Three-dimensional structural labeling microscopy of cilia and flagella.

Locating a molecule within a cell using protein-tagging and immunofluorescence is a fundamental technique in cell biology, whereas in three-dimensional electron microscopy, locating a subunit within a macromolecular complex remains challenging. Recently, we developed a new structural labeling method for cryo-electron tomography by taking advantage of the biotin-streptavidin system, and have intensively used this method to locate a number of proteins and protein domains in cilia and flagella. In this review, we summarize our findings on the three-dimensional architecture of the axoneme, especially the importance of coiled-coil proteins. In addition, we provide an overview of the technical aspects of our structural labeling method.

Characterization of cell response in Chlamydomonas moewusii cultures exposed to the herbicide paraquat: Induction of chlorosis.

The use of herbicides constitutes the principal method of weed control, but the introduction of these compounds into the aquatic environment can provoke severe consequences for non-target organisms such as microalgae. Effects of the widely used herbicide paraquat were assessed on the green freshwater microalga Chlamydomonas moewusii by means of the analysis of its photosynthetic pigment content, using a traditional spectrophotometric technique that provides population bulk measurements, and by means of flow cytometry, which allowed characterizing the microalgal response at a single-cell level. Results obtained reveal that paraquat concentrations above 50nM induce chlorosis in a percentage of microalgal cells depending on herbicide concentration and exposure time, as reflected by a reduced cell chlorophyll autofluorescence and pigment content of the biomass. Cell viability in these cultures was also reduced in a concentration dependent way. The possibility of analysing chlorotic and non-chlorotic subpopulations separately allowed the study of morphological properties and physiological status of both cell types, leading to the conclusion that chlorotic cells are non-viable cells, based on their reduced size and complexity and their inability to be stained in the fluorescein diacetate assay. In the case of non-chlorotic cells, cell viability was reduced with the increase of paraquat concentration. Non-chlorotic cells in these cultures showed an increased size and complexity in comparison with control cells, probably due to a growth inhibition. Chlorophyll fluorescence was the most sensitive parameter since even cells exposed to the lowest concentration assayed, 50nM, although not chlorotic, showed a significantly reduced chlorophyll fluorescence with respect to control cells, reflected also by a reduced chlorophyll content of the biomass.

Evidence for phenotypic plasticity in the Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241.

Life in extreme environments poses unique challenges to photosynthetic organisms. The ability for an extremophilic green alga and its genetic and mesophilic equivalent to acclimate to changes in their environment was examined to determine the extent of their phenotypic plasticities. The Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241 (UWO) was isolated from an ice-covered lake in Antarctica, whereas its mesophilic counterpart C. raudensis Ettl. SAG 49.72 (SAG) was isolated from a meadow pool in the Czech Republic. The effects of changes in temperature and salinity on growth, morphology, and photochemistry were examined in the two strains. Differential acclimative responses were observed in UWO which include a wider salinity range for growth, and broader temperature- and salt-induced fluctuations in F(v)/F(m), relative to SAG. Furthermore, the redox state of the photosynthetic electron transport chain, measured as 1-q(P), was modulated in the extremophile whereas this was not observed in the mesophile. Interestingly, it is shown for the first time that SAG is similar to UWO in that it is unable to undergo state transitions. The different natural histories of these two strains exert different evolutionary pressures and, consequently, different abilities for acclimation, an important component of phenotypic plasticity. In contrast to SAG, UWO relied on a redox sensing and signalling system under the growth conditions used in this study. It is proposed that growth and adaptation of UWO under a stressful and extreme environment poises this extremophile for better success under changing environmental conditions.