Imaging individual events on biomembranes: Study of molecular association and structural dynamics of ion channels by means of genetic tagging and single molcule two-photon fluorescence microscopy

The performance of cellular processes in general requires the coordinated action of a multitude of biomolecules. For describing such processes, it is important to study the sequence of isolated events. Such events can be the association of all involved molecules, their interaction with each other, and the final split of the complex. Using conventional techniques, it is difficult to record the chronology of cellular processes. The development of new techniques was required , based on the study of individual molecules involved in the process. Such methods have been already applied for the study of isolated biomolecules, the final application to the study on living cells, however, had to be accomplished yet. In particular, high background signal of cellular samples complicated such investigations. It was the aim of this project to study single biomolecules in cellular environment during their action. First, we applied "Single Dye Tracing", a technique developed at the Biophysics Institute, which allows to follow the motion of individual fluorescence labeled biomolecules in time. As a first demonstration of the capabilities of this technique, the mobility of individual lipid molecules within the plasma membrane of living smooth muscle cells was studied. All cell membranes are composed of lipids; however, interaction of a variety of different types of lipids defines the microstructure of the membrane. We could show, that some types of lipids are confined to specific domains within the cell membrane, with a typical size of ~0.7m; other types of lipids do not sense those regions. Such domains have become a central area of scientific research, since they modulate the function of proteins, thereby having a dominant regulatory role in cellular metabolism. While this ultrasensitive method of Single Dye Tracing is limited to the study of cells which show a background signal much smaller than the signal of a single fluorophore, the alternative method using two-photon excitation yields high quality images of single molecules for almost all cellular systems. During this project, an apparatus was build up based on two-photon microscopy, and its applicability was validated for the ultrasensitive study of single biomolecules. We could show, that the mobility of individual lipid molecules can be recorded in biomembranes. In addition, the distribution of membrane-proteins could be mapped at high resolution.