Michel Lemullois

Development and functions of the cytoskeleton during ciliogenesis in metazoa

Summary— The different steps of ciliogenesis occurring in quail oviduct were compared to the ciliogenesis pattern described in other metazoan species.

Organization and functions of cytoskeleton in metazoan ciliated cells

Summary— Ciliated cells are characterized by a highly organized cytoskeleton which is connected with the ciliary apparatus. The organization of microtubules, microfilaments, and cytokeratin filaments is described and the relationships of each network with the ciliary apparatus are emphasized. Possible functions of such a complex cytoskeleton are discussed.

The conserved centrosomal protein FOR20 is required for assembly of the transition zone and basal body docking at the cell surface

Summary Within the FOP family of centrosomal proteins, the conserved FOR20 protein has been implicated in the control of primary cilium assembly in human cells. To ascertain its role in ciliogenesis, we have investigated the function of its ortholog, PtFOR20p, in the multiciliated unicellular organism Paramecium. Using combined functional and cytological analyses, we found that PtFOR20p specifically localises at basal bodies and is required to build the transition zone, a prerequisite to their maturation and docking at the cell surface and hence to ciliogenesis. We also found that PtCen2p (one of the two basal body specific centrins, an ortholog of HsCen2) is required to recruit PtFOR20p at the developing basal body and to control its length. By contrast, the other basal-body-specific centrin PtCen3p is not needed for assembly of the transition zone, but is required downstream, for basal body docking. Comparison of the structural defects induced by depletion of PtFOR20p, PtCen2p or PtCen3p, respectively, illustrates the dual role of the transition zone in the biogenesis of the basal body and in cilium assembly. The multiple potential roles of the transition zone during basal body biogenesis and the evolutionary conserved function of the FOP proteins in microtubule membrane interactions are discussed.

Human Cytomegalovirus Infects Caco-2 Intestinal Epithelial Cells Basolaterally Regardless of the Differentiation State

ABSTRACT Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.

Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium

ABSTRACT Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

Evolutionary conservation of an epitope associated with striated rootlets in different epithelial ciliated cells

Summary— In ciliated cells of metazoa, striated rootlets associated with basal bodies anchor the ciliary apparatus to the cytoskeleton. We have used here a monoclonal antibody against a 175 kDa protein associated with the striated rootlets of quail ciliated cells [14], to study ciliated cells of different species. In mussel gill epithelium the antibody recognized a protein of 92 kDa which shows a periodic distribution along the striated rootlets. In frog ciliated palate epithelium, two different rootlets are associated with basal bodies, both are decorated and only one protein of 48 kDa is recognized on immunoblot. The antigen is arranged in a helix around the striated rootlets. In rabbit ciliated oviduct epithelium, we detected the presence of very small and thin rootlets which are weakly labeled. We have shown that an epitope associated with the striated rootlets is preserved through evolution although the molecular weight of the peptide varies. We have also observed the appearance of this epitope on protein associated with junctional complexes in rabbit and cytoskeleton component in quail oviduct.

Paramecium tetraurelia basal body unit isolation for Cryo-electron tomography studies

Objective The Transition Zone (TZ) is defined as the most proximal region of the cilium overlapping with the most distal region of the basal body. This zone has been shown to play a crucial role in cilia biology since it is considered as the site of sorting of proteins that transit to cilia. Protein complexes housed at this zone are found mutated in MKS/NPHP ciliopathies. Although its organization varies from organism to organism, the TZ molecular composition and function are highly conserved. In Paramecium, the TZ is well structured with three distinct plates defined as the terminal, the intermediate and the axosomal plates. In this model, structural and molecular changes of the TZ are observed as anchored basal bodies become ciliated. Therefore, Paramecium appears to be a pertinent model to study the TZ at an ultrastructural level in correlation with its functionality.

C11orf70 mutations causing primary ciliary dyskinesia disrupt a conserved step in the intraflagellar transport-dependent assembly of multiple axonemal dyneins

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomised left-right body asymmetry. PCD is mostly caused by mutations affecting components of the core axoneme structure of motile cilia that are essential for cilia movement. In addition, there is a growing group of PCD genes that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified 2 unrelated families (3 affected children) with mutations in the uncharacterized C11orf70 gene. The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Fluorescently tagged C11orf70 in Paramecium and Chlamydomonas localises mainly in the cytoplasm with a small amount in the ciliary component, its abundance in the axoneme being IFT-dependant. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for IFT-dependant assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.

OFD1 and VFL3/CCDC61 in basal body positioning and docking in Paramecium

Objectives Ciliogenesis is conditioned by a correct positioning/ anchoring of the basal body at the cell surface. In Paramecium we have shown that three conserved proteins FOR20, centrin 2 (CEN2) and centrin 3 (CEN3) participates in this process, with FOR20 and CEN2 being also involved in the transition zone assembly. We established a chronology in basal body assembly: CEN2 is required for FOR20 recruitment, the latter being necessary to recruit CEN3. Our goal now is to integrate others molecules in this cascade.

Centrin localisation in a hypotrich ciliate protozoan paraurostyla

Ciliates are hiehlv differentiated cells covered with many cilia in which the cytoskeleton i< very developped. Among ciliates, the maht characteristic of hypotrichs is the asymetric distribution of their cilia patterned into clusters on-one side of the flattened cell and forming the oral ciliature, specialized in prey prehension and the somatic one, involved in locomotion. In these two tvoes of clusters. the ciliarv bases (basal bodies) are COMected bv a compikx set of links and de&e material. During. binary fission, this ciliature is completely renewed, and the new clusters act as MTOCs for the subpellicular micrombules which constitute the superficial cytoskeleton. In order to elucidate the mechanisms of cvtoskeletal assembly. we have undertaken an analysis of the proteins of the basal bodies clus-mrs. Using specific antibodies (Middendorp et al., 1997, PNAS 94: 9141), we looked for the presence of centrin. Immunofluorescence analysis shows that, in Paraurosf&, the pattern of centrin overlaps that of basal bodies. Ultrastructural immunolocalization allowed us to demonstrate that centrln is one of the main components of basal bodies linkages: in oral clusters, all links are decorated by the antibodies against cent&t, while in somatic ones, the proximal links, but not the median ones, were decorated. These results show that, in Paruurostyla. two kinds of ciliary clusters different in design coexist within the same cell. Depending upon the organisms, centrins may either be localized in close proximity of basal bodies/centrioles or form a contractile cytoskeletal network at the whole cell level. In Parauroqla. the main use of centrins is to built a complex set of linkages in large basal bodies clusters. The characterization and the distribution of the different centrins isofotms in these assemblages are ttttder examination.