SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.

Most eukaryotic cells have a primary cilium which acts as a sensory organelle1. Cilia are assembled by intraflagellar transport (IFT), a process mediated by multimeric IFT particles and molecular motors2. Here we show that lymphoid and myeloid cells, which lack primary cilia, express IFT proteins. IFT20, an IFT component essential for ciliary assembly3 , 4, was found to colocalize with both the MTOC and Golgi and post-Golgi compartments in T-lymphocytes. In antigen-specific conjugates, IFT20 translocated to the immune synapse (IS). IFT20 knockdown resulted in impaired TCR/CD3 clustering and signaling at the IS due to defective polarized recycling. Moreover, IFT20 was required for the inducible assembly of a complex with other IFT components (IFT57, IFT88) and the TCR. The results identify IFT20 as a novel regulator of IS assembly in T-cells and provide the first evidence that IFT is implicated in membrane trafficking in cells lacking primary cilia, thereby opening a new perspective on IFT function beyond its role in ciliogenesis.When naive T-cells encounter antigen presenting cells (APC) bearing cognate MHC-bound peptide antigen, a dynamic rearrangement of membrane and cytosolic molecules occurs at the T-cell:APC contact area. This results in the formation of a highly organized interface known as immune synapse (IS), which acts as a platform for signal integration, fine-tuning and extinction5 , 6. A hallmark of the nascent IS is reorientation of the microtubule organizing center (MTOC) towards the APC7, which ensures targeted delivery of signaling molecules from intracellular pools to the IS8. This includes the TCR/CD3 complex itself, which is carried to the IS through polarized recycling9. Directional movement of structural and regulatory molecules, orchestrated by the MTOC, is a characteristic feature of primary cilia. These structures, present on most non-dividing cells, act as sensory organelles, relaying information from the external environment into the cell through intraflagellar transport (IFT), a process carried out by multimeric IFT particles and molecular motors2 , 10.At variance with most other eukaryotic cell types, hematopoietic cells lack primary cilia10. Surprisingly, IFT20, an IFT component essential for ciliary assembly3 ,4 , was found to be Correspondence should be addressed to CTB. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 November 1. Published in final edited form as:Nat Cell Biol. 2009 November ; 11(11): 1332-1339. doi:10.1038/ncb1977. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript expressed in human cells of both lymphoid (peripheral blood lymphocytes, Jurkat T-lymphoma cells) and myeloid (monocytes, monocyte-derived DC) lineages (Fig.1a). Similar results were obtained in the mouse, where IFT20 was detectable in central (thymus, bone marrow) and peripheral (lymph node, spleen) lymphoid organs (Fig.1a). Immunofluorescence analysis of Jurkat cells and human peripheral blood lymphocytes (PBL) showed that IFT20...

Centrioles are found in the centrosome core and, as basal bodies, at the base of cilia and flagella. Centriole assembly and duplication is controlled by Polo-like-kinase 4 (Plk4): these processes fail if Plk4 is downregulated and are promoted by Plk4 overexpression. Here we show that the centriolar protein Asterless (Asl; human orthologue CEP152) provides a conserved molecular platform, the amino terminus of which interacts with the cryptic Polo box of Plk4 whereas the carboxy terminus interacts with the centriolar protein Sas-4 (CPAP in humans). Drosophila Asl and human CEP152 are required for the centrosomal loading of Plk4 in Drosophila and CPAP in human cells, respectively. Depletion of Asl or CEP152 caused failure of centrosome duplication; their overexpression led to de novo centriole formation in Drosophila eggs, duplication of free centrosomes in Drosophila embryos, and centrosome amplification in cultured Drosophila and human cells. Overexpression of a Plk4-binding-deficient mutant of Asl prevented centriole duplication in cultured cells and embryos. However, this mutant protein was able to promote microtubule organizing centre (MTOC) formation in both embryos and oocytes. Such MTOCs had pericentriolar material and the centriolar protein Sas-4, but no centrioles at their core. Formation of such acentriolar MTOCs could be phenocopied by overexpression of Sas-4 in oocytes or embryos. Our findings identify independent functions for Asl as a scaffold for Plk4 and Sas-4 that facilitates self-assembly and duplication of the centriole and organization of pericentriolar material.

Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. We found that overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly.