Daniel Sandoz

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.

Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells

Summary— The diversity of microtubular networks was analyzed in quail oviduct and in Paramecium cells using conventional and confocal immunofluorescence as well as pre‐ and post‐embedding EM immunocytochemistry with a variety of anti‐tubulin antibodies. The 6‐11B‐1 monoclonal antibody, specific for the post‐translational acetylation of Lys 40 of α‐tubulin [40], and a polyclonal antibody raised against Paramecium axonemal tubulin (anti‐PA tubulin antibody) [16] both decorated stable microtubular arrays in Paramecium ie ciliary axonemes and a set of microtubular bundles associated with the cortex, suggesting that the two antibodies may be directed against the same epitope. However, several differences in the immunocytological patterns yielded by each antibody on the two cell types were evident. For example, in quail, as in all other Metazoa, the anti‐PA tubulin antibody only decorated axonemes enclosed in normal ciliary membrane while it was unreactive on cytoplasmic tubulins. Immunoblotting of peptide maps of axonemal tubulins demonstrated that the epitopes of the two antibodies were indeed completely different. Double immunolabelling of dividing paramecia using a universal anti‐tubulin antibody and the anti‐PA tubulin one revealed that all newly assembled microtubular arrays were first detected by the universal antibody and, only shortly afterwards, by the anti‐PA tubulin one. This provided a strong indication that the anti‐PA tubulin antibody is directed against a post‐translational modification taking place on already assembled microtubules (MTs) (as previously known to be the case for acetylation and detyrosination). In taxol‐treated quail cells undergoing ciliogenesis, massive assembly of MTs and even axonemes occurred in the cytoplasm. These MTs were not decorated by the anti‐PA tubulin antibody however, suggesting that in Metazoa the post‐translational modification can only take place within the ciliary lumen. The present work provides one further mechanism for generating MT immunological and biochemical diversity post‐translationally; this may account for the high multiplicity of tubulin isoforms observed in ciliates which contain very little if any genetic diversity of tubulin genes.

Determination of ciliary polarity precedes differentiation in the epithelial cells of quail oviduct

Summary— In quail oviduct epithelium, as in all metazoan and protozoan ciliated cells, cilia beat in a coordinated cycle. They are arranged in a polarized pattern oriented according to the anteroposterior axis of the oviduct and are most likely responsible for transport of the ovum and egg white proteins from the infundibulum toward the uterus. Orientation of ciliary beating is related to that of the basal bodies, indicated by the location of the lateral basal foot, which points in the direction of the active stroke of ciliary beating. This arrangement of the ciliary cortex occurs as the ultimate step in ciliogenesis and following the oviduct development. Cilia first develop in a random orientation and reorient later, simultaneously with the development of the cortical cytoskeleton. In order to know when the final orientation of basal bodies and cilia is determined in the course of oviduct development, microsurgical reversal of a segment of the immature oviduct was performed. Then, after hormone‐induced development and ciliogenesis, ciliary orientation was examined in the inverted segment and in normal parts of the ciliated epithelium. In the inverted segment, orientation was reversed, as shown by a video recording of the direction of effective flow produced by beating cilia, by the three‐dimensional bending forms of cilia immobilized during the beating cycle and screened by scanning electron microscopy, and by the position of basal body appendages as seen in thin sections by transmission electron microscopy. These results demonstrate that basal body and ciliary orientation are irreversibly determined prior to development by an endogenous signal present early in the cells of the immature oviduct, transmitted to daughter cells during the proliferative phase and expressed at the end of ciliogenesis.

Modification of cell evagination and cell differentiation in quail oviduct hyperstimulated by progesterone

Quail oviduct development is controlled by sex steroid hormones. Estrogens (E) induce cell proliferation, formation of tubular glands by epithelial cell evagination and cell differentiation. Progesterone (P) strongly increases the secretory process in E‐treated quails, but inhibits cell proliferation, cell evagination and differentiation of ciliated cells. The balance between E and P is critical for harmonious development of the oviduct.

Heterogeneity of progesterone receptor expression in epithelial cells of immature and differentiating quail oviduct

The localization of progesterone receptor (PR) in the quail oviduct was investigated before and after the onset of sexual maturation using an immunohistochemical technique. PR was revealed exclusively in nuclei of target cells whatever the hormonal state of the tissue (immature or not, pretreated or not with progesterone).

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.

An helicoidal structure surrounding the cilium axoneme: Visualization by the monoclonal antibody CC-248

Summary— Monoclonal antibody CC‐248 labels cilia differentially on Triton X‐100 permeabilized ciliated epithelium of quail oviduct by indirect immunofluorescence. On isolated ciliated cells, a punctuated staining is seen at the distal region over the bend of cilia. Electron micrographs of immunoperoxidase and immunogold techniques showed that the punctuated fluorescence corresponds to a helical disposition of CC‐248 antigenic sites. This labeling was arranged on the axonemal distal region either as a simple or a double helix externally disposed around the nine microtubular doublets. These results suggest the existence of a detergent insoluble structure in the ciliary matrix that might concern the ciliary skeleton, probably acting as an elastic recoil that keeps the structural integrity of the axoneme during bending. The cross‐reactivity of CC‐248 MAb with the intermediate filament cytoskeleton of ciliated and smooth muscle cells indicates that this structure might be related to the intermediate filament family.

Immunolocalization of laminin during estrogen-induced differentiation of quail oviduct epithelial cells

During estrogen‐induced development of the quail oviduct, tubular glands are formed by evagination of epithelial cells into the stroma. The distribution of laminin was studied during the early stages by means of immunofluorescence and immunoperoxidase techniques. Ultrastructural changes in the basal lamina were studied by electron miscrocopy.