BCAP is a centriolar satellite protein and inhibitor of ciliogenesis.

The centrosome and cilium are organelles with important roles in microtubule organisation, cell division, cell signalling, embryogenesis and tissue homeostasis. The two organelles are mutually exclusive. The centriole/basal body is found at the core of the centrosome (centriole) or at the base of the cilium (basal body) and to change which organelle is present in a cell requires modification to the centriole/basal body both in terms of composition and sub-cellular localisation. While many protein components required for centrosome and cilium biogenesis have been described, there are far fewer known inhibitors of ciliogenesis. Here, we show that a protein called BCAP and labelled in the sequence databases as ODF2-like (ODF2L) is a ciliation inhibitor. We show that it is a centriolar satellite protein. Furthermore, our data suggest that BCAP exists as two isoforms with subtly different roles in inhibition of ciliogenesis. Both are required to prevent ciliogenesis and one additionally controls cilium length after ciliogenesis has completed.

The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4.

The molecular basis of nephronophthisis, the most frequent genetic cause of renal failure in children and young adults, and its association with retinal degeneration and cerebellar vermis aplasia in Joubert syndrome are poorly understood. Using positional cloning, we here identify mutations in the gene CEP290 as causing nephronophthisis. It encodes a protein with several domains also present in CENPF, a protein involved in chromosome segregation. CEP290 (also known as NPHP6) interacts with and modulates the activity of ATF4, a transcription factor implicated in cAMP-dependent renal cyst formation. NPHP6 is found at centrosomes and in the nucleus of renal epithelial cells in a cell cycle-dependent manner and in connecting cilia of photoreceptors. Abrogation of its function in zebrafish recapitulates the renal, retinal and cerebellar phenotypes of Joubert syndrome. Our findings help establish the link between centrosome function, tissue architecture and transcriptional control in the pathogenesis of cystic kidney disease, retinal degeneration, and central nervous system development.

PCM1 conveys centrosome asymmetry to polarized endosome dynamics in regulating daughter cell fate.

Vertebrate radial glia progenitors (RGPs), the principal neural stem cells, balance self-renewal and differentiation through asymmetric cell division (ACD), during which unequal inheritance of centrosomes is observed. Mechanistically, how centrosome asymmetry leads to distinct daughter cell fate remains largely unknown. Here we find that the centrosome protein Pericentriolar Material 1 (Pcm1), asymmetrically distributed at the centrosomes, regulates polarized endosome dynamics and RGP fate. In vivo time-lapse imaging and nanoscale-resolution expansion microscopy of zebrafish embryonic RGPs detect Pcm1 on Notch ligand-containing endosomes, in a complex with the polarity regulator Par-3 and dynein motor. Loss of pcm1 disrupts endosome dynamics, with clonal analysis uncovering increased neuronal production at the expense of progenitors. Pcm1 facilitates an exchange of Rab5b (early) for Rab11a (recycling) endosome markers and promotes the formation of Par-3 and dynein macromolecular complexes on recycling endosomes. Finally, in human-induced pluripotent stem cell-derived brain organoids, PCM1 shows asymmetry and co-localization with PARD3 and RAB11A in mitotic neural progenitors. Our data reveal a new mechanism by which centrosome asymmetry is conveyed by Pcm1 to polarize endosome dynamics and Notch signaling in regulating ACD and progenitor fate.

Identifying an uptake mechanism for the antiepileptic and bipolar disorder treatment valproic acid using the simple biomedical model Dictyostelium.

Valproic acid (VPA) is the most highly prescribed epilepsy treatment worldwide and is also used to prevent bipolar disorder and migraine. Surprisingly, very little is known about its mechanisms of cellular uptake. Here, we employ a range of cellular, molecular and genetic approaches to characterize VPA uptake using a simple biomedical model, Dictyostelium discoideum. We show that VPA is taken up against an electrochemical gradient in a dose-dependent manner. Transport is protein-mediated, dependent on pH and the proton gradient and shows strong substrate structure specificity. Using a genetic screen, we identified a protein homologous to a mammalian solute carrier family 4 (SLC4) bicarbonate transporter that we show is involved in VPA uptake. Pharmacological and genetic ablation of this protein reduces the uptake of VPA and partially protects against VPA-dependent developmental effects, and extracellular bicarbonate competes for VPA uptake in Dictyostelium. We further show that this uptake mechanism is likely to be conserved in both zebrafish (Danio rerio) and Xenopus laevis model systems. These results implicate, for the first time, an uptake mechanism for VPA through SLC4-catalysed activity.

The Polo kinase Plk4 functions in centriole duplication.

The human Polo-like kinase 1 (PLK1) and its functional homologues that are present in other eukaryotes have multiple, crucial roles in meiotic and mitotic cell division. By contrast, the functions of other mammalian Polo family members remain largely unknown. Plk4 is the most structurally divergent Polo family member; it is maximally expressed in actively dividing tissues and is essential for mouse embryonic development. Here, we identify Plk4 as a key regulator of centriole duplication. Both gain- and loss-of-function experiments demonstrate that Plk4 is required--in cooperation with Cdk2, CP110 and Hs-SAS6--for the precise reproduction of centrosomes during the cell cycle. These findings provide an attractive explanation for the crucial function of Plk4 in cell proliferation and have implications for the role of Polo kinases in tumorigenesis.

Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase.

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1-Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation.

Rootletin forms centriole-associated filaments and functions in centrosome cohesion.

After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.

Proteomic characterization of the human centrosome by protein correlation profiling.

The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.

Loss of Both CDKN2A and CDKN2B Allows for Centrosome Overduplication in Melanoma.

Centrosomes duplicate only once in coordination with the DNA replication cycle and have an important role in segregating genetic material. In contrast, most cancer cells have centrosome aberrations, including supernumerary centrosomes, and this correlates with aneuploidy and genetic instability. The tumor suppressors p16 (CDKN2A) and p15 (CDKN2B) (encoded by the familial melanoma CDKN2 locus) inhibit CDK4/6 activity and have important roles in cellular senescence. p16 is also associated with suppressing centrosomal aberrations in breast cancer; however, the role of p15 in centrosome amplification is unknown. Here, we investigated the relationship between p15 and p16 expression, centrosome number abnormalities, and melanoma progression in cell lines derived from various stages of melanoma progression. We found that normal human melanocyte lines did not exhibit centrosome number abnormalities, whereas those from later stages of melanoma did. Additionally, under conditions of S-phase block, p15 and p16 status determined whether centrosome overduplication would occur. Indeed, removal of p15 from p16-negative cell lines derived from various stages of melanoma progression changed cells that previously would not overduplicate their centrosomes into cells that did. Although this study used cell lines in vitro, it suggests that, during clinical melanoma progression, sequential loss of p15 and p16 provides conditions for centrosome duplication to become deregulated with consequences for genome instability.

Alpha-synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome.

Protein aggregates are the pathogenic hallmarks of many different neurodegenerative diseases and include the accumulation of α-synuclein, the main component of Lewy bodies found in Parkinson's disease. Aggresomes are closely-related, cellular accumulations of misfolded proteins. They develop in a juxtanuclear position, adjacent to the centrosome, the microtubule organizing centre of the cell, and share some protein components. Despite the long-standing observation that aggresomes/Lewy bodies and the centrosome sit side-by-side in the cell, no studies have been done to see whether these protein accumulations impede organelle function. We investigated whether the formation of aggresomes affected key centrosome functions: its ability to organise the microtubule network and to promote cilia formation. We find that when aggresomes are present, neuronal cells are unable to organise their microtubule network. New microtubules are not nucleated and extended, and the cells fail to respond to polarity cues. Since neurons are polarised, ensuring correct localisation of organelles and the effective intracellular transport of neurotransmitter vesicles, loss of centrosome activity could contribute to functional deficits and neuronal cell death in Parkinson's disease. In addition, we provide evidence that many cell types, including dopaminergic neurons, cannot form cilia when aggresomes are present, which would affect their ability to receive extracellular signals.

Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos.

BACKGROUND: The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms. RESULTS: We describe zebrafish (Danio rerio) embryos depleted for two uncharacterised, centrosome proteins, Cep70 and Cep131. The phenotype of these embryos resembles that of zebrafish mutants for intraflagellar transport proteins (IFTs), with kidney and ear development affected and left-right asymmetry randomised. These organs and processes are those affected in Bardet-Biedl syndrome and other similar diseases. Like these diseases, the root cause of the phenotype lies, in fact, in dysfunctional cilia, which are shortened but not eliminated in several tissues in the morphants. Centrosomes and basal bodies, on the other hand, are present. Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase) interacting domain. However, we could not detect in yeast two-hybrid assays any interaction with the deacetylase that controls cilium length, HDAC6, or any of the IFTs that we tested. CONCLUSION: Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened. We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

Zebrafish otolith biomineralization requires polyketide synthase.

Deflecting biomineralized crystals attached to vestibular hair cells are necessary for maintaining balance. Zebrafish (Danio rerio) are useful organisms to study these biomineralized crystals called otoliths, as many required genes are homologous to human otoconial development. We sought to identify and characterize the causative gene in a trio of homozygous recessive mutants, no content (nco) and corkscrew (csr), and vanished (vns), which fail to develop otoliths during early ear development. We show that nco, csr, and vns have potentially deleterious mutations in polyketide synthase (pks1), a multi-modular protein that has been previously implicated in biomineralization events in chordates and echinoderms. We found that Otoconin-90 (Oc90) expression within the otocyst is diffuse in nco and csr; therefore, it is not sufficient for otolith biomineralization in zebrafish. Similarly, normal localization of Otogelin, a protein required for otolith tethering in the otolithic membrane, is not sufficient for Oc90 attachment. Furthermore, eNOS signaling and Endothelin-1 signaling were the most up- and down-regulated pathways during otolith agenesis in nco, respectively. Our results demonstrate distinct processes for otolith nucleation and biomineralization in vertebrates and will be a starting point for models that are independent of Oc90-mediated seeding. This study will serve as a basis for investigating the role of eNOS signaling and Endothelin-1 signaling during otolith formation.

Diacylglycerol kinase (DGKA) regulates the effect of the epilepsy and bipolar disorder treatment valproic acid in Dictyostelium discoideum.

Valproic acid (VPA) provides a common treatment for both epilepsy and bipolar disorder; however, common cellular mechanisms relating to both disorders have yet to be proposed. Here, we explore the possibility of a diacylglycerol kinase (DGK) playing a role in regulating the effect of VPA relating to the treatment of both disorders, using the biomedical model Dictyostelium discoideum DGK enzymes provide the first step in the phosphoinositide recycling pathway, implicated in seizure activity. They also regulate levels of diacylglycerol (DAG), thereby regulating the protein kinase C (PKC) activity that is linked to bipolar disorder-related signalling. Here, we show that ablation of the single Dictyostelium dgkA gene results in reduced sensitivity to the acute effects of VPA on cell behaviour. Loss of dgkA also provides reduced sensitivity to VPA in extended exposure during development. To differentiate a potential role for this DGKA-dependent mechanism in epilepsy and bipolar disorder treatment, we further show that the dgkA null mutant is resistant to the developmental effects of a range of structurally distinct branched medium-chain fatty acids with seizure control activity and to the bipolar disorder treatment lithium. Finally, we show that VPA, lithium and novel epilepsy treatments function through DAG regulation, and the presence of DGKA is necessary for compound-specific increases in DAG levels following treatment. Thus, these experiments suggest that, in Dictyostelium, loss of DGKA attenuates a common cellular effect of VPA relating to both epilepsy and bipolar disorder treatments, and that a range of new compounds with this effect should be investigated as alternative therapeutic agents.This article has an associated First Person interview with the first author of the paper.

Skipping in a hybrid polyketide synthase. Evidence for ACP-to-ACP chain transfer.

A tetraketide synthase containing a loading module (LM), the extension modules erythromycin module 1, rapamycin module 2, and erythromycin module 2 (LM-Ery1-Rap2-Ery2-TE), when expressed in Saccharopolyspora erythraea strain JC2, produced as previously reported a mixture of tetraketide lactones (minor products) and triketide lactones (major products). Several alternative plausible mechanisms by which this "skipping" phenomenon might occur may be proposed. Site-directed mutagenesis of the ketosynthase (KS) and acylcarrier protein (ACP) domains in the interpolated module has shown that skipping within the hybrid PKS involves passage of the growing polyketide through the interpolated module, by direct ACP-to-ACP transfer of the polyketide chain.

Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression.

Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation. Nine genes are currently known to be associated with the condition, all of which encode centrosomal or spindle pole proteins. MCPH is associated with a reduction in proliferation of neural progenitors during fetal development. The cellular mechanisms underlying the proliferation defect, however, are not fully understood. The zebrafish retinal neuroepithelium provides an ideal system to investigate this question. Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size. Imaging studies reveal a dramatic rise in the fraction of proliferating cells in mitosis in all cases, and time-lapse microscopy points to a failure of progression through prometaphase. There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones.

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.

Polarization and orientation of retinal ganglion cells in vivo.

In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.

Increasing the efficiency of heterologous promoters in actinomycetes.

An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.

Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase.

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ-containing loading modules are shown here to contain a key arginine residue at their active site. Site-specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13-methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex-tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1-TE, in which the 4'-phosphopante-theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro-ducts in detectable amounts.