Osmoregulation in Chlamydomonas reinhardtii
Osmoregulation in green freshwater flagellates involves contractile vacuoles (CVs). CVs also serve as sensor for the water status of a cell and allows studying osmoregulation in living cells at the cellular level. On the one hand we are therefore studying the cell biology of the CV, which might even shed light on the evolution of the plant vacuolar system (see Becker 2007, Hoef-Emden and Becker 2009, 2013 for a detailed discussion). On the other hand we want to use the CV as sensor to investigate basic mechanisms of osmoregulation (sensing the environment, signal transduction) at the cellular level.
Towards this aims, we have now identified new proteins involved in CV function using an insertional mutagenesis approach (e.g. sec6, a component of the exocyst complex is deleted in the Osmo75 mutant, Komsic-Buchmann et al. 2012, other mutants are currently characterized). In addition, we are now establishing the proteome of the CV. A membrane fraction containing only the CV marker protein MIP1 (an aquaporin) has been isolated and is currently investigated in collaboration with Prof. Dr. M. Schroda (University of Kaiserslautern).
We are especially interested in the water expulsion process, since in contrast to the ciliate system, no water expulsion pore has so far been found in Chlamydomonas. We plan to investigate the structure of the CV – plasma membrane interface during systole (water expulsion step) using TIRF microscopy (in collaboration with Prof. Dr. K.-F. Lechtreck, University of Athens, Georgia, USA) and electron tomography (in collaboration with Prof. Dr. U. Lütz-Meindl, University of Salzburg).
In addition, using the CV as a sensor we have now established a basic concept for adaptation of Chlamydomonas cells to environments of different osmotic strength (Komsic-Buchmann et al. 2014). Chlamydomonas cells use mainly the size of the CV to compensate increased water uptake during cell growth, and the contraction interval to adapt to media of different osmotic strength. Currently we analyze the transcriptomes of Chlamydomonas adapted to media of different osmotic strength to identify additional proteins involved in osmoregulation. In the future we want to develop this project into a systems biology approach of CV function, with the aim to use CV function as a model system to understand quantitative aspects of membrane fusion in the endomembrane system and exocytosis.