Centrosomes are the major microtubule organising center in animal cells, participating in cell division, cell polarity and migration. The number of centrosomes in the cell is highly controlled, so that there is one centrosome at each pole of the mitotic spindle. More than a century ago, Theodor Boveri postulated that multiple centrosomes could lead to multipolar mitosis, leading to abnormal divisions and cancer[28]. In the last decade it has been shown that multiple cancers have abnormal centrosome number and structure [29]. Moreover, changes in centrosome number lead to tumour formation and microcephaly in flies and mice [30]. Each centrosome is composed of two centrioles. Centriole duplication involves the controlled assembly of a single centriole close to the one that already exists. Removal of centrioles from human cells with laser ablation leads to the ectopic formation of several centrioles, called de novo formation [7]. It is thus possible that centrioles control when and where new will form and that this is deregulated in cancer. Knowing how centrioles control their own formation is critical to understand centriole number regulation in normal and cancer cells. PLK4 is a kinase that is upregulated in bad prognosis breast cancer and changes in its levels are associated with liver and lung cancer in mice. We have shown that Plk4 is a master regulator of centriole biogenesis, providing us with a strategy to investigate centriole number control [1, 2]. Plk4 overrexpression induces the formation of an excess of centrioles both in the absence of a pre-existing centriole, (as in Drosophila non-fertilized eggs), and in the presence of centrioles, (Drosophila fertilized eggs). However, assembly is faster in the presence of centrioles suggesting that they recruit PLK4. This would sequester Plk4 from the cytoplasm and promote new centriole assembly next to the pre-existing one. To test how centriole formation is regulated in space and time there is a strong need for an experimental system amenable to live imaging of centrosomes and where we can genetically and chemically manipulate components. We will take advantage of our newly developed technique [3], by which the internal cellular components from a single syncytial Drosophila embryo can be isolated and analyzed by live imaging. This technique was recently developed for the analysis of cell cycle progression, and we want to extend it to quantitatively and mechanistically study centrosome formation. This system is tractable for: genetics, imaging (the extract forms a thin layer with ideal optical properties) and manipulations (the extract is an open system to which components can be added). We will define the best conditions and markers to follow centriole formation along the cell cycle in extracts from embryos and eggs. We will manipulate the number of centrosomes and their localization in the extract to investigate how those structures regulate their own biogenesis, by adding exogenously purified centrosomes and/or by laser ablation of existing centrosomes. This system will allow an unprecedented amount of manipulations to understand centrosome formation. As a proof of concept we will test a critical hypothesis regarding PLK4 and centrosome number control. Given the presence of centrosomes, since PLK4 is recruited there, the levels of active PLK4 will build up, allowing the formation of new centrioles. If no centrioles are present, PLK4 may stochastically accumulate in the cytoplasm activating itself, leading to the de novo formation of centrioles. We will add wild type and mutant versions of PLK4 fluorescently tagged to the extract, in the presence and absence of centrosomes. We will also take advantage of the great genetic potential of Drosophila, making extracts from mutant embryos and eggs. This project will develop an innovative, powerful and unique system to study centrosome formation and function, processes that are poorly characterized and deregulated in cancer. Moreover, this project will test a critical hypothesis regarding the role of the tumor-related kinase PLK4 in centrosome formation, which will allows us to finish a critical manuscript. We have all the tools, expertise and knowledge necessary to develop this project. This project represents a great opportunity for: i) scientific synergy between two laboratories in Portugal; ii) the best occasion for the principal investigator to start her own independent project, an essential step for the development of her career as a scientist; iii) an opportunity to answer a very important question that will lead to a major publication, build the reputation of both the PI and the host laboratory, which is essential to further apply to international grants, which is so important at this time.

• Project/scholarship ID

  133854

• Reference

  EXPL/BIM-ONC/0830/2013

• FundRef URI

  http://www.fct.pt/apoios/projectos/consulta/vglobal_projecto.phtml.en?idProjecto=133854&idElemConcurso=7645

• Funder

  FCT - Fundação para a Ciência e a Tecnologia, I.P.

• Funder's country

  Portugal

• Funding program

  3599-PPCDT

• Funding amount

  49,925.00 €

• Start date

  2014-02-03

• End date

  2015-08-02