How is the PCM organised?
The PCM appears to be structurally weakened in the absence of Cnn. Living colchicine-treated WT or cnn mutant embryos expressing a GFP-centriole marker (green) and RFP-PCM markers (red - as indicated above each panel). In the WT embryos the centrioles remain centered within the PCM. In cnn embryos, the centrioles cannot maintain their central positioning within the PCM. By analogy with how certain bacteria such as Shigella and Listeria organise an actin cloud around themselves, this striking observation suggests that the PCM cloud surrounding the centrioles is stucturally weakened in cnn embryos (see Lucas and Raff, 2007).
Many proteins are concentrated in the PCM, but it is unclear how they interact with one another to form the PCM. One protein that has been strongly implicated in organising the PCM is Pericentrin. It shares a centrosomal-targeting domain (the PACT domain) with another centrosomal protein AKAP450, which has also been implicated in PCM organisation. In flies, there is a single PACT domain protein, which we call Drosophila Pericentrin-like protein (D-PLP). Surprisingly, we showed that D-PLP is highly concentrated in centrioles and basal bodies and is present at much lower levels in the PCM. Moreover, D-PLP-deficient mutants have a relatively subtle defect in PCM recruitment, but completely lack functional cilia (Martinez-Campos et al., 2004).
To better understand how D-PLP functions in PCM recruitment and cilia formation we immunoprecipitated (i/p’d) the protein from embryo extracts and studied the precipitates by western blotting to see if any other known centriolar or centrosomal components are normally bound to D-PLP. We found a strong interaction with the PCM component Centrosomin (Cnn). Like D-PLP, Cnn is required for efficient PCM recruitment. Cnn is weakly related to two human centrosomal proteins—Cdk5/Rap2 and Myomegalin. Interestingly, like human SAS-4 (CenpJ), when Cdk5/Rap2 is mutated, it causes primary autosomal recessive microcephaly. Mutations in human Pericentrin have been linked to Seckel syndrome and osteodysplastic primordial dwarfism type II (MOPD II), which are disorders associated with a small brain and small stature.
To analyse how Cnn and D-PLP might cooperate to recruit the PCM, we examined the behaviour of the centrioles and various PCM markers in living Cnn-deficient or D-PLP-deficient mutant embryos. We found that, in embryos lacking Cnn, the centrioles can recruit PCM but cannot maintain their connection to the PC. As a result, the centrioles “rocket” around inside the embryo, and often lose their connection to the spindle poles during mitosis (Lucas and Raff, 2007). This results in severe mitotic defects in the embryos and in defects in centriole segregation in somatic cells. When colchicine was injected into Cnn-deficient embryos, the centrioles now maintain their connection to the PCM (as the centrioles are no longer propelled around the embryo by the polymerisation of MTs). Interestingly, however, whereas the centrioles in colchicine-injected WT embryos can maintain their position at the centre of the PCM, this is not the case in colchicine-injected embryos that lack Cnn (see pictures above). This result is important as, by analogy with the actin-based rocketing of certain bacteria, it suggests that the PCM is structurally weakened in the absence of Cnn. In embryos lacking D-PLP, the PCM also appears to be structurally weakened, but to a lesser extent than in embryos lacking Cnn. Taken together, these findings indicate that Cnn and D-PLP cooperate to provide mechanical strength to the PCM.
A limitation to these studies is that we are only investigating the functions of and interactions between proteins that have already been implicated in PCM recruitment. To overcome this limitation we performed a genome-wide RNAi screen in Drosophila S2R+ cells to identify all proteins required for PCM recruitment and centriole duplication (Dobbelaere et al., 2008). This screen identified just 9 proteins required for centriole duplication, 11 required for PCM recruitment, 9 required for both processes, and 3 required for centriole/centrosome separation. Encouragingly, this list includes virtually every protein previously identified in flies as being required for these processes, as well as several fly proteins related to centrosomal proteins that had been implicated in these processes in other species and several new proteins that we showed were bona fide centrosomal components by tagging with GFP. Thus, we believe we are approaching a complete inventory of the proteins required for centriole duplication and PCM recruitment. We are currently studying all of these proteins to better understand how the PCM is organised.