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Biology of centrosomes and Cilia

Keywords : centrosome, cilia, pericentriolar material, stem cells, microtubule

Group leader : Renata Basto

Renata Basto

Our lab is interested in understanding how centrosomes (organelle that functions as the main microtubule (MT) organizing center) and cilia (organelle essential for motility or sensory perception) regulate various cellular processes and how these influence development, proliferation and the establishment of diseases.

Fig. 1Fig. 1The two centrosomes of the cell (shown in yellow) nucleate and organize the MT network (red) to assemble a bipolar mitotic spindle. The chromosomes are shown in blue.

At the ultra structural level, the centrosome comprises a pair of centrioles (barrel shape microtubule (MT) structures) surrounded by a matrix of proteins, the pericentriolar material or PCM. The PCM is the site of MT nucleation during somatic mitosis and it is now well established that centrosomes are not essential for mitosis but increase the efficiency of mitotic spindle assembly and cytokinesis. Centrosomes have, however, essential roles in polarity establishment and asymmetric cell division. For example, during fertilization of the C. elegans embryo, the centrosome, brought in by the sperm, specifies the posterior of the embryo. In addition, spindle positioning during asymmetric cell division also relies on the presence of functional centrosomes.

Fig. 2Fig. 2Electron microscopy (EM) picture of a cross section of Drosophila sperm tail flagellum.
 

In differentiated cells, centrioles can localize near the plasma membrane and behave as basal bodies. Basal bodies nucleate two types of MT structures: motile cilia or flagella and primary cilia. In recent years, we have realised how important these structures are. For example, primary cilia are crucial for mechano, chemo or photo perception in all animals whereas motile cilia or flagella are essential for movement of fluids in our body or sperm motility.

Centrosome and cilia dysfunction are associated with a variety of human diseases such as microcephaly, lissencefaly, cancer or infertility. It is therefore, essential, to characterize the basic mechanisms that regulate centriole replication and cilia assembly in the context of a developing organism to understand how mutations in centrosome, basal body and cilia components can lead to the establishment of disease.

We use a variety of different approaches that include molecular and cell biology methods, genetics and tissue culture to answer to these questions.

 

Fig. 3Fig. 3Paramecium tetraurelia stained with basal body markers.

 

Our work on centriole replication is primarily focused on the characterization of centriole replication mutants in the fruit fly Drosophila melanogaster. We are also using other model organisms such as the multiciliated Paramecium to investigate the role of centriole replication components in basal body duplication and cilia assembly.

Another area of research in the lab investigates how centrosomes and MTs influence spindle positioning during stem cell divisions. Stem cells divide asymmetrically to generate another stem cell (self-renewal) and a differentiating cell. Asymmetric cell division defects can lead to an imbalance between self-renewal and differentiation, which can contribute to over-proliferation and tumour formation. We use Drosophila neural stem cells (neuroblasts) to investigate the fundamental mechanisms that regulate these divisions.

Movie 1: Wild-type neuroblast co-expressing RFP-Tubulin to visualize the mitotic spindle and GFP-Pon, an adaptor protein that binds to Numb, a cell fate determinant. This cell divides asymmetrically and produces a large neuroblast and a small cell, the ganglion mother cell. GFP-Pon is only inherited by the GMC.

Movie 2: DSas-4 mutant neuroblast. This cell does not contain centrosomes and the spindle fails to be aligned along the polarity axis. The cell divides symmetrically and the two daughter cells inherit GFP-Pon.

We have recently characterized Drosophila fly lines that contain supernumerary centrosomes. We have noticed that during cell division, cells with extra centrosomes manage to assemble a functional bipolar spindle. Moreover, we have shown that centrosome amplification can also lead to over-proliferation and tumour formation. We are currently investigating which mechanism can either facilitate or inhibit tumour formation when extra centrosomes are present.

Movie 3: Wild-type cell co-expressing RFP-Tubulin to visualize the mitotic spindle and GFP-DSas-4 to visualize the centrosomes. The two centrosomes assemble a bipolar mitotic spindle and after division each cell inherits a single centrosome.

Movie 4: A cell with three centrosomes. As the cell enter into mitosis, the extra centrosomes cluster and form the poles of a bipolar spindle. Centrosome clustering is an efficient process since we rarely observed defects in cell division.

Key publications

  • Year of publication : 2014

  • Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs) such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT) functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.

  • Year of publication : 2013

  • Mutations in ASPM are the most frequent cause of microcephaly, a disorder characterized by reduced brain size at birth. ASPM is recognized as a major regulator of brain size, yet its role during neural development remains poorly understood. Moreover, the role of ASPM proteins in invertebrate brain morphogenesis has never been investigated. Here, we characterized the function of the Drosophila ASPM orthologue, Asp, and found that asp mutants present severe defects in brain size and neuroepithelium morphogenesis. We show that size reduction depends on the mitotic function of Asp, whereas regulation of tissue shape depends on an uncharacterized function. Asp interacts with myosin II regulating its polarized distribution along the apico-basal axis. In the absence of Asp, mislocalization of myosin II results in interkinetic nuclear migration and tissue architecture defects. We propose that Asp regulates neuroepithelium morphogenesis through myosin-II-mediated structural and mechanical processes to maintain force balance and tissue cohesiveness.

  • Centrosome amplification is a hallmark of human tumours. In flies, extra centrosomes cause spindle position defects that result in the expansion of the neural stem cell (NSC) pool and consequently in tumour formation. Here we investigated the consequences of centrosome amplification during mouse brain development and homeostasis. We show that centrosome amplification causes microcephaly due to inefficient clustering mechanisms, where NSCs divide in a multipolar fashion producing aneuploid cells that enter apoptosis. Importantly, we show that apoptosis inhibition causes the accumulation of highly aneuploid cells that lose their proliferative capacity and differentiate, thus depleting the pool of progenitors. Even if these conditions are not sufficient to halt brain development, they cause premature death due to tissue degeneration. Our results support an alternative concept to explain the etiology of microcephaly and show that centrosome amplification and aneuploidy can result in tissue degeneration rather than overproliferation and cancer.

  • Year of publication : 2011

  • The LIM-domain protein Ajuba localizes at sites of epithelial cell-cell adhesion and has also been implicated in the activation of Aurora-A (Aur-A). Despite the expected importance of Ajuba, Ajuba-deficient mice are viable, which has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insights into the function of Ajuba, we investigated its role in Drosophila, where a single gene (jub) encodes a protein closely related to Ajuba and LIMD1. We identified a key function in neural stem cells, where Jub localizes to the centrosome. In these cells, mutation in jub leads to centrosome separation defects and aberrant mitotic spindles, which is a phenotype similar to that of aur-A mutants. We show that in jub mutants Aur-A activity is not perturbed, but that Aur-A recruitment and maintenance at the centrosome is affected. As a consequence the active kinase is displaced from the centrosome. On the basis of our studies in Drosophila neuroblasts, we propose that a key function of Ajuba, in these cells, is to maintain active Aur-A at the centrosome during mitosis.

  • Year of publication : 2008

  • Centrosome amplification is a common feature of many cancer cells, and it has been previously proposed that centrosome amplification can drive genetic instability and so tumorigenesis. To test this hypothesis, we generated Drosophila lines that have extra centrosomes in approximately 60% of their somatic cells. Many cells with extra centrosomes initially form multipolar spindles, but these spindles ultimately become bipolar. This requires a delay in mitosis that is mediated by the spindle assembly checkpoint (SAC). As a result of this delay, there is no dramatic increase in genetic instability in flies with extra centrosomes, and these flies maintain a stable diploid genome over many generations. The asymmetric division of the larval neural stem cells, however, is compromised in the presence of extra centrosomes, and larval brain cells with extra centrosomes can generate metastatic tumors when transplanted into the abdomens of wild-type hosts. Thus, centrosome amplification can initiate tumorigenesis in flies.