A Highlights from MBoC Selection: Ccdc11 is a novel centriolar satellite protein essential for ciliogenesis and establishment of left–right asymmetry

The establishment of left–right (L-R) asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt L-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here we demonstrate that Ccdc11 is a novel component of centriolar satellites—cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer’s vesicle and results in aberrant L-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia and implicate centriolar satellite–associated proteins as a new class of proteins in the pathology of L-R patterning and congenital heart disease.     

Primary ciliogenesis is a crucial step for multiciliated cell determinism in the respiratory epithelium

The alteration of the mucociliary clearance is a major hallmark of respiratory diseases related to structural and functional cilia abnormalities such as chronic obstructive pulmonary diseases (COPD), asthma and cystic fibrosis. Primary cilia and motile cilia are the two principal organelles involved in the control of cell fate in the airways. We tested the effect of primary cilia removal in the establishment of a fully differentiated respiratory epithelium. Epithelial barrier integrity was not altered while multiciliated cells were decreased and mucous-secreting cells were increased. Primary cilia homeostasis is therefore paramount for airway epithelial cell differentiation. Primary cilia-associated pathophysiologic implications require further investigations in the context of respiratory diseases.     

The motile cilium in development and disease: emerging new insights

In this paper, I review a collection of recently published papers that have provided significant new information about the biogenesis and functions of motile cilia. In vertebrates, the activity of motile cilia has been associated with a fascinating diversity of developmental and physiological processes. Despite the importance, much remains to be learned about the genetic control and cellular events that are involved in the differentiation of motile cilia. We also need to better understand the mechanisms by which cilia‐driven fluid flow is able to influence such a variety of developmental and physiological processes. The Foxj1 family of proteins has now been definitively established as master regulators of motile ciliogenesis. 1 , 2 Identification of the Kintoun/PF13 protein has shed light on the assembly of dynein arms, 3 whereas live imaging of ciliary motility has led to the discovery of an intriguing new role for motile cilia in otolith formation in the ear. 4     

Selection of nonmotile Tetrahymena with Ficoll underlayers.

The mechanisms regulating the development of cilia in Tetrahymena are poorly understood but might be revealed through the study of ciliogenesis mutants. Failure to regenerate cilia after dibucaine deciliation results in continued absence of motility. Therefore, to isolate ciliogenesis mutants efficiently, methods for separating motile and nonmotile cells are essential. We examined the efficacy of Ficoll underlayers for these separations. Ciliates of T. thng type IV) were mixed with Ficoll and added as underlayers to separatory funnels containing growth medium. At 27 C most of the cells remained motile and were found in the top layer; at 37 C, there was a time-dependent increase in the number of nonmotile cells and the number of cells in the Ficoll layer. After 150 min at 37 C, most of the cells became nonmotile and were found in the Ficoll layer. Other studies indicated that at 37 C, the cells remained alive and capable of regenerating cilia when deciliated. Thus, it is clear that the Ficoll underlayer effectively separates the majority of nonmotile cells from the majority of motile cells. Evidently, however, at 37 C wild-type T. thermophila exhibit temperature-sensitive phenotypic variability with regard to motility which should be minimized when selecting for mutations affecting motility and ciliogenesis.     

Mitotic control by mRNA splicing regulators ensures primary cilia formation

The biogenesis of the primary cilium is coordinated with cell cycle exit/re-entry in most types of cells. After serum starvation, the cilia-generating cells enter quiescence and produce the primary cilium; upon re-addition of serum, they re-enter the cell cycle and resorb the cilium. We previously identified novel mechanisms to link cell cycle progression and ciliogenesis by high-content genome-wide RNAi cell-based screening. In the present study, we pay attention to reveal the impact of mRNA splicing on cilia assembly after mitosis of cell cycle. We demonstrate that splicing regulators such as SON and XAB2 play an important role in mitosis exit, and thus affect ciliogenesis in G1/G0 phases. Knockdown of the splicing regulators in hTERT-RPE1 cells caused abnormal G2/M arrest under both serum addition and serum starvation, indicating defects in mitosis exit. Moreover, the knockdown cells failed to assemble the cilia under serum starvation and an inhibition of mRNA splicing using SSA, a spliceosome inhibitor, also revealed ciliogenesis defect. Finally, we show that the SSA-treated zebrafish display abnormal vascular development as a ciliary defect. These findings suggest the pivotal role of mRNA splicing regulators in cilia assembly and underscore the importance of mitotic regulation in ciliogenesis.     

The toxic effect of cytostatics on primary cilia frequency and multiciliation

The primary cilium is considered as a key component of morphological cellular stability. However, cancer cells are notorious for lacking primary cilia in most cases, depending upon the tumour type. Previous reports have shown the effect of starvation and cytostatics on ciliogenesis in normal and cancer cells although with limited success, especially when concerning the latter. In this study, we evaluated the presence and frequency of primary cilia in breast fibroblasts and in triple‐negative breast cancer cells after treatment with cytostatics finding that, in the case of breast fibroblasts, primary cilia were detected at their highest incidence 72 hours after treatment with 120 nM doxorubicin. Further, multiciliated cells were also detected after treatment with 80 nM doxorubicin. On the other hand, treatment with taxol increased the number of ciliated cells only at low concentrations (1.25 and 3.25 nM) and did not induce multiciliation. Interestingly, triple‐negative breast cancer cells did not present primary cilia after treatment with either doxorubicin or taxol. This is the first study reporting the presence of multiple primary cilia in breast fibroblasts induced by doxorubicin. However, the null effect of these cytostatics on primary cilia incidence in the evaluated triple negative breast carcinomas cell lines requires further research.     

Development of a multiciliated cell

Multiciliated cells (MCC) are evolutionary conserved, highly specialized cell types that contain dozens to hundreds of motile cilia that they use to propel fluid directionally. To template these cilia, each MCC produces between 30 and 500 basal bodies via a process termed centriole amplification. Much progress has been made in recent years in understanding the pathways involved in MCC fate determination, differentiation, and ciliogenesis. Recent studies using mammalian cell culture systems, mice, Xenopus, and other model organisms have started to uncover the mechanisms involved in centriole and cilia biogenesis. Yet, how MCC progenitor cells regulate the precise number of centrioles and cilia during their differentiation remains largely unknown. In this review, we will examine recent findings that address this fundamental question.     

Selection of Nonmotile Tetrahymena with Ficoll Underlayers*,†

The mechanisms regulating the development of cilia in Tetrahymena are poorly understood but might be revealed through the study of ciliogenesis mutants. Failure to regenerate cilia after dibucaine deciliation results in continued absence of motility. Therefore, to isolate ciliogenesis mutants efficiently, methods for separating motile and nonmotile cells are essential. We examined the efficacy of Ficoll underlayers for these separations. Ciliates of T. thermophila strain mpr^-/mpr (6 mp sens IV) (6-methyl purine-sensitive; mating type IV) were mixed with Ficoll and added as underlayers to separatory funnels containing growth medium. At 27 C most of the cells remained motile and were found in the top layer; at 37 C, there was a time-dependent increase in the number of nonmotile cells and the number of cells in the Ficoll layer. After 150 min at 37 C, most of the cells became nonmotile and were found in the Ficoll layer. Other studies indicated that at 37 C, the cells remained alive and capable of regenerating cilia when deciliated. Thus, it is clear that the Ficoll underlayer effectively separates the majority of nonmotile cells from the majority of motile cells. Evidently, however, at 37 C wild-type T. thermophila exhibit temperature-sensitive phenotypic variability with regard to motility which should be minimized when selecting for mutations affecting motility and ciliogenesis.     

CCRK depletion inhibits glioblastoma cell proliferation in a cilium‐dependent manner

Loss of primary cilia is frequently observed in tumour cells, including glioblastoma cells, and proposed to benefit tumour growth, but a causal link has not been established. Here, we show that CCRK (cell cycle‐related kinase) and its substrate ICK (intestinal cell kinase) inhibit ciliogenesis. Depletion of CCRK leads to accumulation of ICK at ciliary tips, altered ciliary transport and inhibition of cell cycle re‐entry in NIH3T3 fibroblasts. In glioblastoma cells with deregulated high levels of CCRK, its depletion restores cilia through ICK and an ICK‐related kinase MAK, thereby inhibiting glioblastoma cell proliferation. These results indicate that inhibition of ciliogenesis might be a mechanism used by cancer cells to provide a growth advantage.     

GEMC1 is a critical regulator of multiciliated cell differentiation

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1‐deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.     

MDM1 is a microtubule-binding protein that negatively regulates centriole duplication

Mouse double-minute 1 (Mdm1) was originally identified as a gene amplified in transformed mouse cells and more recently as being highly up-regulated during differentiation of multiciliated epithelial cells, a specialized cell type having hundreds of centrioles and motile cilia. Here we show that the MDM1 protein localizes to centrioles of dividing cells and differentiating multiciliated cells. 3D-SIM microscopy showed that MDM1 is closely associated with the centriole barrel, likely residing in the centriole lumen. Overexpression of MDM1 suppressed centriole duplication, whereas depletion of MDM1 resulted in an increase in granular material that likely represents early intermediates in centriole formation. We show that MDM1 binds microtubules in vivo and in vitro. We identified a repeat motif in MDM1 that is required for efficient microtubule binding and found that these repeats are also present in CCSAP, another microtubule-binding protein. We propose that MDM1 is a negative regulator of centriole duplication and that its function is mediated through microtubule binding.     

Development of cilia in embryos of the turbellarian Macrostomum

Electron microscopy of Macrostomum hystricinum raised in culture shows that ciliogenesis in the worm's epidermal blastomeres begins in embryos 39–41 h old with kinetosomal and de novo genesis of presumptive basal bodies, which are morphologically distinguishable from centrioles of the mitotic apparatus, and proceeds by the migration of basal bodies to the apical plasma membrane of the cells and their production there of ciliary axonemes by an age of 51–53 h when the bastomeres emerge between yolk cells on the embryo's surface. Ciliogenesis continues throughout development with the addition of cilia virtually one by one to the expanding epidermal cells' surfaces. At no time in ciliogenesis are stages seen that might show derivation of these multiciliated cells from the primitive monociliated cell type presumably present in the ancestors of the Turbellaria.     

Putative sensory structures in marine bryozoans

Abstract. SEM studies of 21 species of marine bryozoans demonstrated that the abfrontal side of the tentacles bears a row of mono- or multiciliated cells, which are presumably sensory. In stenolaemates, the abfrontal cells, as well as the cells at the tentacle tips and the laterofrontal cells, are monociliated. In the 17 gymnolaemate species studied, each tentacle tip bears at least 3 multiciliated cells, each with a tuft of 5–7 stiff cilia of various lengths. On the abfrontal tentacle surface, mono- and multiciliated cells alternate, but all species studied have multiciliated cells at the base and the tip of each tentacle. In live animals, single cilia perform occasional flicks, whereas the tufts of 7–15 cilia on the multiciliated cells are immotile. Length and number of abfrontal cilia vary between species. Two types of multiciliated, putative sensory organs were found on the introvert of some gymnolaemates. One has an apical knob surrounded by a ring of cilia; the other has an apical tuft of cilia. The ultrastructure of the sensory cells of tentacles and introvert was studied in Rhamphostomella ovata . Our observations on both fixed and living material all suggest that these cells are primitive mechanoreceptors. The few species lacking ciliary structures on the introvert have long proximal ciliary tufts on the abfrontal tentacle surface.