Tracking single molecules in the live cell plasma membrane-Do's and Don't's.
In recent years, the development of fast and highly sensitive microscopy has changed the way of thinking of cell biologists: it became more and more important to study the structural origin for cellular function, and industry turned its attention to the improvement of the required instruments. Optical microscopy has now reached a milestone in sensitivity by resolving the signal of a single, fluorescence-labeled biomolecule within a living cell. First steps towards these pioneering studies were set by methods developed in the late eighties for tracking single biomolecules labeled with fluorescent latex spheres or gold-particles. Meanwhile, a time-resolution of milliseconds for imaging weakly fluorescent cellular structures like small organelles, vesicles, or even single molecules is state-of-the-art. The advances in the fields of microscopy brought new cell biological questions into reach. The investigation of a single fluorescent molecule-or simultaneously of an ensemble of individual molecules-provides principally new information, which is generally hidden in ensemble-averaged signals of molecules. In this paper we describe strategies how to make use of single molecule trajectories for deducing information about nanoscopic structures in a live cell context. In particular, we focus our discussion on elucidating the plasma membrane organization by single molecule tracking. A diffusing membrane constituent--e.g. a protein or a lipid--experiences a manifold of interactions on its path: the most rapid interactions represent the driving force for free diffusion; stronger or correlated interactions can be frequently observed as subdiffusive behavior. Correct interpretation of the data has the potential to shine light on this enigmatic organelle, where membrane rafts, protein microdomains, fences and pickets still frolic through the text-book sketches. We summarize available analytical models and point out potential pitfalls, which may result in quantitative or three even qualitative misinterpretations.
Wieser
Stefan
S
Biophysics Institute, Johannes-Kepler-University Linz, Altenbergerstr.69, A-4040 Linz, Austria.
Schütz
Gerhard J
GJ
D000465:Algorithms; D002462:Cell Membrane / Q000502:physiology*; D004058:Diffusion; D008563:Membrane Lipids / Q000502:physiology*; D021962:Membrane Microdomains / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D015398:Signal Transduction
D008563:Membrane Lipids; D008565:Membrane Proteins