Anne Fleury

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.

Power and limits of laser scanning confocal microscopy

In confocal microscopy, the object is illuminated and observed so as to rid the resulting image of the light from out‐of‐focus planes. Imaging may be performed in the reflective or in the fluorescence mode. Confocal microscopy allows accurate and nondestructive optical sectioning in a plane perpendicular or parallel to the optical axis of the microscope. Further digital three‐dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. Several examples illustrating each of these possibilities are given. Three‐dimensional reconstitution of nuclear components using a cubic representation and a ray‐tracing based method are also given. Instrumental and experimental factors can introduce some bias into the acquisition of the 3‐D data set: self‐shadowing effects of thick specimens, spherical aberrations due to the sub‐optimum use of the objective lenses and photobleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for 3‐D reconstruction. We delineate each of these problems and indicate to what extent they can be solved. Some tips are given for the practice of confocal microscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image, how to prevent artificial flattening of the specimens. Finally, future prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV‐lasers and a possible means to attenuate photodamage to biological specimens.

Isolation and characterization of monoclonal antibodies to cytoskeletal and membrane proteins of the Paramecium cortex

Monoclonal antibodies have been generated against whole cortical fragments of Paramecium to provide tools for the analysis of cortex morphogenesis as well as for the biochemical dissection of this complex membrane-cytoskeleton structure. Of 80 positive hybridomas in ELISA tests, 17 monoclonal were characterized by immunoblotting, immunofluorescence tests on sectioned as well as on Triton-permeabilized cells (including confocal microscopy), and by EM immunocytochemistry on permeabilized cells. Five classes of monoclonals were obtained directed, respectively, against the epiplasm (or elements tightly associated with it), the trichocysts, the microtubules, the surface membranes, and poorly defined intracellular antigens. Of these, the newest and most promising appear to be a set of monoclonals decorating both intensely and sharply some specific parts of the epiplasm (outer periphery, outer central part or core). These antibodies therefore provide the first demonstration of a molecular heterogeneity of composition at the level of individual epiplasmic scales in Paramecium. In addition, they offer powerful tools to follow the biogenesis of these structures during cell division. Finally, they have allowed the identification of a number of previously uncharacterized protein components of the cortex.

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out‐of‐locus light and provides a greater resolution than the wide‐field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction‐limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two‐dimensional image. Thus, confocal scanning optical microscopy allows accurate non‐invasive optical sectioning and further three‐dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three‐dimensional imaging.

Microtubule dynamics and morphogenesis in Paramecium: I. Deployment and dynamic properties of a cortical network of acetylated microtubules in relation to the invariance of a morphogenetic field

Summary The cortex of Paramecium behaves as a mosaic of territories expressing different morphogenetic behavior in the course of cell division, being made up of both hyperduplicated and invariant regions, i.e. regions transmitted unchanged from one generation to the next. In this paper, we analyze the dynamics of an extensive network of acetylated microtubules associated with a specific region of the interphase cell, the A-paratene area. The A-paratene-associated microtubules exhibit stability properties similar in behavior to microtubules observed in epithelial cells: they are cold labile but nocodazole resistant. When the cell divides, at least a large part of the A-paratene area behaves as an invariant region. The A-paratene-associated microtubules depolymerize in two steps, accompanying and following the basal body wave, respectively; then, they reassemble after cell separation. Although the invariant region and the A-paratene area do not strictly coincide and microtubule dynamics involve other mechanisms in addition to acetylation, these results strongly suggest that the deployment of this network might correlate with the invariance property of this territory and could be related to its morphogenetic history.

Interspecific immunological cross-reactions among cortical proteins of four ciliates

Summary Many ciliates contain a highly organized membrane skeleton called the epiplasm, which underlies the cortical membranes. The epiplasms of the different species studied here exhibit a marked heterogeneity with regard to the size and number of their constituent proteins. Three monoclonal antibodies (Mabs) raised against the epiplasm of Pseudomicrothorax dubius were tested on Paramecium tetraurelia, Tetrahymena pyriformis and Euplotes aediculatus , and showed cross-reactivity in most cases. In the cross-reactions, the Mab 2A6F3 labels an epitope shared by proteins localized to the edges of the epiplasm or epiplasm-like structures, both around basal bodies and peripherally to the alveoli. Additionally, it is the first antibody that labels the argyrome of Euplotes aediculatus by immunofluorescence. The other two Mabs, 4B5F3 and 1E11F5, are specific for an epitope common to nearly all the epiplasmic proteins of P. dubius , but they cross-react weakly with P. tetraurelia and T. pyriformis , which is consistent with the evolutionary relationships among these species. However, these Mabs are highly cross-reactive on euglenids: it implies either a genetic homology or a functional convergence of these proteins. Regardless of their origin, the epiplasmic proteins constitute a new class of cytoskeletal elements.

Transmission of surface pattern through a dedifferentiated stage in the ciliate Paraurostyla. Evidence from the analysis of microtubule and basal body deployment

ABSTRACT. During conjugation of the hypotrich ciliate Paraurostyla weissei, the two partners fuse to form a transient dedifferentiated stage, the zygocyst, which later redifferentiates into a vegetative cell. Immunocytochemical studies have been performed to follow the deployment of microtubules and basal bodies during the entire cycle of conjugation. They show that a superficial lattice persists during the whole zygocyst stage, after most of the infraciliature of the exconjugants has been disassembled. These superficial microtubules display different immunocytochemical properties in the mature zygocyst and during its morphogenesis, suggesting that some transient chemical modifications of the microtubules are associated with the morphogenetic activity. In the zygocyst, the superficial microtubules retain the specific orientation characteristic of the ventral and the dorsal sides of the recipient cell, respectively. In the course of subsequent morphogenesis of the zygocyst, these specific cellular territories differentiate into the ventral and dorsal sides of the new cell. Although our experiments do not resolve the question of whether superficial microtubules play an active or merely a passive role in the transmission of surface pattern, they show that no complete breakdown in cell polarity occurs, even through a profound dedifferentiated stage. Thus, the overall surface pattern appears to be retained, in a simplified form, through the conjugation cycle.

A dynamical model for post-translational modifications of microtubules

In many cases, post‐translational modifications constitute indirect markers of non‐dynamic microtubules. The correlation between stability of microtubular systems and post‐translational modifications suggests that they may act as a signal in patterning mechanisms. However, a crucial question remains as to how the repertoire of distinctly modified microtubules is generated. We propose here an ubiquitous mechanism of spatial and temporal differentiation of microtubules. In this model, the diversity of post‐translational modifications results from a dynamical pathway separation which is ensured by biochemical switches between self‐regulated mechanisms of differentiation. Thus, it does not require any hypothetical subcellular compartmentalization of enzymatic activities responsible for the various post‐translational modifications. These results are discussed in relation to experimental evidences for a temporal and spatial regulation of microtubule modifications.

Comparison of the electrophoretic behaviour of tubulins from distantly related ciliates

Differences in gel electrophoretic mobility were used to compare tubulin subunits from a variety of distantly related ciliate species. The relative electrophoretic mobility of ciliate tubulins is shown to vary depending upon the electrophoretic conditions used to separate them. Tubulin migration is altered by changing the pH of the separating gel: the positions of the tubulin subunits at pH 8.1 are apparently reversed when compared with their migration at pH 9.3. Examination of the tubulin electrophoretic behaviour reveals considerable differences among species of ciliates belonging to different taxonomic groups. Such an electrophoretic diversity is in good agreement with the large evolutionary distances within the phylum derived from rRNA sequence analyses. Our results allow us to group the ciliates analyzed in clusters which can be fitted quite satisfactorily into the traditional phylogenetic schemes.

Microtubule dynamics and morphogenesis in Paramecium: II. Modelling of the conversion of a transient molecular signal into a morphogenetical process

Summary In Paramecium, the deployment of an acetylated microtubular network is restricted to a part of the ventral cell surface, the A-paratene zone. Upon cell division, this microtubular network depolymerizes; it is reassembled only after cell separation. Convergent evidences suggest that one of the primary signals for cortical morphogenesis could be a Ca2+ wave. Correlatively, calcium ions inhibit the enzyme which acetylates microtubules and prevent microtubule stabilization by associated proteins (MAPs). All these data have been gathered into a model which shows how a transient signal (the calcium wave) may be converted into a permanent morphological modification of the assembly state of the A-paratene-associated microtubules. In this model, biochemical switches between acetylation and depolymerization pathways acting on stabilized microtubules occur beyond the assembly/disassembly step of unstabilized microtubules. Accordingly, only the inhibitory effect of Ca2+ on the acetylation enzyme has a triggering action on the dynamics of the microtubules whereas its indirect effect on MAPs is irrelevant in this respect. This model also interprets why the A-paratene-associated microtubules resists nocodazole but not cold treatment. From this analysis, we suggest that the various control mechanisms which act at the level of each modification enzyme would make both the origin and the biological significance of the diversity of microtubules regulative.