Identification of a novel Wnt5a-CK1ɛ-Dvl2-Plk1-mediated primary cilia disassembly pathway

Non‐motile primary cilium is an antenna‐like structure whose defect is associated with a wide range of pathologies, including developmental disorders and cancer. Although mechanisms regulating cilia assembly have been extensively studied, how cilia disassembly is regulated remains poorly understood. Here, we report unexpected roles of Dishevelled 2 (Dvl2) and interphase polo‐like kinase 1 (Plk1) in primary cilia disassembly. We demonstrated that Dvl2 is phosphorylated at S143 and T224 in a manner that requires both non‐canonical Wnt5a ligand and casein kinase 1 epsilon (CK1ε), and that this event is critical to interact with Plk1 in early stages of the cell cycle. The resulting Dvl2–Plk1 complex mediated Wnt5a–CK1ε–Dvl2‐dependent primary cilia disassembly by stabilizing the HEF1 scaffold and activating its associated Aurora‐A (AurA), a kinase crucially required for primary cilia disassembly. Thus, via the formation of the Dvl2–Plk1 complex, Plk1 plays an unanticipated role in primary cilia disassembly by linking Wnt5a‐induced biochemical steps to HEF1/AurA‐dependent cilia disassembly. This study may provide new insights into the mechanism underlying ciliary disassembly processes and various cilia‐related disorders.     

A novel interaction between kinase activities in regulation of cilia formation

Background

The primary cilium is an extension of the cell membrane that encloses a microtubule-based axoneme. Primary cilia are essential for transmission of environmental cues that determine cell fate. Disruption of primary cilia function is the molecular basis of numerous developmental disorders. Despite their biological importance, the mechanisms governing their assembly and disassembly are just beginning to be understood. Cilia growth and disassembly are essential events when cells exit and reenter into the cell cycle. The kinases never in mitosis-kinase 2 (Nek2) and Aurora A (AurA) act to depolymerize cilia when cells reenter the cell cycle from G₀.

Results

Coexpression of either kinase with its kinase dead companion [AurA with kinase dead Nek2 (Nek2 KD) or Nek2 with kinase dead AurA (AurA KD)] had different effects on cilia depending on whether cilia are growing or shortening. AurA and Nek2 are individually able to shorten cilia when cilia are growing but both are required when cilia are being absorbed. The depolymerizing activity of each kinase is increased when coexpressed with the kinase dead version of the other kinase but only when cilia are assembling. Additionally, the two kinases act additively when cilia are assembling but not disassembling. Inhibition of AurA increases cilia number while inhibition of Nek2 significantly stimulates cilia length. The complex functional relationship between the two kinases reflects their physical interaction. Further, we identify a role for a PP1 binding protein, PPP1R42, in inhibiting Nek2 and increasing ciliation of ARPE-19 cells.

Conclusion

We have uncovered a novel functional interaction between Nek2 and AurA that is dependent on the growth state of cilia. This differential interdependence reflects opposing regulation when cilia are growing or shortening. In addition to interaction between the kinases to regulate ciliation, the PP1 binding protein PPP1R42 directly inhibits Nek2 independent of PP1 indicating another level of regulation of this kinase. In summary, we demonstrate a complex interplay between Nek2 and AurA kinases in regulation of ciliation in ARPE-19 cells.

     

Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

We show here that human embryonic stem (ES) and induced pluripotent stem cell–derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide—in particular, saturated, long-chain C_16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C_24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C_24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation.     

Calmodulin activation of Aurora-A kinase (AURKA) is required during ciliary disassembly and in mitosis

The centrosomal Aurora-A kinase (AURKA) regulates mitotic progression, and overexpression and hyperactivation of AURKA commonly promotes genomic instability in many tumors. Although most studies of AURKA focus on its role in mitosis, some recent work identified unexpected nonmitotic activities of AURKA. Among these, a role for basal body-localized AURKA in regulating ciliary disassembly in interphase cells has highlighted a role in regulating cellular responsiveness to growth factors and mechanical cues. The mechanism of AURKA activation involves interactions with multiple partner proteins and is not well understood, particularly in interphase cells. We show here that AURKA activation at the basal body in ciliary disassembly requires interactions with Ca(2+) and calmodulin (CaM) and that Ca(2+)/CaM are important mediators of the ciliary disassembly process. We also show that Ca(2+)/CaM binding is required for AURKA activation in mitosis and that inhibition of CaM activity reduces interaction between AURKA and its activator, NEDD9. Finally, mutated derivatives of AURKA impaired for CaM binding and/or CaM-dependent activation cause defects in mitotic progression, cytokinesis, and ciliary resorption. These results define Ca(2+)/CaM as important regulators of AURKA activation in mitotic and nonmitotic signaling.     

纤毛及纤毛相关疾病研究进展

纤毛是一种以细胞微管为主形成的突出于细胞表面的结构,分布于哺乳动物体内的大多数细胞。近年来研究发现,很多人类疾病都与纤毛结构、长度的失调相关,所以有关纤毛的研究是目前研究的热点领域。越来越多的证据证明,纤毛除了提供流体推动力参与细胞的运动功能之外,还具有信号传导的功能,在细胞生命活动的各个方面发挥着多种关键作用。它参与调控细胞生理活动、增殖与分化以及动物个体发育。因此,深入地探索纤毛调控机理对基础生物学理论的发展和人类纤毛相关疾病的攻克有重要意义。该文简要介绍了纤毛的结构、组装与解聚的机制、参与信号传导的功能以及纤毛缺陷同人类疾病的关系。     

Primary cilium: an elaborate structure that blocks cell division?

A primary cilium is a microtubule-based membranous protrusion found in almost all cell types. A primary cilium has a “9 + 0” axoneme that distinguishes this ancient organelle from the canonical motile “9 + 2” cilium. A primary cilium is the sensory center of the cell that regulates cell proliferation and embryonic development. The primary ciliary pocket is a specialized endocytic membrane domain in the basal region. The basal body of a primary cilium exists as a form of the centriole during interphase of the cell cycle. Although conventional thinking suggests that the cell cycle regulates centrosomal changes, recent studies suggest the opposite, that is, centrosomal changes regulate the cell cycle. In this regard, centrosomal kinase Aurora kinase A (AurA), Polo-like kinase 1 (Plk1), and NIMA related Kinase (Nek or Nrk) propel cell cycle progression by promoting primary cilia disassembly which indicates a non-mitotic function. However, the persistence of primary cilia during spermatocyte division challenges the dominate idea of the incompatibility of primary cilia and cell division. In this review, we demonstrate the detailed structure of primary cilia and discuss the relationship between primary cilia disassembly and cell cycle progression on the background of various mitotic kinases.     

Aurora A kinase activity influences calcium signaling in kidney cells

Most studies of Aurora A (AurA) describe it as a mitotic centrosomal kinase. However, we and others have recently identified AurA functions as diverse as control of ciliary resorption, cell differentiation, and cell polarity control in interphase cells. In these activities, AurA is transiently activated by noncanonical signals, including Ca(2+)-dependent calmodulin binding. These and other observations suggested that AurA might be involved in pathological conditions, such as polycystic kidney disease (PKD). In this paper, we show that AurA is abundant in normal kidney tissue but is also abnormally expressed and activated in cells lining PKD-associated renal cysts. PKD arises from mutations in the PKD1 or PKD2 genes, encoding polycystins 1 and 2 (PC1 and PC2). AurA binds, phosphorylates, and reduces the activity of PC2, a Ca(2+)-permeable nonselective cation channel and, thus, limits the amplitude of Ca(2+) release from the endoplasmic reticulum. These and other findings suggest AurA may be a relevant new biomarker or target in the therapy of PKD.     

Trichoplein and Aurora A block aberrant primary cilia assembly in proliferating cells

The primary cilium is an antenna-like organelle that modulates differentiation, sensory functions, and signal transduction. After cilia are disassembled at the G0/G1 transition, formation of cilia is strictly inhibited in proliferating cells. However, the mechanisms of this inhibition are unknown. In this paper, we show that trichoplein disappeared from the basal body in quiescent cells, whereas it localized to mother and daughter centrioles in proliferating cells. Exogenous expression of trichoplein inhibited primary cilia assembly in serum-starved cells, whereas ribonucleic acid interference-mediated depletion induced primary cilia assembly upon cultivation with serum. Trichoplein controlled Aurora A (AurA) activation at the centrioles predominantly in G1 phase. In vitro analyses confirmed that trichoplein bound and activated AurA directly. Using trichoplein mutants, we demonstrate that the suppression of primary cilia assembly by trichoplein required its ability not only to localize to centrioles but also to bind and activate AurA. Trichoplein or AurA knockdown also induced G0/G1 arrest, but this phenotype was reversed when cilia formation was prevented by simultaneous knockdown of IFT-20. These data suggest that the trichoplein-AurA pathway is required for G1 progression through a key role in the continuous suppression of primary cilia assembly.     

Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors

Primary cilia are displayed during the G(0)/G(1) phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1(T94E) mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G(1) length, cell cycle entry and fate determination of these cells during corticogenesis.     

A chemical screen identifies class a g-protein coupled receptors as regulators of cilia

Normal cilia length and motility are critical for proper cellular function. Prior studies of the regulation of ciliary structure and length have primarily focused on the intraflagellar transport machinery and motor proteins required for ciliary assembly and disassembly. However, several mutants with abnormal length flagella highlight the importance of signaling proteins as well. In this study, an unbiased chemical screen was performed to uncover signaling pathways that are critical for ciliogenesis and length regulation using flagella of the green alga Chlamydomonas reinhardtii as a model. The annotated Sigma LOPAC1280 chemical library was screened for effects on flagellar length, motility, and severing as well as cell viability. Assay data were clustered to identify pathways regulating flagella. The most frequent target found to be involved in flagellar length regulation was the family of dopamine binding G-protein coupled receptors (GPCRs). In mammalian cells, cilium length could indeed be altered with expression of the dopamine D1 receptor. Our screen thus reveals signaling pathways that are potentially critical for ciliary formation, resorption, and length maintenance, which represent candidate targets for therapeutic intervention of disorders involving ciliary malformation and malfunction.     

Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors

Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.     

The ciliary membrane‐associated proteome reveals actin‐binding proteins as key components of cilia

Primary cilia are sensory, antennae‐like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane‐associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high‐stoichiometric presence of actin‐binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F‐actin causes further enrichment of the actin‐binding and actin‐related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin‐binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.     

Cyclic expression of the voltage‐gated potassium channel KV10.1 promotes disassembly of the primary cilium

The primary cilium, critical for morphogenic and growth factor signaling, is assembled upon cell cycle exit, but the links between ciliogenesis and cell cycle progression are unclear. K_V10.1 is a voltage‐gated potassium channel frequently overexpressed in tumors. We have previously reported that expression of K_V10.1 is temporally restricted to a time period immediately prior to mitosis in healthy cells. Here, we provide microscopical and biochemical evidence that K_V10.1 localizes to the centrosome and the primary cilium and promotes ciliary disassembly. Interference with K_V10.1 ciliary localization abolishes not only the effects on ciliary disassembly, but also K_V10.1‐induced tumor progression in vivo. Conversely, upon knockdown of K_V10.1, ciliary disassembly is impaired, proliferation is delayed, and proliferating cells show prominent primary cilia. Thus, modulation of ciliogenesis by K_V10.1 can explain the influence of K_V10.1 expression on the proliferation of normal cells and is likely to be a major mechanism underlying its tumorigenic effects.     

CYLD mediates ciliogenesis in multiple organs by deubiquitinating Cep70 and inactivating HDAC6

Cilia are hair-like organelles extending from the cell surface with important sensory and motility functions. Ciliary defects can result in a wide range of human diseases known as ciliopathies. However, the molecular mechanisms controlling ciliogenesis remain poorly defined. Here we show that cylindromatosis (CYLD), a tumor suppressor protein harboring deubiquitinase activity, plays a critical role in the assembly of both primary and motile cilia in multiple organs. CYLD knockout mice exhibit polydactyly and various ciliary defects, such as failure in basal body anchorage and disorganization of basal bodies and axenomes. The ciliary function of CYLD is partially attributed to its deconjugation of the polyubiquitin chain from centrosomal protein of 70 kDa (Cep70), a requirement for Cep70 to interact with γ-tubulin and localize at the centrosome. In addition, CYLD-mediated inhibition of histone deacetylase 6 (HDAC6), which promotes tubulin acetylation, constitutes another mechanism for the ciliary function of CYLD. Small-molecule inhibitors of HDAC6 could partially rescue the ciliary defects in CYLD knockout mice. These findings highlight the importance of protein ubiquitination in the modulation of ciliogenesis, identify CYLD as a crucial regulator of this process, and suggest the involvement of CYLD deficiency in ciliopathies.     

Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins

Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras superfamily of GTPases, is required for ciliary structure but has poorly defined ciliary functions. In this paper, we further characterize the role of Arl13b in cilia by examining mutant cilia in vitro and determining the localization and dynamics of Arl13b within the cilium. Previously, we showed that mice lacking Arl13b have abnormal Sonic hedgehog (Shh) signaling; in this study, we show the dynamics of Shh signaling component localization to the cilium are disrupted in the absence of Arl13b. Significantly, we found Smoothened (Smo) is enriched in Arl13b-null cilia regardless of Shh pathway stimulation, indicating Arl13b regulates the ciliary entry of Smo. Furthermore, our analysis defines a role for Arl13b in regulating the distribution of Smo within the cilium. These results suggest that abnormal Shh signaling in Arl13b mutant embryos may result from defects in protein localization and distribution within the cilium.     

The centrosomal kinase Plk1 localizes to the transition zone of primary cilia and induces phosphorylation of nephrocystin-1

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.     

HDAC2 promotes loss of primary cilia in pancreatic ductal adenocarcinoma

Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells.     

Heteromerization of Ciliary G Protein-Coupled Receptors in the Mouse Brain

Nearly every cell type in the mammalian body projects from its cell surface a primary cilium that provides important sensory and signaling functions. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and adenylyl cyclase type 3, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling. Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin-concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling.