Intracellular motility and motor proteins in desmids

The proposed project is based on results of earlier investigations, obtained by the group of the applicant during preceding FWF funded projects on function, structure and distribution of cyto-skeletal elements by different cell biological and biochemical methods in unicellular green algae of the family Desmidiaceae. Aim of the new project is to gain general information about regulation of intracellular motion and the participation of motor proteins in plants. This should be achieved through experimental influence and immunocytochemical investigations of the extraordinary motility mechanisms e.g. different nuclear- and chloroplast movements in desmids like Micrasterias, Pleurenterium and Closterium. The following questions are to be addressed during the project: Which motor proteins are responsible for intracellular movements in these algae? Where are these motors localized during development dependent stages of motions? How is the activity of the corresponding motor regulated? Is it possible to gain new insights about the biological relevance of myosins in Micrasterias using molecular biological methods, so far not employed in desmids? Are the microtubule-centers, which have been described during nuclear migration on the basis of electron microscopical investigations microtubuli organizing centers? How is the distribution of actin and myosin altered when the pattern of cytoplasmic streaming is changed? Which motors are involved in mitosis of Micrasterias? Elucidation of these questions will be obtained on the one hand by the use of different inhibitors, microinjection of antibodies, experimental influence on intracellular ion concentration and phosphorylation combined with exact measurements of the motions in vivo. On the other hand immunodetection on Western blots for possible motor proteins like myosin, kinesin, dynein, dynamin with various antibodies and in a second step localization at ultrathin sections of high pressure frozen, freeze substituted cells during different stages of the movements will be carried out. All methods necessary for these investigations have already been employed at the objects, respectively adapted for Desmidiaceae in previous projects. During an international cooperation in a part of the project molecular biological methods shall be employed in desmids for the first time. The demanded equipment for the investigations is mostly available at the Institute of Plant Physiology at the University of Salzburg, an absolute requirement for the fulfillment of this project is the granting of a full time contract of employment-- post-doc position and a full-time laboratory assistance as well as funds for chemicals.

During this three years project it has been investigated which proteins function as "motors" for motility processes such as nuclear and chloroplast translocation or cytoplasmic streaming in different unicellular green algae. By using these algae as model systems insight into force generation during intracellular motility processes in plant cells have been obtained. Different motility processes known to depend on filamentous protein structures such as actin microfilaments or microtubules have been investigated during the course of this project. For example both, the bi-directional nuclear migration during cell growth in the alga Micrasterias and the circular nuclear movement in Pleurenterium during which the nucleus revolves around the cell centre of the fully developed cell with accelerating velocity have been investigated. In addition chloroplast migrations, motility processes during nuclear division as well as force generation during cytoplasmic streaming occurring in different streaming patters in these algae have been studied. A postdoc and a part-time laboratory assistant have been involved in the experiments for this project together with the project leader. Biochemical, immunocytochemical and immunoelectron microscopical investigations revealed participation of two different "motors" in bi-directional nuclear migration of Micrasterias. Kinesin-like proteins are involved in force generation at the microtubule system around the migrating nucleus and myosin-like proteins bind to the co- localized actin filament aggregation. By using specific inhibitors it has been shown in living cells that inactivation of the "kinesin-motor" results in complete cessation, whereas inhibition of the "myosin-motor", though inducing changes in the course of the motion does not entirely stop the migration. In addition, inhibiting the "myosin motor" causes marked alterations in cell shape formation and in cytoplasmic streaming. Extensive digital time-lapse analyses in living cells combined with immunobiochemical experiments indicated an involvement of two different "microtubule motors" in circular nuclear migration of Pleurenterium. Both dynein-like proteins and kinesin-like proteins have been detected in protein extracts of these algae. From the effects of different specific inhibitors on the course of the movement it has been concluded that dyneins are more important for force generation whereas kinesins seem to be responsible for keeping the nucleus at its cytoplasmic track during the revolutions. The results did not provide any hint for participation of an actin filament associated motor in Pleurenterium. Chondramides, a novel class of drugs commercially used as antifungal agents and cytostatica by inducing actin filament polymerization have been used for elucidating actin filament function in intracellular motility processes during the course of the project. The results have given insight into changes of actin-dependent processes by overstabilization of actin filaments and disturbance of the actin turnover. In cooperation with a group at the University of Jena laser forceps and microfocused infrared laser beams have been used in order to obtain information on forces and mechanic properties of organelle tracks during different processes of intracellular motility in desmids.