Centrosome Biogenesis

The formation of centrosomes is highly regulated. They duplicate once every cell cycle, with one centriole, the “daughter”, forming close to an already existing one, the “mother”, so that their number remains stable.

Figure 1. The canonical centriole duplication cycle. Electron microscopy micrographs of HeLa cells showing distinct steps of centriole duplication (also represented diagrammatically). The mother centriole is represented in dark green showing appendages. Daughter centrioles are shown in light green. At mitotic exit–early G1 phase, centrioles in a centrosome loose their orthogonal configuration. There might be an intercentriole link at this stage. Next, duplication starts in late G1–S phase with the nucleation of daughter centrioles (see electron micrograph; the arrowhead shows a procentriole). Note that the axis of the daughter intercepts the parent. The procentrioles elongate fully by late G2 phase or by the beginning of G1 phase of the next cell cycle. Last, maturation and separation of the two centrosomes occur at the G2–M transition by the acquisition of maturation markers, the recruitment of pericentriolar material (PCM; orange) and an increase in microtubule-organizing centre (MTOC) activity. From Bettencourt-Dias & Glover (2007), please see review for references.

To provide new perspectives on cell proliferation we screened by RNAi all the protein kinases from fruit flies. Amongst several novel players, we identified SAK/PLK4, a kinase implicated in tumourigenesis. We showed in flies and humans that SAK/PLK4 is essential for centriole duplication. Strikingly, SAK/PLK4 can trigger centriole formation in the absence of a mother, i.e., de novo, showing the mother centriole is not a bona-fide “template” in daughter formation. Instead, the mother centriole is a platform for recruitment of regulatory molecules, such as SAK/PLK4, hence triggering the assembly of daughter centrioles close by.

Figure 2. Overexpression of SAK leads to de novo centrosome formation in Drosophila eggs. The Drosophila egg provides a “living test tube” in which to study centriole biogenesis. This is because it contains a maternal dowry of sufficient proteins necessary to make 2*13 centrosomes in the rapid rounds of mitosis in its syncytial stage of development. Additionally, because centrioles are naturally eliminated from the oocyte during development and then provided by the sperm in the form of a basal body, this has allowed us to study centriole biogenesis in a cytoplasm that either contained centrioles, the embryo, or lacked them, the unfertilized egg. We found that 100% of unfertilised eggs overexpressing SAK/PLK4 exhibited de novo centriole formation (right image), something that is never observed in wild type eggs (left), where only the polar bodies are observed. This was followed by massive centriole amplification. Ultrastructural analysis showed that these centrioles were structurally normal suggesting that SAK/PLK4 alone is able to induce both canonical and de novo formation of centrioles.

Both these processes require the activity of DSAS-6 and DSAS-4, two regulators of canonical duplication. Overexpression of DSAS-6 also results in the de novo formation of MTOCs, however, it is only able to specify tube-like structures, not the whole centriole. Downregulation of DSAS-6 results in centriolar structures that fail to close and elongate along all 9 axis of symmetry. Taken together our results show that centriole biogenesis is a template-free self-assembly process that is locally triggered by association of SAK with the existing centriole. Our results suggest that SAK recruits DSAS-6; D-SAS6 organizes a carthweel structure that has nine fold information and ensures the maintenance of that symmetry. This work suggests misregulation of centriole duplication regulators may generate some of the centrosome abnormalities observed in cancer. In that case, those molecules may be used in the diagnostic, prognostic and as targets in cancer treatment.

Figure 3. SAS-6 is necessary for the 9 fold symmetry. A. During spermatogenesis, each stem cell division produces a gonial cell that undertakes four rounds of mitosis to produce a cyst of 16 primary spermatocytes. These are connected through 15 ring canals as result of incomplete cytokinesis. Each of these cells has four centrioles (red bars) and undertakes a prolonged G2 phase, where centrioles grow approximately 10X. Centrioles within the centrosome remain close together in a V-shape. During this phase all centrioles within each cell migrate to the membrane and form a small cilium. Afterwards they migrate again to a position closer to the nucleus, in preparation for meiosis. Meiotic divisions produce a cyst of 64 interconnected spermatids, each with one centriole. Early spermatids have a single nucleus (white sphere) and a mitochondrial derivative (Nebenkern, black sphere) of similar sizes. The spermatid centriole differentiates into a basal body to organize the flagellar axoneme of the sperm. B. We found that the centrioles present in DSAS-6 mutant primary spermatocytes were shorter and had failed to complete the assembly of the 9-fold symmetrical cylindrical structure such that adjacent centriolar triplets were missing. C. DSAS-6 mutant spermatids also developed abnormal axonemes, some missing 1 to 5 adjacent axonemal MT doublets, others presenting 1 or 2 supernumerary doublets. These abnormal axonemal structures are likely to reflect corresponding defects in the underlying centrioles/basal bodies from which the axonemes are extended.

Figure 4. Model for centriole biogenesis: Self-assembly and modularity. SAK/PLK4 triggers centriole formation close to the mother centriole (A.) or de novo in the absence of this structure (B.). One of the first steps reported in centriole assembly is the formation of an electron dense material, which may be the PCM, playing a role in the microtubule driven recruitment of centriole components. C. The cartwheel defines the nine-fold centriolar symmetry. SAS-6 localises to the hub and spokes and has a very important role in the assembly of the cartwheel and centriole biogenesis. D. Centriole microtubules are then tethered to this structure, a process that may be controlled by bld10/CEP135, and SAS-4. Microtubules may assemble first as singlets, then doublets and then triplets (not depicted here). Although we know SAK/PLK4 triggers centriole biogenesis, it is not clear whether it promotes cartwheel assembly, or regulates the PCM activity in centriole formation or through both. Please see our reviews on centrosomes for references (more).