Centriolar satellites are assembly points for proteins implicated in human ciliopathies, including oral-facial-digital syndrome 1.

Ciliopathies are caused by mutations in genes encoding proteins required for cilia organization or function. We show through colocalization with PCM-1, that OFD1 (the product of the gene mutated in oral-facial-digital syndrome 1) as well as BBS4 and CEP290 (proteins encoded by other ciliopathy genes) are primarily components of centriolar satellites, the particles surrounding centrosomes and basal bodies. RNA interference experiments reveal that satellite integrity is mutually dependent upon each of these proteins. Upon satellite dispersal, through mitosis or forced microtubule depolymerization, OFD1 and CEP290 remain centrosomal, whereas BBS4 and PCM-1 do not. OFD1 interacts via its fifth coiled-coil motif with the N-terminal coiled-coil domain of PCM-1, which itself interacts via its C-terminal non-coiled-coil region with BBS4. OFD1 localization to satellites requires its N-terminal region, encompassing the LisH motif, whereas expression of OFD1 C-terminal constructs causes PCM-1 and CEP290 mislocalization. Moreover, in embryonic zebrafish, OFD1 and BBS4 functionally synergize, determining morphogenesis. Our observation that satellites are assembly points for several mutually dependent ciliopathy proteins provides a further possible explanation as to why the clinical spectrum of OFD1, Bardet-Biedl and Joubert syndromes overlap. Furthermore, definition of how OFD1 and PCM-1 interact helps explain why different OFD1 mutations lead to clinically variable phenotypes.

Studying Centriole Duplication and Elongation in Human Cells.

Centrioles assemble centrosomes and cilia/flagella, which are microtubule-based structures with key roles in cell division, polarity, motility, and signaling. Centriole biogenesis is a tightly regulated process, and deregulation of centriole numbers and structure can have dramatic consequences for cellular function and integrity. However, their small size poses a challenge to study them. Here, we describe protocols that allow the identification and assessment of true centrioles and that provide straightforward strategies to study the role of new candidate proteins in centriole duplication and elongation.

Phenotypic Screen with TSC-Deficient Neurons Reveals Heat-Shock Machinery as a Druggable Pathway for mTORC1 and Reduced Cilia.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficient neurons. To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors of the heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.

Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis.

Centrosome abnormalities are a typical hallmark of human cancers. However, the origin and dynamics of such abnormalities in human cancer are not known. In this study, we examined centrosomes in Barrett's esophagus tumorigenesis, a well-characterized multistep pathway of progression, from the premalignant condition to the metastatic disease. This human cancer model allows the study of sequential steps of progression within the same patient and has representative cell lines from all stages of disease. Remarkably, centrosome amplification was detected as early as the premalignant condition and was significantly expanded in dysplasia. It was then present throughout malignant transformation both in adenocarcinoma and metastasis. The early expansion of centrosome amplification correlated with and was dependent on loss of function of the tumor suppressor p53 both through loss of wild-type expression and hotspot mutations. Our work shows that centrosome amplification in human tumorigenesis can occur before transformation, being repressed by p53. These findings suggest centrosome amplification in humans can contribute to tumor initiation and progression.

PLK4 trans-Autoactivation Controls Centriole Biogenesis in Space.

Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions.

OFD1 and flotillins are integral components of a ciliary signaling protein complex organized by polycystins in renal epithelia and odontoblasts.

Mutation of the X-linked oral-facial-digital syndrome type 1 (OFD1) gene is embryonic lethal in males and results in craniofacial malformations and adult onset polycystic kidney disease in females. While the OFD1 protein localizes to centriolar satellites, centrosomes and basal bodies, its cellular function and how it relates to cystic kidney disease is largely unknown. Here, we demonstrate that OFD1 is assembled into a protein complex that is localized to the primary cilium and contains the epidermal growth factor receptor (EGFR) and domain organizing flotillin proteins. This protein complex, which has similarity to a basolateral adhesion domain formed during cell polarization, also contains the polycystin proteins that when mutant cause autosomal dominant polycystic kidney disease (ADPKD). Importantly, in human ADPKD cells where mutant polycystin-1 fails to localize to cilia, there is a concomitant loss of localization of polycystin-2, OFD1, EGFR and flotillin-1 to cilia. Together, these data suggest that polycystins are necessary for assembly of a novel flotillin-containing ciliary signaling complex and provide a molecular rationale for the common renal pathologies caused by OFD1 and PKD mutations.

The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation.

Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

Pix1 and Pix2 are novel WD40 microtubule-associated proteins that colocalize with mitochondria in Xenopus germ plasm and centrosomes in human cells.

In many animals, the germ line develops from a distinct mitochondria-rich region of embryonic cytoplasm called the germ plasm. However, the protein composition of germ plasm and its formation remain poorly understood, except in Drosophila. Here, we show that Xpat, a recently identified protein component of Xenopus germ plasm, interacts via its C-terminal domain with a novel protein, xPix1. Xpat and xPix1 are co-expressed in ovaries, eggs and early embryos and colocalize to the mitochondrial cloud and germ plasm in stage I and stage VI oocytes, respectively. Although Xpat appears unique to Xenopus, Pix proteins, which contain an N-terminal WD40 domain and C-terminal coiled-coil, are widely conserved. In humans, two proteins, Pix1 and Pix2, are expressed at varying levels in different cancer cell lines. Importantly, as well as localizing to mitochondria, human Pix proteins localize to centrosomes and associate with microtubules in vitro and in vivo. Although, Pix proteins are stably expressed through the cell cycle, Pix2 concentrates on microtubule structures in mitosis and microinjection of Pix antibodies interferes with cell division. Based on these data, we propose that Pix1 and Pix2 are microtubule-associated adaptor proteins that likely contribute to a range of developmental and cell division processes.