Plasma membrane domains in characean green algae

The plasma membranes are thought to display a lateral compartmentalization into distinct domains. Among them lipid rafts are considered to be small (10-200 nm in diameter), highly dynamic regions which are enriched in sterols and sphingolipids and harbor specific proteins. Lipid rafts have been the subject of numerous studies in animal and fungal cells which showed their involvement in a multitude of processes, including endocytosis, secretion, signal transduction, development and pathogen perception. Far less is known about lateral organization of biological membranes in plant and algae. In internodal cells of the characean green algae, stable plasma membrane domains with a diameter of up to one m can be visualized by the fluorescent endocytic tracers FM 1-43 and FM 4-64 and by the sterol-specific probe filipin suggesting clustering of lipid rafts. During the course of this project we want to verify or discard the following hypotheses: (1) FM- and filipin-stained plasma membrane domains in characean internodal cells have a protein and lipid composition similar to that of lipid rafts. (2) The large plasma membrane domains develop by fusion of small sterol-enriched areas and correspond to elaborate plasma membrane invaginations (charasomes) in internodal cells of the genus Chara but not in Nitella. (3) Plasma membrane domains are involved in local proton extrusion and carbon uptake required for efficient photosynthesis. For our study we combine biochemical and molecular biological methods with confocal laser scanning microscopy and electron microscopy. The research proposed in this application will extend our knowledge about basic processes in membrane compartmentation and transmembrane transport. It will help to substantiate key findings obtained with other organisms but could also open up new possibilities of critically testing the membrane raft hypothesis.

The plasma membrane harbors numerous transporters for the exchange of ions and metabolites. The green alga Chara australis increases the plasma membrane area by the formation of convoluted plasma membrane domains. These charasomes are involved in the acidification of the cell surface which facilitates carbon uptake required for photosynthesis.Our finding that charasomes can be stained by fluorescent plasma membrane dyes in live cells greatly expedites studies about the formation and degradation of these organelles. We show that charasome formation requires the existence of a pH banding pattern, i. e. a regular alternation of alkaline and acid bands along the cell surface which is probably generated by local activation/deactivation of proton pumps in the charasome-free, smooth plasma membrane. Once charasomes are formed, their distribution determines the pH banding pattern under steady state conditions because of the increase in membrane area equipped with proton pumps. Interestingly, charasome formation requires photosynthesis but charasomes can also develop far apart from chloroplasts suggesting the involvement of a mobile signal. We also show that the charasome tubules are derived from the trans Golgi network, an organelle involved both in endo- and exocytosis. Growth of charasomes requires fusion of trans Golgi network-derived vesicles with the plasma membrane and local inhibition of clathrin-dependent membrane recycling. Drug treatments suggest that the local inhibition of membrane recycling occurs via inactivation of PIP3 and/or PIP4 kinases, enzymes involved in signal transduction. Darkness- or drug-induced arrest of photosynthesis causes the release of this inactivation so that charasome membranes can be degraded via clathrin coated vesicles. The analysis of the cortical cytoskeleton indicates that the inner surface membrane of charasomes is depleted of actin and microtubule nucleators or binding proteins. Consistently, we show that charasome degradation can occur without the participation of the cytoskeleton. Biochemical and molecular biological data obtained during the course of this project suggest that C. australis possesses two isoforms of the plasma membrane proton pump and that only one, probably the charasome proton pump, is degraded upon dark incubation. The analysis of C. australis clathrin, a protein involved in the formation and degradation of charasomes, revealed interesting peculiarities in the sequences of the light chains. Finally, we provide the first descriptions of ARA6 and ARA7 in green algae, two RAB5 GTPases involved in vesicular trafficking. The charasome- and trans Golgi network-associated ARA6 is possibly involved in charasome formation.Our findings indicate that the study of charasomes yields not only important information about cellular strategies to improve photosynthesis in an aquatic environment but also that charasomes are an interesting model to study endo- and exocytosis.