Analysis of mucilage formation in algae by energy filter TEM

Mucilage production by algae has been subject to biological investigations for both environmental and economic reasons. Despite their negative impact on humans and their economic interests, slime sheaths secreted by algae provide a variety of ecological advantages for the respective organism. Mucilage investments may protect algae against unfavorable environmental conditions such as drought, low temperature or UV-irradiation and may have important function in algal reproduction, motility, and interaction of organisms in aquatic systems. Whereas numerous studies have dealt so far with chemical composition of mucilage as well as with environmental factors inducing slime production only little is known about the intracellular basis of mucilage formation and the mechanisms by which mucilage excretion may be achieved. The present project is intended to study structural aspects of intracellular mucilage formation and excretion in two particular groups of fresh water green algae (Desmidiaceae and Mesotaeniaceae) by means of new energy filtering techniques in transmission electron microscopy (TEM). This method for the first time allows depiction of biological ultrastructure without using heavy metals for contrast enhancement and thus yields more reliable and comparable results than conventional TEM. The goals of the project may be subdivided into three topics: 1. to clarify how mucilage formation is achieved inside the cells, how it is packaged into vesicles, which vesicle populations are responsible for mucilage transport and how vesicle trafficking to the sites of excretion is achieved. 2. to analyze how efficient mucilage excretion may be accomplished at the cell surface. The structural basis for two different excretion mechanisms shall be investigated by comparing two algal systems: excretion through particular highly differentiated cell wall pores on the one hand and mucilage extrusion through a multi-layered cell wall at the other hand. Development and possible opening and closing mechanisms of the cell wall pores will be analyzed. 3. to find out how ecologically relevant environmental conditions such as low temperature and increased UV-irradiation influence intracellular mucilage formation and excretion. Algae collected at their natural habitats during wintertime as well as kept at low temperature under laboratory conditions will be studied. In cooperation with the GSF-Research Center (Germany) the impact of UV- irradiation of different wavelengths produced in a sun-simulator on mucilage formation will be investigated.

Understanding of intracellular mucilage production and excretion is important both for ecological and economic reasons. Despite their negative impact on human health and economic interests, slime sheaths secreted by algae provide a variety of ecological advantages for the algae themselves. They may protect algae against unfavourable environmental conditions, such as drought or extreme temperature, may establish a microclimate for reproduction, and are important for motility and interaction of organisms in aquatic systems. This project was intended to analyse mucilage production, to follow the mucilage secretion pathway and to study mechanisms of fast mucilage release in two different unicellular fresh water green algae by means of modern electron microscopy and by immuno and biochemical techniques. Moreover, extreme environmental conditions were simulated in the laboratory and their impact on mucilage production was investigated. Whereas the cell biological studies were done at the University of Salzburg experiments with UV simulation were carried out at the GSF Research Centre in Munich, Germany and biochemical analyses were performed in a co-operation with the University of Rouen, France. Two different and alternative modes of mucilage excretion were shown by digital time-lapse video sequences: mucilage release all over the cell surface which leads to huge protective mucilage investments and excretion of mucilage from one of the cell poles which allows directed movement of the cells in respect to light. Intracellular compartments were defined as "mucilage" vesicles by raising an antibody against external algal mucilage and using it at the electron microscopic level. Their intracellular production and maturation was investigated and it was studied at which areas of the cell they become excreted. In contrast to earlier reports in the literature it could be shown that pores in the cell walls of the investigated algae are not the only mucilage excreting organs and that mucilage can be also released directly through the cell wall. By use of new electron microscopic techniques and adaptation of the respective preparation methods for algal cells organelle interactions and digestions processes of organelles and mucilage vesicles could be visualized for the first time. It was shown that low temperature around the freezing point did not stop intracellular mucilage excretion which allows the cells to cover themselves with mucilage e.g. in winter at their natural habitats. Different inhibitors were used to influence intracellular conditions and to find out how intracellular mucilage production is achieved.