Primary Cilia Disassembly Research Articles

Distinct Roles of TRAPPC8 and TRAPPC12 in Ciliogenesis via Their Interactions With OFD1.

The transport protein particle (TRAPP) complex was initially identified as a tethering factor for COPII vesicle. Subsequently, three forms (TRAPPI, II, and III) have been found and TRAPPIII has been reported to serve as a regulator in autophagy. This study investigates a new role of mammalian TRAPPIII in ciliogenesis. We found a ciliopathy protein, oral-facial-digital syndrome 1 (OFD1), interacting with the TRAPPIII-specific subunits TRAPPC8 and TRAPPC12. TRAPPC8 is necessary for the association of OFD1 with pericentriolar material 1 (PCM1). Its depletion reduces the extent of colocalized signals between OFD1 and PCM1, but does not compromise the structural integrity of centriolar satellites. The interaction between TRAPPC8 and OFD1 inhibits that between OFD1 and TRAPPC12, suggesting different roles of TRAPPIII-specific subunits in ciliogenesis and explaining the differences in cilium lengths in TRAPPC8-depleted and TRAPPC12-depleted hTERT-RPE1 cells. On the other hand, TRAPPC12 depletion causes increased ciliary length because TRAPPC12 is required for the disassembly of primary cilia. Overall, this study has revealed different roles of TRAPPC8 and TRAPPC12 in the assembly of centriolar satellites and demonstrated a possible tethering role of TRAPPIII during ciliogenesis.

Rab7 regulates primary cilia disassembly through cilia excision.

The primary cilium is a sensory organelle that protrudes from the cell surface. Primary cilia undergo dynamic transitions between assembly and disassembly to exert their function in cell signaling. In this study, we identify the small GTPase Rab7 as a novel regulator of cilia disassembly. Depletion of Rab7 potently induced spontaneous ciliogenesis in proliferating cells and promoted cilia elongation during quiescence. Moreover, Rab7 performs an essential role in cilia disassembly; knockdown of Rab7 blocked serum-induced ciliary resorption, and active Rab7 was required for this process. Further, we demonstrate that Rab7 depletion significantly suppresses cilia tip excision, referred to as cilia ectocytosis, which has been identified as required for cilia disassembly. Mechanically, the failure of F-actin polymerization at the site of excision of cilia tips caused suppression of cilia ectocytosis on Rab7 depletion. Overall, our results suggest a novel function for Rab7 in regulating cilia ectocytosis and cilia disassembly via control of intraciliary F-actin polymerization.

Fbxo41 Promotes Disassembly of Neuronal Primary Cilia

Neuronal primary cilia are signaling organelles with crucial roles in brain development and disease. Cilia structure is decisive for their signaling capacities but the mechanisms regulating it are poorly understood. We identify Fbxo41 as a novel Skp1/Cullin1/F-box (SCF) E3-ligase complex subunit that targets to neuronal centrioles where its accumulation promotes disassembly of primary cilia, and affects sonic hedgehog signaling, a canonical ciliary pathway. Fbxo41 targeting to centrioles requires its Coiled-coil and F-box domains. Levels of Fbxo41 at the centrioles inversely correlate with neuronal cilia length, and mutations that disrupt Fbxo41 targeting or assembly into SCF-complexes also disturb its function in cilia disassembly and signaling. Fbxo41 dependent cilia disassembly in mitotic and post-mitotic cells requires rearrangements of the actin-cytoskeleton, but requires Aurora A kinase activation only in mitotic cells, highlighting important mechanistical differences controlling cilia size between mitotic and post-mitotic cells. Phorbol esters induce recruitment of overexpressed Fbxo41 to centrioles and cilia disassembly in neurons, but disassembly can also occur in absence of Fbxo41. We propose that Fbxo41 targeting to centrosomes regulates neuronal cilia structure and signaling capacity in addition to Fbxo41-independent pathways controlling cilia size.

The correlative hypotheses between Pitchfork and Kif3a in palate development

It is well known that dysfunction of primary cilia during embryonic development causes a range of developmental disorders such as cleft lip and palate, lung, kidney and heart disease. Both Pitchfork and Kinesin family member 3a (Kif3a) are associating with primary cilia, but whether there is a correlation between them are still inconclusive. Our research confirmed that Pitchfork over-expression induced lateral cleft palate and primary cilia disassembly during palate development. We also demonstrated that Sonic hedgehog (Shh) and Patched1 (Ptc1) expression levels were altering in the over-expressed Pitchfork group during palate development. Then we observed by consulting a vast amount of literature that specific knockout of the Kif3a also induced lateral cleft palate and expended the expression domains of Shh and Gli1 during palate development. Furthermore, loss of the Kif3a results in disassembly of the primary cilia and eventually leads to abnormal palatal development. Finally, we found that both Pitchfork and Kif3a are accumulating at the basal body and ciliary necklace during the early phase of cilia assembly and disassembly and both of them are involved in ciliary transport. Based on the above evidence, we hypotheses that there may be a potential correlation between Pitchfork and Kif3a, that could regulate primary cilia disassembly during palate development.

Failure to reabsorb the primary cilium induces cellular senescence.

Aurora kinase A (AURKA) is necessary for proper primary cilium disassembly before mitosis. We found that depletion of caveolin-1 expression promotes primary cilia formation through the proteasomal-dependent degradation of aurora kinase A and induces premature senescence in human fibroblasts. Down-regulation of intraflagellar transport-88, a protein essential for ciliogenesis, inhibits premature senescence induced by the depletion of caveolin-1. In support of these findings, we showed that alisertib, a pharmacological inhibitor of AURKA, causes primary cilia formation and cellular senescence by irreversibly arresting cell growth. Suppression of primary cilia formation limits cellular senescence induced by alisertib. The primary cilium must be disassembled to free its centriole to form the centrosome, a necessary structure for mitotic spindle assembly and cell division. We showed that the use of the centriole to form primary cilia blocks centrosome formation and mitotic spindle assembly and prevents the completion of mitosis in cells in which cellular senescence is caused by the inhibition of AURKA. We also found that AURKA is down-regulated and primary cilia formation is enhanced when cellular senescence is promoted by other senescence-inducing stimuli, such as oxidative stress and UV light. Thus, we propose that impaired AURKA function induces premature senescence by preventing reabsorption of the primary cilium, which inhibits centrosome and mitotic spindle formation and consequently prevents the completion of mitosis. Our study causally links the inability of the cell to disassemble the primary cilium, a microtubule-based cellular organelle, to the development of premature senescence, a functionally and pathologically relevant cellular state.-Jeffries, E. P., Di Filippo, M., Galbiati, F. Failure to reabsorb the primary cilium induces cellular senescence.

Primary Cilia as Signaling Hubs in Health and Disease.

Primary cilia detect extracellular cues and transduce these signals into cells to regulate proliferation, migration, and differentiation. Here, the function of primary cilia as signaling hubs of growth factors and morphogens is in focus. First, the molecular mechanisms regulating the assembly and disassembly of primary cilia are described. Then, the role of primary cilia in mediating growth factor and morphogen signaling to maintain human health and the potential mechanisms by which defects in these pathways contribute to human diseases, such as ciliopathy, obesity, and cancer are described. Furthermore, a novel signaling pathway by which certain growth factors stimulate cell proliferation through suppression of ciliogenesis is also described, suggesting novel therapeutic targets in cancer.

Mechanical loading induces primary cilia disassembly in tendon cells via TGF\u03b2 and HDAC6

This study used isolated human tenocytes to test the hypothesis that cyclic mechanical strain directly stimulates primary cilia disassembly, and to elucidate the mechanisms involved. Cells were seeded onto flexible membranes and strained at 0–3%; 1 Hz, for up to 24 hours. Cilia length and prevalence progressively reduced with increasing strain duration but showed full recovery within 2 hours of strain removal. The response to loading was not influenced by actin organisation as seen in other cell types. However, the loading response could be recreated by treatment with TGFβ. Furthermore, treatment with the HDAC6 inhibitor Tubacin, or a TGFβ receptor inhibitor both prevented strain induced cilia disassembly. These data are the first to describe primary cilia expression in isolated tenocytes, showing that mechanical strain regulates cilia expression independent of changes in tendon extracellular matrix. Furthermore, we show that cilia disassembly is mediated by the activation of TGFβ receptors leading to activation of HDAC6. Previous studies have shown that cilia are required for TGFβ signalling and that tendon mechanosignalling is mediated by TGFβ. The present study therefore suggests a novel feedback mechanism whereby cilia disassembly inhibits prolonged TGFβ activation in response to continuous cyclic loading.

Autophagy alteration prevents primary cilium disassembly in RPE1 cells

Primary cilium is a microtubule structure that emanates from the surface of most human cells. Primary cilia assemble during the resting stage (G0 phase) and disassemble with cell cycle progression. Defects associated with the control of the assembly or disassembly of the primary cilium have been implicated in various human diseases, including ciliopathy and cancer. Although studies have suggested the interplay between activation of autophagy and ciliogenesis, any direct mechanism between autophagy abatement and disassembly of primary cilium remains elusive. In this study, we found that the gradual abatement in autophagy during serum-restimulation was a dynamic process and significantly correlated with the disassembly of primary cilium in human retinal pigmented epithelial (RPE1) cells. Although autophagy activity was gradually decreased during serum-restimulation, the alteration in autophagy under the same condition prevented the disassembly of the primary cilium. Autophagy inhibitors such as chloroquine, U18666A and 3-methyladenine (3-MA) retained both the number of ciliated cells and cilium length. In contrast, rapamycin treatment during serum-restimulation maintained the number of ciliated cells with shortened cilia. Taken together, alteration in autophagy during serum-restimulation prevent the disassembly of the primary cilium, and autophagy modulators may serve as useful compounds for studying mechanistic details related to the disassembly of the primary cilium and ciliopathy.

Oncoprotein CIP2A promotes the disassembly of primary cilia and inhibits glycolytic metabolism.

In most mammalian cells, the primary cilium is a microtubule-enriched protrusion of the plasma membrane and acts as a key coordinator of signaling pathways during development and tissue homeostasis. The primary cilium is generated from the basal body, and cancerous inhibitor of protein phosphatase 2A (CIP2A), the overexpression of which stabilizes c-MYC to support the malignant growth of tumor cells, is localized in the centrosome. Here, we show that CIP2A overexpression induces primary cilia disassembly through the activation of Aurora A kinase, and CIP2A depletion increases ciliated cells and cilia length in retinal pigment epithelium (RPE1) cells. CIP2A depletion also shifts metabolism toward the glycolytic pathway by altering the expression of metabolic genes related to glycolysis. However, glycolytic activation in CIP2A-depleted cells does not depend on cilia assembly, even though enhanced cilia assembly alone activates glycolytic metabolism. Collectively, these data suggest that CIP2A promotes primary cilia disassembly and that CIP2A depletion induces metabolic reprogramming independent of primary cilia.

The Fungal Metabolite Brefeldin A Inhibits Dvl2-Plk1-Dependent Primary Cilium Disassembly.

The primary cilium is a non-motile microtubule-based organelle that protrudes from the surface of most human cells and works as a cellular antenna to accept extracellular signals. Primary cilia assemble from the basal body during the resting stage (G0 phase) and simultaneously disassemble with cell cycle re-entry. Defective control of assembly or disassembly causes diverse human diseases including ciliopathy and cancer. To identify the effective compounds for studying primary cilium disassembly, we have screened 297 natural compounds and identified 18 and 17 primary cilium assembly and disassembly inhibitors, respectively. Among them, the application of KY-0120, identified as Brefeldin A, disturbed Dvl2-Plk1-mediated cilium disassembly via repression of the interaction of CK1ɛ-Dvl2 and the expression of Plk1 mRNA. Therefore, our study may suggest useful compounds for studying the cellular mechanism of primary cilium disassembly to prevent ciliopathy and cancer.

The electric fence to cell-cycle progression: Do local changes in membrane potential facilitate disassembly of the primary cilium?: Timely and localized expression of a potassium channel may set the conditions that allow retraction of the primary cilium.

Kv10.1 is a voltage-gated potassium channel relevant for tumor biology, but the underlying mechanism is still unclear. We propose that Kv10.1 plays a role coordinating primary cilium disassembly with cell cycle progression through localized changes of membrane potential at the ciliary base. Most non-dividing cells display a primary cilium, an antenna-like structure important for cell physiology. The cilium is disassembled when the cell divides, which requires an increase of Ca2+ concentration and a redistribution of phospholipids in its basal region, both of which would be facilitated by local hyperpolarization. Cells lacking Kv10.1 show impaired ciliary disassembly and delayed entrance into mitosis. Kv10.1 is predominantly expressed during G2/M, a critical period for ciliary resorption, and localizes to the ciliary base and vesicles associated with the centrosome. This could explain the influence of Kv10.1 in cell proliferation, as well as phenotypic features of patients carrying gain of function mutations in the gene.

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.

Cilium assembly and disassembly.

The primary cilium is an antenna-like, immotile organelle present on most types of mammalian cells, which interprets extracellular signals that regulate growth and development. Although once considered a vestigial organelle, the primary cilium is now the focus of considerable interest. We now know that ciliary defects lead to a panoply of human diseases, termed ciliopathies, and the loss of this organelle may be an early signature event during oncogenic transformation. Ciliopathies include numerous seemingly unrelated developmental syndromes, with involvement of the retina, kidney, liver, pancreas, skeletal system and brain. Recent studies have begun to clarify the key mechanisms that link cilium assembly and disassembly to the cell cycle, and suggest new possibilities for therapeutic intervention.

Modulation of primary cilia length by melanin-concentrating hormone receptor 1

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1) is a class A G-protein-coupled receptor (GPCR). The MCH-MCHR1 system has been implicated in the regulation of feeding, emotional processing, and sleep in rodents. Recent work revealed that MCHR1 is selectively expressed in neuronal primary cilia of the central nervous system. Cilia have various chemosensory functions in many types of cell, and ciliary dysfunction is associated with ciliopathies such as polycystic kidney disease and obesity. Although dynamic modulation of neuronal cilia length is observed in obese mice, the functional interaction of neuronal ciliary GPCR and its endogenous ligand has not yet been elucidated. We report here that MCH treatment significantly reduced cilia length in hTERT-RPE1 cells (hRPE1 cells) transfected with MCHR1. Quantitative analyses indicated that MCH-induced cilia shortening progressed in a dose-dependent manner with an EC50 lower than 1nM when cells were treated for 6h. Although the assembly and disassembly of primary cilia are tightly coupled to the cell cycle, cell cycle reentry was not a determinant of MCH-induced cilia shortening. We confirmed that MCH elicited receptor internalization, Ca2+ mobilization, ERK and Akt phosphorylation, and inhibition of cyclic AMP accumulation in MCHR1-expressing hRPE1 cells. Among these diverse pathways, we revealed that Gi/o-dependent Akt phosphorylation was an important component in the initial stage of MCH-induced cilia length shortening. Furthermore, induction of fewer cilia by Kif3A siRNA treatment significantly decreased the MCH-mediated phosphorylation of Akt, indicating the functional importance of the MCHR1-Akt pathway in primary cilia. Taken together, the present data suggest that the MCH-MCHR1 axis may modulate the sensitivity of cells to external environments by controlling the cilia length. Therefore, further characterization of MCHR1 as a ciliary GPCR will provide a potential molecular mechanism to link cilia length control with obesity.

Nek2 activation of Kif24 ensures cilium disassembly during the cell cycle.

Many proteins are known to promote ciliogenesis, but mechanisms that promote primary cilia disassembly before mitosis are largely unknown. Here we identify a mechanism that favours cilium disassembly and maintains the disassembled state. We show that co-localization of the S/G2 phase kinase, Nek2 and Kif24 triggers Kif24 phosphorylation, inhibiting cilia formation. We show that Kif24, a microtubule depolymerizing kinesin, is phosphorylated by Nek2, which stimulates its activity and prevents the outgrowth of cilia in proliferating cells, independent of Aurora A and HDAC6. Our data also suggest that cilium assembly and disassembly are in dynamic equilibrium, but Nek2 and Kif24 can shift the balance toward disassembly. Further, Nek2 and Kif24 are overexpressed in breast cancer cells, and ablation of these proteins restores ciliation in these cells, thereby reducing proliferation. Thus, Kif24 is a physiological substrate of Nek2, which regulates cilia disassembly through a concerted mechanism involving Kif24-mediated microtubule depolymerization.

Autophagy Regulates Formation of Primary Cilia in Mefloquine-Treated Cells.

Primary cilia have critical roles in coordinating multiple cellular signaling pathways. Dysregulation of primary cilia is implicated in various ciliopathies. To identify specific regulators of autophagy, we screened chemical libraries and identified mefloquine, an anti-malaria medicine, as a potent regulator of primary cilia in human retinal pigmented epithelial (RPE) cells. Not only ciliated cells but also primary cilium length was increased in mefloquine-treated RPE cells. Treatment with mefloquine strongly induced the elongation of primary cilia by blocking disassembly of primary cilium. In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux. Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells. Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.

The Microtubule-Depolymerizing Activity of a Mitotic Kinesin Protein KIF2A Drives Primary Cilia Disassembly Coupled with Cell Proliferation

SUMMARYThe primary cilium is an antenna-like, microtubule-based organelle on the surface of most vertebrate cells for receiving extracellular information. Although primary cilia form in the quiescent phase, ciliary disassembly occurs when quiescent cells re-enter the proliferative phase. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for cell-proliferation-coupled primary cilia disassembly. Here, we report that kinesin superfamily protein 2A (KIF2A), phosphorylated at T554 by PLK1, exhibits microtubule-depolymerizing activity at the mother centriole to disassemble the primary cilium in a growth-signal-dependent manner. KIF2A-deficient hTERT-RPE1 cells showed the impairment of primary cilia disassembly following growth stimulation. It was also found that the PLK1-KIF2A pathway is constitutively active in cells from patients with premature chromatid separation (PCS) syndrome and is responsible for defective ciliogenesis in this syndrome. These findings provide insights into the roles of the PLK1-KIF2A pathway in physiological cilia disassembly and cilia-associated disorders.

PDGFRβ and oncogenic mutant PDGFRα D842V promote disassembly of primary cilia through a PLCγ- and AURKA-dependent mechanism.

Primary cilia are microtubule-based sensory organelles projecting from most quiescent mammalian cells, which disassemble in cells cultured in serum-deprived conditions upon re-addition of serum or growth factors. Platelet-derived growth factors (PDGF) are implicated in deciliation, but the specific receptor isoforms and mechanisms involved are unclear. We report that PDGFRβ promotes deciliation in cultured cells and provide evidence implicating PLCγ and intracellular Ca(2+) release in this process. Activation of wild-type PDGFRα alone did not elicit deciliation. However, expression of constitutively active PDGFRα D842V mutant receptor, which potently activates PLCγ (also known as PLCG1), caused significant deciliation, and this phenotype was rescued by inhibiting PDGFRα D842V kinase activity or AURKA. We propose that PDGFRβ and PDGFRα D842V promote deciliation through PLCγ-mediated Ca(2+) release from intracellular stores, causing activation of calmodulin and AURKA-triggered deciliation.

Morphological and molecular changes associated with Pitchfork during mouse palate development.

Mammalian palate development is regulated by complex processes. Many cellular and molecular events, such as cell proliferation, apoptosis, cell migration and the epithelial mesenchymal transition, regulate proper palate development and some abnormalities in palate development lead to cleft palate. Various developmental disorders, such as cleft palate and disorders of the lung, kidney and heart, are known to be associated with ciliary defects. Pitchfork, a mouse embryonic node gene, is associated with ciliary targeting complexes located at the basal body during primary cilia disassembly. To determine the function of Pitchfork during palate development, we examine Pitchfork expression patterns and morphological changes in the developing secondary palate after Pitchfork over-expression. From embryonic day 12.5 (E12.5) to E13.5 in mice, Pitchfork was highly expressed in the developing mouse secondary palate. Morphological differences were observed in vitro in cultured palates in the Pitchfork over-expression group compared with the control group. Pitchfork over-expression induced primary cilia disassembly during palate development. Sonic hedgehog and Patched1 expression levels and palatine rugae morphology were altered in the over-expressed Pitchfork group during palate development. Thus, the proper expression levels of Pitchfork might play a pivotal role in normal secondary palate morphogenesis.

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.