p53/p21 pathway activation contributes to the ependymal fate decision downstream of GemC1.

Multiciliated ependymal cells and adult neural stem cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here, we show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, is induced, without affecting cell survival. Genotoxic stress is not sufficient by itself to induce ependymal cell differentiation, although the absence of p53 or p21 in progenitors hinders differentiation by maintaining cell division. Activation of the p53-p21 pathway downstream of GemC1 leads to cell-cycle slowdown/arrest, which permits timely onset of ependymal cell differentiation in progenitor cells.

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

Ependymal cell differentiation, from monociliated to multiciliated cells.

Primary and motile cilia differ in their structure, composition, and function. In the brain, primary cilia are immotile signalling organelles present on neural stem cells and neurons. Multiple motile cilia are found on the surface of ependymal cells in all brain ventricles, where they contribute to the flow of cerebrospinal fluid. During development, monociliated ependymal progenitor cells differentiate into multiciliated ependymal cells, thus providing a simple system for studying the transition between these two stages. In this chapter, we provide protocols for immunofluorescence staining of developing ependymal cells in vivo, on whole mounts of lateral ventricle walls, and in vitro, on cultured ependymal cells. We also provide a list of markers we currently use to stain both types of cilia, including proteins at the ciliary membrane and tubulin posttranslational modifications of the axoneme.

Centriole amplification by mother and daughter centrioles differs in multiciliated cells.

The semi-conservative centrosome duplication in cycling cells gives rise to a centrosome composed of a mother and a newly formed daughter centriole. Both centrioles are regarded as equivalent in their ability to form new centrioles and their symmetric duplication is crucial for cell division homeostasis. Multiciliated cells do not use the archetypal duplication program and instead form more than a hundred centrioles that are required for the growth of motile cilia and the efficient propelling of physiological fluids. The majority of these new centrioles are thought to appear de novo, that is, independently from the centrosome, around electron-dense structures called deuterosomes. Their origin remains unknown. Using live imaging combined with correlative super-resolution light and electron microscopy, we show that all new centrioles derive from the pre-existing progenitor cell centrosome through multiple rounds of procentriole seeding. Moreover, we establish that only the daughter centrosomal centriole contributes to deuterosome formation, and thus to over ninety per cent of the final centriole population. This unexpected centriolar asymmetry grants new perspectives when studying cilia-related diseases and pathological centriole amplification observed in cycling cells and associated with microcephaly and cancer.

Mechanism for astral microtubule capture by cortical Bud6p priming spindle polarity in S. cerevisiae.

BACKGROUND: Budding yeast is a unique model to dissect spindle orientation in a cell dividing asymmetrically. In yeast, this process begins with the capture of pole-derived astral microtubules (MTs) by the polarity determinant Bud6p at the cortex of the bud in G(1). Bud6p couples MT growth and shrinkage with spindle pole movement relative to the contact site. This activity resides in N-terminal sequences away from a domain linked to actin organization. Kip3p (kinesin-8), a MT depolymerase, may be implicated, but other molecular details are essentially unknown. RESULTS: We show that Bud6p and Kip3p play antagonistic roles in controlling the length of MTs contacting the bud. The stabilizing role of Bud6p required the plus-end-tracking protein Bim1p (yeast EB1). Bim1p bound Bud6p N terminus, an interaction that proved essential for cortical capture of MTs in vivo. Moreover, Bud6p influenced Kip3p dynamic distribution through its effect on MT stability during cortical contacts via Bim1p. Coupling between Kip3p-driven depolymerization and shrinkage at the cell cortex required Bud6p, Bim1p, and dynein, a minus-end-directed motor helping tether the receding plus ends to the cell cortex. Validating these findings, live imaging of the interplay between dynein and Kip3p demonstrated that both motors decorated single astral MTs with dynein persisting at the plus end in association with the site of cortical contact during shrinkage at the cell cortex. CONCLUSIONS: Astral MT shrinkage linked to Bud6p involves its direct interaction with Bim1p and the concerted action of two MT motors-Kip3p and dynein.

Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability.

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting ß-tubulin, suggesting Mgr function is required for tubulin stability. Instability of ß-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic ß-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.