Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During <em>ex vivo</em> Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement de Rennes)

CRBM, CNRS, University Montpellier

Introduction to Xenopus laevis as a molecular and histological model for genetic studies.

Several serine-threonine kinases related to the Ipl1p kinase in budding yeast, termed aurora kinases, have been cloned recently. Their characterization revealed them to be important regulators of mitotic functions, including (i) the separation of the centrosome, (ii) assembly of the spindles, and (iii) segregation of the chromosomes. The Perspective by Descamps and Prigent delves into the latest observations on aurora kinases in humans and the specific roles of each kinase within the process of mitosis.

Two mammalian mitotic aurora kinases: who's who?

Eukaryotic cells must possess mechanisms for condensing and decondensing chromatin. Chromatin condensation is particularly evident during mitosis and cell death induced by apoptosis, whereas chromatin decondensation is necessary for replication, repair, recombination and transcription. Histones are among the numerous DNA-binding proteins that control the level of DNA condensation, and post-translational modification of histone tails plays a critical role in the dynamic condensation/decondensation that occurs during the cell cycle. Phosphorylation of Ser10 in the tails of histone H3 has been extensively studied in many organisms. Interestingly, this modification is involved in both transcription and cell division, two events requiring opposite alterations in the degree of chromatin compaction. How does one and the same modification of histone H3 fulfil such roles? For instance, in interphase, phosphorylation of H3 correlates with chromatin relaxation and gene expression, whereas in mitosis it correlates with chromosome condensation. What is the kinase and under what circumstances does Ser10 becomes phosphorylated? Most importantly, what are the consequences of phosphorylation of this residue?

Phosphorylation of serine 10 in histone H3, what for?

Aurora kinases control multiple events during cell cycle progression and are essential for mitotic and meiotic bipolar spindle assembly and function. There are three Aurora kinases in mammals, some of which have oncogenic properties and all of which are overexpressed in multiple cancers. Pharmaceutical companies quickly made these kinases priority targets for the development of inhibitors to be used as cancer treatments. In this review, we focus on Aurora A, against which several inhibiting compounds have been discovered and made available; however, even though some of these compounds underwent clinical trials, none have yet gone beyond Phase III trials. The varying efficiencies and particularities of these drugs raise several questions that are explored in this review: is Aurora A even a good target? What biomarkers can we use to measure its activity in vivo? How can we improve the Aurora A-inhibiting drugs?

Aurora A Kinase Is a Priority Pharmaceutical Target for the Treatment of Cancers.

During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper.

Aurora A kinase activity is required to maintain an active spindle assembly checkpoint during prometaphase.

In metazoans, organisms arising from a fertilized egg, the embryo will develop through multiple series of cell divisions, both symmetric and asymmetric, leading to differentiation. Aurora A is a serine threonine kinase highly involved in such divisions. While intensively studied at the cell biology level, its function in the development of a whole organism has been neglected. Here we investigated the pleiotropic effect of Aurora A loss-of-function in Drosophila larval early development. We report that Aurora A is required for proper larval development timing control through direct and indirect means. In larval tissues, Aurora A is required for proper symmetric division rate and eventually development speed as we observed in central brain, wing disc and ring gland. Moreover, Aurora A inactivation induces a reduction of ecdysteroids levels and a pupariation delay as an indirect consequence of ring gland development deceleration. Finally, although central brain development is initially restricted, we confirmed that brain lobe size eventually increases due to additive phenotypes: delayed pupariation and over-proliferation of cells with an intermediate cell-identity between neuroblast and ganglion mother cell resulting from defective asymmetric neuroblast cell division.

Size matters! Aurora A controls Drosophila larval development.

During mitosis, the cell sequentially constructs two microtubule-based spindles to ensure faithful segregation of chromosomes. A bipolar spindle first pulls apart the sister chromatids, then a central spindle further separates them away. Although the assembly of the first spindle is well described, the assembly of the second remains poorly understood. We report here that the inhibition of Aurora A leads to an absence of the central spindle resulting from a lack of nucleation of microtubules in the midzone. In the absence of Aurora A, the HURP (also known as DLGAP5) and NEDD1 proteins that are involved in nucleation of microtubules fail to concentrate in the midzone. HURP is an effector of RanGTP, whereas NEDD1 serves as an anchor for the γ-tubulin ring complex (γTURC). Interestingly, Aurora A phosphorylates HURP and NEDD1 during assembly of the initial bipolar spindle. We show here that the expression of a NEDD1 isoform mimicking phosphorylation by Aurora A is sufficient to restore microtubule nucleation in the midzone under conditions of Aurora A inhibition. These results reveal a new control mechanism of microtubule nucleation by Aurora A during assembly of the central spindle.

Claude Prigent

Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement de Rennes)

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis