Maintaining cell polarity through vegetative cell pattern dedifferentiation: cytoskeleton and morphogenesis in the hypotrich ciliate Sterkiella histriomuscorum.

The morphological differentiation of ciliates is achieved through the development of a submembraneous cytoskeleton in which the cilia are anchored. In most hypotrich ciliates, this cytoskeleton is mainly constructed of microtubules. In these species, cells pass through vegetative cell pattern dedifferentiated stages during their biological cycle. In order to investigate the behaviour of the cytoskeleton during these stages, we analysed the reorganization of the cytoskeleton during the sexual cycle of Sterkiella histriomuscorum by microscopy. Sterkiella exconjugants transiently dedifferentiate to form zygocysts devoid of ciliature and infraciliature. Immunofluorescence images obtained with antibodies directed against pericentrosomal material and tubulin showed that the cells resorb their ciliature and basal bodies, but retain their submembraneous microtubular cytoskeleton during the whole process and that the body plan is maintained through vegetative cell pattern dedifferentiation: the cell polarity remains printed on the cell surface by the microtubular cytoskeleton which in turn could mark the sites of basal body assembly during zygocyst morphogenesis. The results are discussed in terms of mechanisms of cell patterning.

Localization of centrins in the hypotrich ciliate Paraurostyla weissei.

Centrins are ubiquitous cytoskeletal proteins that are generally associated with the centrosome and form large cytoskeletal networks in protists. To obtain more data on the respective role of different centrin proteins, we studied their distribution and behavior in one ciliate species, Paraurostyla weissei, using specific antibodies. In this species, only two major proteins of 21 and 24 kDa corresponding to centrins, were identified by 1D and 2D electrophoresis. Immunofluorescence analysis showed that these two proteins displayed non-overlapping localization in the interphase cell and during morphogenesis. Both centrin proteins localize on the fibrous network linking the oral basal bodies in the interphase cell and in the form of marginal dots, which correspond to the proximal ends of the striated rootlets; the 21 kDa centrin was also detected within the basal bodies, whereas the 24 kDa centrin allowed identifying new structures, the frontal dashes. During morphogenesis, the 21 kDa centrin locates at the basal bodies, while the 24 kDa centrin is detected along the striated rootlets and in close association with the basal bodies pairs. These data are discussed in terms of the potential roles of the two centrins in different cellular functions.

Cysteine proteases and cell differentiation: excystment of the ciliated protist Sterkiella histriomuscorum.

The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.