processing, folding and translocation of newly synthesized glycoproteins. It is postulated that defective quality control of proteins initiates endoplasmic reticulum-associated degradation (ERAD), which disrupts hepatic homeostasis in patients with PRKCSH or Sec63 mutations. However, the precise molecular mechanisms are not known. Here, we show that over-expression or depletion of PRKCSH in zebrafish embryos leads to pronephric cysts, abnormal body curvature and situs inversus. Identical phenotypic changes are induced by depletion or over-expression of TRPP2. Increased PRKCSH levels ameliorate developmental abnormalities caused by over-expressed TRPP2, whereas excess TRPP2 can compensate the loss PRKCSH, indicating that the proteins share a common signaling pathway. PRKCSH binds the C-terminal domain of TRPP2, and both proteins co-localize within the ER. Furthermore, PRKCSH interacts with Herp, and inhibits Herp-mediated ubiquitination of TRPP2. Our findings suggest that PRKCSH functions as a chaperone-like molecule, which prevents ERAD of TRPP2. Dysequilibrium between TRPP2 and PRKCSH may lead to cyst formation in PCLD patients with PRKCSH mutations, and thereby account for the overlapping manifestations observed in PCLD and autosomal dominant polycystic kidney disease.
Human Molecular Genetics 10/2009; 19(1):16-24. · 7.64 Impact Factor
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Kassiani Skouloudaki,
Michael Puetz,
Matias Simons,
Jean-Remy Courbard, Christopher Boehlke,
Björn Hartleben,
Christina Engel,
Marcus J Moeller,
Christoph Englert,
Frank Bollig,
Tobias Schäfer,
Haribaskar Ramachandran,
Marek Mlodzik,
Tobias B Huber,
E Wolfgang Kuehn,
Emily Kim,
Albrecht Kramer-Zucker,
Gerd Walz
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ABSTRACT: Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila. Vertebrates express 4 Fat molecules, Fat1-4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates.
Proceedings of the National Academy of Sciences 06/2009; 106(21):8579-84. · 9.68 Impact Factor
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Michael Köttgen,
Björn Buchholz,
Miguel A Garcia-Gonzalez,
Fruzsina Kotsis,
Xiao Fu,
Mara Doerken, Christopher Boehlke,
Daniel Steffl,
Robert Tauber,
Tomasz Wegierski,
Roland Nitschke,
Makoto Suzuki,
Albrecht Kramer-Zucker,
Gregory G Germino,
Terry Watnick,
Jean Prenen,
Bernd Nilius,
E Wolfgang Kuehn,
Gerd Walz
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ABSTRACT: The primary cilium has evolved as a multifunctional cellular compartment that decorates most vertebrate cells. Cilia sense mechanical stimuli in various organs, but the molecular mechanisms that convert the deflection of cilia into intracellular calcium transients have remained elusive. Polycystin-2 (TRPP2), an ion channel mutated in polycystic kidney disease, is required for cilia-mediated calcium transients but lacks mechanosensitive properties. We find here that TRPP2 utilizes TRPV4 to form a mechano- and thermosensitive molecular sensor in the cilium. Depletion of TRPV4 in renal epithelial cells abolishes flow-induced calcium transients, demonstrating that TRPV4, like TRPP2, is an essential component of the ciliary mechanosensor. Because TRPV4-deficient zebrafish and mice lack renal cysts, our findings challenge the concept that defective ciliary flow sensing constitutes the fundamental mechanism of cystogenesis.
The Journal of General Physiology 10/2008; 132(3):i2. · 3.84 Impact Factor
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Michael Köttgen,
Björn Buchholz,
Miguel A. Garcia-Gonzalez,
Fruzsina Kotsis,
Xiao Fu,
Mara Doerken, Christopher Boehlke,
Daniel Steffl,
Robert Tauber,
Tomasz Wegierski,
Roland Nitschke,
Makoto Suzuki,
Albrecht Kramer-Zucker,
Gregory G. Germino,
Terry Watnick,
Jean Prenen,
Bernd Nilius,
E. Wolfgang Kuehn,
Gerd Walz
[show abstract]
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ABSTRACT: The primary cilium has evolved as a multifunctional cellular compartment that decorates most vertebrate cells. Cilia sense
mechanical stimuli in various organs, but the molecular mechanisms that convert the deflection of cilia into intracellular
calcium transients have remained elusive. Polycystin-2 (TRPP2), an ion channel mutated in polycystic kidney disease, is required
for cilia-mediated calcium transients but lacks mechanosensitive properties. We find here that TRPP2 utilizes TRPV4 to form
a mechano- and thermosensitive molecular sensor in the cilium. Depletion of TRPV4 in renal epithelial cells abolishes flow-induced
calcium transients, demonstrating that TRPV4, like TRPP2, is an essential component of the ciliary mechanosensor. Because
TRPV4-deficient zebrafish and mice lack renal cysts, our findings challenge the concept that defective ciliary flow sensing
constitutes the fundamental mechanism of cystogenesis.
The Journal of Cell Biology 08/2008; 182(3):437-447. · 10.26 Impact Factor
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E Wolfgang Kuehn,
Marc N Hirt,
Anne-K John,
Petra Muehlenhardt, Christopher Boehlke,
Michael Pütz,
Albrecht G Kramer-Zucker,
Mikhail Bashkurov,
Philipp S van de Weyer,
Fruzsina Kotsis,
Gerd Walz
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ABSTRACT: Inherited mutations in genes encoding for ciliary proteins lead to a broad spectrum of human diseases, such as polycystic kidney disease (PKD), situs inversus and retinitis pigmentosa. In the human kidney, autosomal dominant PKD (ADPKD) is caused by mutations in PKD1 (PC1), or PKD2 (TRPP2). Both are necessary for ciliary mechanotransduction, whereby bending of the cilium elicits a calcium response in the cell. We have previously shown that overexpression of mutated forms of the chemosensor kidney injury molecule 1 (Kim1) abolishes the flow response in ciliated MDCK cells. Here we identify Kim1 as an endogenous ciliary protein. Kim1 co-precipitates with TRPP2. Mutational analysis reveals that the interaction between Kim1 and TRPP2 requires the ciliary sorting motif in the N-terminus of TRPP2, and the presence of a highly conserved tyrosine in the intracellular tail of Kim1, which has previously been shown to play a role in ciliary flow sensing. These data support the notion that TRPP2 functionally interacts with ciliary chemosensors.
Biochemical and Biophysical Research Communications 01/2008; 364(4):861-6. · 2.48 Impact Factor
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ABSTRACT: Primary cilia have been shown to play an important role in embryonic development as well as in postnatal life. Dysfunctional cilia are associated with situs inversus, retinal abnormalities, impaired mucociliary clearance, infertility, hydrocephalus, and congenital renal cysts. In autosomal dominant polycystic kidney disease, mutations of the ciliary proteins polycystin1 or the transient receptor potential (TRP) channel family protein polycystin2 (TRPP2) cause progressive cyst formation and destruction of the kidney. Primary cilia act as flow sensors and respond to flow-mediated bending with a prolonged intracellular calcium increase, which appears to require an intact polycystin protein complex. We have established a novel flow chamber system, which allows us to study renal epithelial cells by live cell imaging. We show that MDCK cells respond to flow by a delayed increase in intracellular calcium and that this response requires these cells to be ciliated. We show that a novel interactor of TRPP2, kidney injury molecule-1 (Kim1), which is expressed at low levels in the normal kidney and upregulated after ischemia, in renal cell cancer and in PKD is targeted to primary cilia when stably expressed in MDCK cells. We demonstrate that expression of tyrosine mutant Kim1, lacking a conserved tyrosine in the intracellular tail, abolishes the calcium increase in response to flow in a dominant negative manner. These results establish Kim1 as a novel regulatory molecule of flow-induced calcium signaling.
Pflügers Archiv - European Journal of Physiology 04/2007; 453(6):819-29. · 4.46 Impact Factor
