E. Wolfgang Kuehn

Primary cilia regulate mTORC1 activity and cell size through Lkb1

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.

Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development

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.

mTOR and rapamycin in the kidney: signaling and therapeutic implications beyond immunosuppression

The immunosuppressive drug rapamycin has helped to identify a large signaling network around the target of rapamycin (TOR) protein that integrates information on nutrient availability and growth factors to control protein synthesis and cell size. Studies using rapamycin in animal models of kidney disease indicate that mTOR deregulation has a role in glomerular disease, polycystic kidney disease, and renal cancer. The role of mTOR activation in podocytes is context dependent, and indirect evidence suggests that mTOR may have a role in chronic podocyte loss. Several lines of evidence show that cyst formation in polycystic kidney disease (PKD) involves mTOR activation and its upstream regulator TSC. Polycystin 1 regulates mTOR activity through different pathways, and TSC intersects with the primary cilium, a crucial cell organelle in the pathogenesis of PKD. Data from hamartoma syndromes provide clear evidence that mutation of members of the mTOR network results in renal cancers. The detailed analysis of renal cell carcinomas has revealed a positive feedback loop involving VHL and mTOR. Rapamycin and its derivatives have been approved for the treatment of advanced renal cancer and are being investigated for the treatment of PKD. Discrepancies exist between the effects of rapamycin in animal models and the clinical experience with patients, precluding the widespread use of mTOR inhibitors in kidney disease. The details of mTOR signaling in the kidney need to be clarified to hopefully develop targeted treatments for renal disease in the future.

ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.

Inhibition of mTORC1 by Astrin and Stress Granules Prevents Apoptosis in Cancer Cells

Mammalian target of rapamycin complex 1 (mTORC1) controls growth and survival in response to metabolic cues. Oxidative stress affects mTORC1 via inhibitory and stimulatory inputs. Whereas downregulation of TSC1-TSC2 activates mTORC1 upon oxidative stress, the molecular mechanism of mTORC1 inhibition remains unknown. Here, we identify astrin as an essential negative mTORC1 regulator in the cellular stress response. Upon stress, astrin inhibits mTORC1 association and recruits the mTORC1 component raptor to stress granules (SGs), thereby preventing mTORC1-hyperactivation-induced apoptosis. In turn, balanced mTORC1 activity enables expression of stress factors. By identifying astrin as a direct molecular link between mTORC1, SG assembly, and the stress response, we establish a unifying model of mTORC1 inhibition and activation upon stress. Importantly, we show that in cancer cells, apoptosis suppression during stress depends on astrin. Being frequently upregulated in tumors, astrin is a potential clinically relevant target to sensitize tumors to apoptosis.

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum.

Ca2+ is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca2+ influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca2+‐dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca2+ concentration in the endoplasmic reticulum (ER). ER‐resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca2+ ATPase by increasing the ER Ca2+ permeability. This results in diminished cytosolic and mitochondrial Ca2+ signals upon stimulation of inositol 1,4,5‐trisphosphate receptors and reduces Ca2+ release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca2+ release and augments sensitivity to apoptosis. Our findings indicate an important function of ER‐resident TRPP2 in the modulation of intracellular Ca2+ signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.

Differential role of Rab proteins in ciliary trafficking: Rab23 regulates Smoothened levels

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.

von Hippel-Lindau: A Tumor Suppressor Links Microtubules to Ciliogenesis and Cancer Development

Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic renal cell carcinoma. The tumor suppressor role of the protein pVHL is based on its ability to target transcription factors of the hypoxia-inducible factor family for degradation, but other functions of pVHL are less clearly defined. New findings show that pVHL is necessary for cilia formation. pVHL interacts with PAR proteins, a complex that specifies the membrane domains of polarized epithelial cells, and directs the orientation of growing microtubules. Loss of pVHL results in aberrant orientation of newly formed microtubules and prevents ciliogenesis. These results add to a growing body of evidence linking cilia and the cell cycle and suggest that the tumor suppressor role of pVHL may involve previously unrecognized pathways.

Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly.

Crystallographic analysis of the B core complex of the intraflagellar transport machinery provides insight into the molecular basis of ciliogenesis defects caused by several specific IFT mutations.

The ciliary flow sensor and polycystic kidney disease

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.