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.     

The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries

Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes.     

The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.     

Morphofunctional characteristics of the striated centrosome rootlets of cultured embryonic pig kidney cells

The analysis of the centrosome structure in PK-cells has shown that the striated rootlets occur in 25-30% of the cells. Most frequently, the centrosome contains 1-2 rootlets, oriented either to the active or to the nonactive centriole. Occasionally, they occur in both the centrioles simultaneously. The presence of striated rootlets does not correlate with the presence of a primary cilium in the centrosome: there occur centrosomes with the primary cilium but without striated rootlets, and conversely: with striated rootlets but without the primary cilium.     

Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells,but a “sleeping beauty” in cultured cells?

Primary cilium development along with other components of the centrosome in mammalian cells was analysed ultrastructurally and by immunofluorescent staining with anti-acetylated tubulin antibodies. We categorized two types of primary cilia, nascent cilia that are about 1microm long located inside the cytoplasm, and true primary cilia that are several microm long and protrude from the plasma membrane. The primary cilium is invariably associated with the older centriole of each diplosome, having appendages at the distal end and pericentriolar satellites with cytoplasmic microtubules emanating from them. Only one cilium per cell is formed normally through G(0), S and G(2)phases. However, in some mouse embryo fibroblasts with two mature centrioles, bicilates were seen. Primary cilia were not observed in cultured cells where the mature centriole had no satellites and appendages (Chinese hamster kidney cells, line 237, some clones of l-fibroblasts). In contrast to primary cilia, striated rootlets were found around active and non-active centrioles with the same frequency. In proliferating cultured cells, a primary cilium can be formed several hours after mitosis, in fibroblasts 2-4 h after cell division and in PK cells only during the S-phase. In interphase cells, formation of the primary cilium can be stimulated by the action of metabolic inhibitors and by reversed depolymerization of cytoplasmic microtubules with cold or colcemid treatments. In mouse renal epithelial cells in situ, the centrosome was located near the cell surface and mature centrioles in 80% of the cells had primary cilium protruding into the duct lumen. After cells were explanted and subcultured, the centrosome comes closer to the nucleus and the primary cilium was depolymerized or reduced. Later primary cilia appeared in cells that form islets on the coverslip. However, the centrosome in cultured ciliated cells was always located near the cell nucleus and primary cilium never formed a characteristic distal bulb. A sequence of the developmental stages of the primary cilium is proposed and discussed. We also conclude that functioning primary cilium does not necessarily operate in culture cells, which might explain some of the contradictory data on cell ciliation in vitro reported in the literature.     

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.     

CONFOCAL ANALYSIS OF PRIMARY CILIA STRUCTURE AND COLOCALIZATION WITH THE GOLGI APPARATUS IN CHONDROCYTES AND AORTIC SMOOTH MUSCLE CELLS

Detyrosinated and acetylated α-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium—centrosome and centrosome—Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.     

The importance of a single primary cilium

The centrosome is the main microtubule-organizing center in animal cells, and helps to influence the morphology of the microtubule cytoskeleton in interphase and mitosis. The centrosome also templates the assembly of the primary cilium, and together they serve as a nexus of cell signaling that provide cells with diverse organization, motility, and sensory functions. The majority of cells in the human body contain a solitary centrosome and cilium, and cells have evolved regulatory mechanisms to precisely control the numbers of these essential organelles. Defects in the structure and function of cilia lead to a variety of complex disease phenotypes termed ciliopathies, while dysregulation of centrosome number has long been proposed to induce genome instability and tumor formation. Here, we review recent findings that link centrosome amplification to changes in cilium number and signaling capacity, and discuss how supernumerary centrosomes may be an important aspect of a set of cilia-related disease phenotypes.     

CPAP promotes timely cilium disassembly to maintain neural progenitor pool

A mutation in the centrosomal‐P4.1‐associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms of NPCs maintenance remain unclear. Here, we report an unexpected role for the cilium in NPCs maintenance and identify CPAP as a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly, CPAP provides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, and OFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutated CPAP fails to localize at the ciliary base associated with inefficient CDC recruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re‐entry leading to premature differentiation of patient iPS‐derived NPCs. Aberrant CDC function also promotes premature differentiation of NPCs in Seckel iPS‐derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.     

The PCM–basal body/primary cilium coalition

The centrosome is an organelle that acts as a microtubule-organizing center (MTOC) throughout the cell cycle. Within the centrosome are often two components that each have an ability to organize microtubule structures: the centriole that has the potential to function as a basal body and nucleate a cilium or a flagellum and a mass of protein material that in the presence of a centriole is commonly referred to as the pericentriolar material (PCM) that organizes cytoplasmic and spindle microtubule arrays. One characteristic of a large variety of cells is the ability to express a non-motile primary cilium. It is now appreciated that the function of the primary cilium is integral to a variety of essential cell functions and defects affecting this structure underlie a variety of human disease. While the function of the primary cilium and manner in which a basal body organizes a primary cilium has received extensive attention there is now a need to explore the inter-relationship between the PCM and the basal body/primary cilium. It is this latter topic that is the focus of this review where we show that the PCM is integrated with the centriole to form a coalition that is essential for both the expression and function of the primary cilium as well as the organization and function of the cellular environment that surrounds it.     

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.     

CEP90 Is Required for the Assembly and Centrosomal Accumulation of Centriolar Satellites,Which Is Essential for Primary Cilia Formation

Centriolar satellites are PCM-1-positive granules surrounding centrosomes. Proposed functions of the centriolar satellites include protein targeting to the centrosome, as well as communication between the centrosome and surrounding cytoplasm. CEP90 is a centriolar satellite protein that is critical for spindle pole integrity in mitotic cells. In this study, we examined the biological functions of CEP90 in interphase cells. CEP90 physically interacts with PCM-1 at centriolar satellites, and this interaction is essential for centrosomal accumulation of the centriolar satellites and eventually for primary cilia formation. CEP90 is also required for BBS4 loading on centriolar satellites and its localization in primary cilia. Our results imply that the assembly and transport of centriolar satellites are critical steps for primary cilia formation and ciliary protein recruitment.     

The Centriolar Satellite Protein AZI1 Interacts with BBS4 and Regulates Ciliary Trafficking of the BBSome

Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer''s vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.     

Primary cilium mechanotransduction of tensile strain in 3D culture: Finite element analyses of strain amplification caused by tensile strain applied to a primary cilium embedded in a collagen matrix

Human adipose-derived stem cells (hASC) exhibit multilineage differentiation potential with lineage specification that is dictated by both the chemical and mechanical stimuli to which they are exposed. We have previously shown that 10% cyclic tensile strain increases hASC osteogenesis and cell-mediated calcium accretion. We have also recently shown that primary cilia are present on hASC and that chemically-induced lineage specification of hASC concurrently results in length and conformation changes of the primary cilia. Further, we have observed cilia length changes in hASC cultured within a collagen I gel in response to 10% cyclic tensile strain. We therefore hypothesize that primary cilia may play a key mechanotransduction role for hASC exposed to tensile strain. The goal of this study was to use finite element analysis (FEA) to determine strains occurring within the ciliary membrane in response to 10% tensile strain applied parallel, or perpendicular, to cilia orientation. To elucidate the mechanical environment experienced by the cilium, several lengths were modeled and evaluated based on cilia lengths measured on hASC grown under varied culture conditions. Principal tensile strains in both hASC and ciliary membranes were calculated using FEA, and the magnitude and location of maximum principal tensile strain determined. We found that maximum principal tensile strain was concentrated at the base of the cilium. In the linear elastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane from 150% to 200%, while applying strain parallel to the cilium resulted in much higher strains, approximately 400%. In the hyperelastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane around 30%, while applying strain parallel to the cilium resulted in much higher strains ranging from 50% to 70%. Interestingly, FEA results indicated that primary cilium length was not directly related to ciliary membrane strain. Rather, it appears that cilium orientation may be more important than cilium length in determining sensitivity of hASC to tensile strain. This is the first study to model the effects of tensile strain on the primary cilium and provides newfound insight into the potential role of the primary cilium as a mechanosensor, particularly in tensile strain and potentially a multitude of other mechanical stimuli beyond fluid shear.     

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

The ciliopathy-linked protein CCDC66 is only known for its ciliary functions. This study reveals that CCDC66 also has extensive non-ciliary functions, localizing to the spindle poles, spindle midzone, central spindle and midbody throughout cell division, where it regulates mitosis and cytokinesis by promoting microtubule nucleation and organization.     

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase‐4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.     

Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

Background

The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by β-arrestins, βarr1 and βarr2, which control both their signalling and endocytosis, suggesting that βarrs may also function at primary cilium.

Methodology/Principal Findings

In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.

Conclusions/Significance

Our results show that βarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”.     

Regulation of basal body and ciliary functions by Diversin

The centrosome is essential for the formation of the cilia and has been implicated in cell polarization and signaling during early embryonic development. A number of Wnt pathway components were found to localize at the centrosome, but how this localization relates to their signaling functions is unclear. In this study, we assessed a role for Diversin, a putative Wnt pathway mediator, in developmental processes that involve cilia. We find that Diversin is specifically localized to the basal body compartment near the base of the cilium in Xenopus multi-ciliated skin cells. Overexpression of Diversin RNA disrupted basal body polarization in these cells, suggesting that tightly regulated control of Diversin levels is crucial for this process. In cells depleted of endogenous Diversin, basal body structure appeared abnormal and this was accompanied by disrupted polarity, shortened or absent cilia and defective ciliary flow. These results are consistent with the involvement of Diversin in processes that are related to the acquisition of cell polarity and require ciliary functions.     

Analysis of the orientation of primary cilia in growth plate cartilage: a mathematical method based on multiphoton microscopical images

The chondrocytic primary cilium has been hypothesized to act as a mechano-sensor, analogously to primary cilium of cells in epithelial tissues. We hypothesize that mechanical inputs during growth, sensed through the primary cilium, result in directed secretion of the extracellular matrix, thereby establishing tissue anisotropy in growth plate cartilage. The cilium, through its orientation in three-dimensional space, is hypothesized to transmit to the chondrocyte the preferential direction for matrix secretion. This paper reports on the application of classical mathematical methods to develop an algorithm that addresses the particular challenges relative to the assessment of the orientation of the primary cilium in growth plate cartilage, based on image analysis of optical sections visualized by multiphoton microscopy. Specimens are prepared by rapid cold precipitation-based fixation to minimize possible artifactual post-mortem alterations of ciliary orientation. The ciliary axoneme is localized by immunocytochemistry with antibody acetylated-alpha-tubulin. The method is applicable to investigation of ciliary orientation in different zones of the growth plate, under either normal or altered biomechanical environments. The methodology is highly flexible and adaptable to other connective tissues where tissue anisotropy and directed secretion of extracellular matrix components are hypothesized to depend on the tissue's biomechanical environment during development and growth.