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.     

FOP Is a Centriolar Satellite Protein Involved in Ciliogenesis

Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner) is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G₁, when FOP protein levels are lowest, and most labeled in G₂. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.     

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.     

A ciliopathy complex builds distal appendages to initiate ciliogenesis

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.     

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies.     

CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease

Primary cilia are nonmotile organelles implicated in signaling and sensory functions. Understanding how primary cilia assemble could shed light on the many human diseases caused by mutations in ciliary proteins. The centrosomal protein CP110 is known to suppress ciliogenesis through an unknown mechanism. Here, we report that CP110 interacts with CEP290--a protein whose deficiency is implicated in human ciliary disease--in a discrete complex separable from other CP110 complexes involved in regulating the centrosome cycle. Ablation of CEP290 prevents ciliogenesis without affecting centrosome function or cell-cycle progression. Interaction with CEP290 is absolutely required for the ability of CP110 to suppress primary cilia formation. Furthermore, CEP290 and CP110 interact with Rab8a, a small GTPase required for cilia assembly. Depletion of CEP290 interferes with localization of Rab8a to centrosomes and cilia. Our results suggest that CEP290 cooperates with Rab8a to promote ciliogenesis and that this function is antagonized by CP110.     

A non‐canonical function of Plk4 in centriolar satellite integrity and ciliogenesis through PCM1 phosphorylation

Centrioles are the major constituents of the animal centrosome, in which Plk4 kinase serves as a master regulator of the duplication cycle. Many eukaryotes also contain numerous peripheral particles known as centriolar satellites. While centriolar satellites aid centriole assembly and primary cilium formation, it is unknown whether Plk4 plays any regulatory roles in centriolar satellite integrity. Here we show that Plk4 is a critical determinant of centriolar satellite organisation. Plk4 depletion leads to the dispersion of centriolar satellites and perturbed ciliogenesis. Plk4 interacts with the satellite component PCM1, and its kinase activity is required for phosphorylation of the conserved S372. The nonphosphorylatable PCM1 mutant recapitulates phenotypes of Plk4 depletion, while the phosphomimetic mutant partially rescues the dispersed centriolar satellite patterns and ciliogenesis in cells depleted of PCM1. We show that S372 phosphorylation occurs during the G1 phase of the cell cycle and is important for PCM1 dimerisation and interaction with other satellite components. Our findings reveal that Plk4 is required for centriolar satellite function, which may underlie the ciliogenesis defects caused by Plk4 dysfunction.     

Cilia born out of shock and stress

EMBO J (2013) 32 23, 3029–3040 10.1038/emboj.2013.223; published online October112013Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins.Centrioles are barrel-shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule-organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle-dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule-dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM-1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet-Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: First, in cycling cells they may serve to sequester essential ciliary proteins (Stowe et al, 2012). Second, upon initiation of the ciliogenesis programme, centriolar satellite components seem to promote the recruitment of specific ciliary proteins to basal bodies (Ferrante et al, 2006; Lopes et al, 2011; Stowe et al, 2012).In a new study in The EMBO Journal, Villumsen et al (2013) now describe how stress–response pathways conspire to control ciliogenesis. The authors observed that specific environmental stresses, such as ultraviolet light radiation (UV) or heat shock, but not ionizing radiation (IR), trigger rapid displacement of PCM-1, AZI1 and CEP290 from centriolar satellites. However, OFD1 remained associated with satellites, indicating that centriolar satellites persist despite UV-induced removal of PCM-1. This might come as some surprise, since PCM-1 depletion by RNA interference (RNAi) is thought to disrupt satellite integrity (Kim et al, 2008; Lopes et al, 2011); however, satellite loss upon PCM-1 RNAi may be a consequence of prolonged depletion of PCM-1, while acute PCM-1 displacement by stress might only ‘remodel'' centriolar satellites. It is also possible that not all satellites are created equal, and they do vary in protein composition (Kim et al, 2008; Staples et al, 2012). If so, UV-induced PCM-1 removal may disrupt some, but not all satellites.A good candidate regulator of centriolar satellite remodelling was the stress-activated MAP kinase p38, and indeed, Villumsen et al (2013) found p38 MAPK activity to be stimulated by both UV and heat shock but not IR in U2OS cells, mirroring those very stress pathways that also cause displacement of AZI1 and PCM-1 from satellites. Furthermore, p38 MAPK was essential for UV-induced dispersal of PCM-1 and AZI1. The authors then tested the hypothesis that stress-induced centriolar satellite remodelling could involve changes in the interactome of AZI1, and—consistent with an earlier proteomics study (Akimov et al, 2011)—identified PCM-1, CEP290 and the mindbomb E3 ubiquitin protein ligase 1 (MIB1) as the main AZI1 binding partners. GFP-MIB1 localized to centriolar satellites and mono-ubiquitylated AZI1, PCM-1 and CEP290 in cycling cells. In response to UV, both ubiquitylation of these proteins and MIB1 activity were reduced; notably, UV-induced MIB1 inactivation was independent of p38 MAPK activity, indicating that these two enzymes may act via distinct pathways (Figure 1A).Open in a separate windowFigure 1(A) Regulation of centriolar satellite remodelling. (B) Schematic summary of how centriolar satellite remodelling might facilitate ciliogenesis. See text for details.What could be the purpose of MIB1-dependent ubiquitylation of these satellite proteins? It certainly does not seem to regulate subcellular targeting, as in MIB1-depleted cells, AZI1 and PCM-1 both localised normally to centriolar satellites and could still be displaced by UV. Instead, ubiquitylation seems to suppress the interaction between AZI1 and PCM-1, consistent with the observation that UV, a condition that also reduces their ubiquitylation, enhances the binding of AZI1 to PCM-1.PCM-1, CEP290 and AZI1 all participate in ciliogenesis (Kim et al, 2008; Wilkinson et al, 2009; Stowe et al, 2012), raising the possibility that MIB1 might also affect this process. Indeed, serum starvation, which is known to promote cilia formation, attenuated MIB1 activity. Furthermore, MIB1 overexpression reduced the ciliogenesis observed in serum-starved cells, while MIB1 depletion in proliferating cells triggered a marked increase in the proportion of cells that formed cilia; this seems to reflect a direct effect of MIB1 on ciliogenesis, since neither MIB1 depletion nor overexpression altered cell cycle progression. Taken together, downregulation of MIB1 enzymatic activity appears to be a pre-requisite for efficient ciliogenesis, regardless of whether it is triggered by physiological ciliogenesis-promoting signals or by environmental stresses, making MIB1 a novel negative regulator of cilia formation.The recent discovery of ciliopathy-associated mutations in constituents of the DNA damage response signalling pathway pointed to a connection between DNA damage and ciliogenesis (Chaki et al, 2012). With the new link between UV and centriolar satellites, the authors next asked if UV radiation might affect ciliogenesis. Remarkably, UV and heat shock both triggered cilia assembly in RPE-1 cells in a p38 MAPK-dependent manner. MIB1 depletion further enhanced ciliogenesis after UV radiation, again implying an additive effect of p38 MAPK signalling and MIB1 suppression (Figure 1A).While finer details on the precise role of centriolar satellite components in cilia formation are still lacking, a more coherent picture is finally starting to emerge. In cycling cells, ubiquitination by MIB1 could serve to limit the interaction between AZI1 and PCM-1 on centriolar satellites (Figure 1B). Under these conditions PCM-1 may bind and sequester CEP290, an essential ciliogenic protein, thereby precluding untimely cilia formation (Stowe et al, 2012). Both during normal and stress-induced ciliogenesis programs, remodelling of centriolar satellites creates a permissive environment for cilia formation, and a key step in this process is downregulation of MIB1 activity. While it remains to be established how the latter is achieved, it is clear that MIB1 inactivation causes loss of ubiquitylation and increased binding between AZI1 and PCM-1. Preferential interaction of PCM-1 with AZI1 could in turn facilitate release of CEP290 from centriolar satellites and its subsequent accumulation at the centrosome. Once CEP290 reaches the optimum concentration at the centriole/basal body, it could serve to tether AZI1–PCM-1 complexes. PCM-1 could then concentrate Rab8 GTPase near centrosomes, allowing CEP290 to recruit Rab8 into the cilium, where it acts to extend the ciliary membrane (Kim et al, 2008).Collectively, the findings reported here provide strong experimental support to the notion that centriolar satellites are negative regulators of ciliogenesis in proliferating cells. Their role is central to limit untimely formation of cilia in cells. Environmental strains elicit stress–response pathways that converge to relieve the ciliogenesis block imposed by satellites. It is tempting to speculate that stress-induced cilia might serve as signalling platforms and contribute to checkpoint activation or perhaps initiation of repair mechanisms, but more work is needed to establish the true purpose of ciliogenesis in this context. It is of considerable interest that a recent study reports that autophagy, another stress-induced pathway, selectively removes OFD1 from satellites to promote ciliogenesis (Tang et al, 2013). Therefore stress-mediated centriolar satellite remodelling seems to be an evolving theme in the control of ciliogenesis.     

Human microcephaly protein CEP135 binds to hSAS‐6 and CPAP,and is required for centriole assembly

Centrioles are cylindrical structures that are usually composed of nine triplets of microtubules (MTs) organized around a cartwheel‐shaped structure. Recent studies have proposed a structural model of the SAS‐6‐based cartwheel, yet we do not know the molecular detail of how the cartwheel participates in centriolar MT assembly. In this study, we demonstrate that the human microcephaly protein, CEP135, directly interacts with hSAS‐6 via its carboxyl‐terminus and with MTs via its amino‐terminus. Unexpectedly, CEP135 also interacts with another microcephaly protein CPAP via its amino terminal domain. Depletion of CEP135 not only perturbed the centriolar localization of CPAP, but also blocked CPAP‐induced centriole elongation. Furthermore, CEP135 depletion led to abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. Overexpression of a CEP135 mutant lacking the proper interaction with hSAS‐6 had a dominant‐negative effect on centriole assembly. We propose that CEP135 may serve as a linker protein that directly connects the central hub protein, hSAS‐6, to the outer MTs, and suggest that this interaction stabilizes the proper cartwheel structure for further CPAP‐mediated centriole elongation.     

EB1 is required for primary cilia assembly in fibroblasts

EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and different cellular organelles [1, 2]. EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network [3, 4]. We previously showed that EB1 localizes to the flagellar tip and proximal region of the basal body in Chlamydomonas[5], but the function of EB1 in the cilium/flagellum is unknown. We depleted EB1 from NIH3T3 fibroblasts by using siRNA and found that EB1 depletion causes a approximately 50% reduction in the efficiency of primary cilia assembly in serum-starved cells. Expression of dominant-negative EB1 also inhibited cilia formation, and expression of mutant dominant-negative EB1 constructs suggested that binding of EB1 to p150(Glued) is important for cilia assembly. Finally, expression of a C-terminal fragment of the centrosomal protein CAP350, which removes EB1 from the centrosome but not MT plus ends [6], also inhibited ciliogenesis. We conclude that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts.     

A Highlights from MBoC Selection: Ccdc11 is a novel centriolar satellite protein essential for ciliogenesis and establishment of left–right asymmetry

The establishment of left–right (L-R) asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt L-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here we demonstrate that Ccdc11 is a novel component of centriolar satellites—cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer’s vesicle and results in aberrant L-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia and implicate centriolar satellite–associated proteins as a new class of proteins in the pathology of L-R patterning and congenital heart disease.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly

We have identified Talpid3/KIAA0586 as a component of a CP110-containing protein complex important for centrosome and cilia function. Talpid3 assembles a ring-like structure at the extreme distal end of centrioles. Ablation of Talpid3 resulted in an aberrant distribution of centriolar satellites involved in protein trafficking to centrosomes as well as cilia assembly defects, reminiscent of loss of Cep290, another CP110-associated protein. Talpid3 depletion also led to mislocalization of Rab8a, a small GTPase thought to be essential for ciliary vesicle formation. Expression of activated Rab8a suppressed cilia assembly defects provoked by Talpid3 depletion, suggesting that Talpid3 affects cilia formation through Rab8a recruitment and/or activation. Remarkably, ultrastructural analyses showed that Talpid3 is required for centriolar satellite dispersal, which precedes the formation of mature ciliary vesicles, a process requiring Cep290. These studies suggest that Talpid3 and Cep290 play overlapping and distinct roles in ciliary vesicle formation through regulation of centriolar satellite accretion and Rab8a.     

PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis

The microtubule-organizing activity of the centrosome oscillates during the cell cycle, reaching its highest level at mitosis. At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar matrix (PCM) and a robust increase in microtubule-organizing activity. It is known that PLK1 is critical for the initiation of centrosome maturation. In this paper, we report that pericentrin (PCNT), a PCM protein, was specifically phosphorylated by PLK1 during mitosis. Phosphoresistant point mutants of PCNT did not recruit centrosomal proteins, such as CEP192, GCP-WD (γ-complex protein with WD repeats), γ-tubulin, Aurora A, and PLK1, into the centrosome during mitosis. However, centrosomal recruitment of CEP215 depended on PCNT irrespective of its phosphorylation status. Furthermore, ectopic expression of PLK1-PCNT fusion proteins induced the centrosomal accumulation of CEP192, GCP-WD, and γ-tubulin even in interphase cells, mimicking centrosome maturation. Based on these results, we propose that PLK1-mediated phosphorylation of PCNT initiates centrosome maturation by organizing the spindle pole-specific PCM lattice.     

Hook2 is involved in the morphogenesis of the primary cilium

Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localizes at the Golgi apparatus and centrosome/basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilizes pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP::Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation.     

Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.     

Double life of centrioles: CP110 in the spotlight

Centrioles lead an important double life: they can give rise to the centrosome or convert to basal bodies and template cilia. Little is known about the control of centriole fate. Spektor and colleagues have now identified a centriolar complex, composed of CP110 and CEP97, which inhibits centriole to basal body conversion, preventing cilia formation. This work paves the way to understanding centriole and cilia biogenesis, which are two processes misregulated in human diseases, such as cancer and polycystic kidney disease.