title: Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.
abstract: A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.
authors.affiliation: 1Institute of Experimental Botany,Academy of Sciences of the Czech Republic,Rozvojová 263,165 02 Prague 6,Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Laňková
authors.affiliation: 2J. Heyrovský Institute of Physical Chemistry,Academy of Sciences of the Czech Republic,Dolejškova 2155/3,182 23 Prague 8,Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Humpolíčková
authors.affiliation: 3Department of Experimental Plant Biology, Faculty of Science,Charles University,Viničná 5,128 44 Prague 2,Czech Republic.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Vosolsobě
authors.affiliation: 1Institute of Experimental Botany,Academy of Sciences of the Czech Republic,Rozvojová 263,165 02 Prague 6,Czech Republic.
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Cit
authors.affiliation: 1Institute of Experimental Botany,Academy of Sciences of the Czech Republic,Rozvojová 263,165 02 Prague 6,Czech Republic.
authors.forename: Jozef
authors.initials: J
authors.lastname: Lacek
authors.affiliation: 1Institute of Experimental Botany,Academy of Sciences of the Czech Republic,Rozvojová 263,165 02 Prague 6,Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Čovan
authors.affiliation: 1Institute of Experimental Botany,Academy of Sciences of the Czech Republic,Rozvojová 263,165 02 Prague 6,Czech Republic.
authors.forename: Milada
authors.initials: M
authors.lastname: Čovanová
authors.affiliation: 2J. Heyrovský Institute of Physical Chemistry,Academy of Sciences of the Czech Republic,Dolejškova 2155/3,182 23 Prague 8,Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Hof
authors.affiliation: 1Institute of Experimental Botany,Academy of Sciences of the Czech Republic,Rozvojová 263,165 02 Prague 6,Czech Republic.
authors.forename: Jan
authors.identifier: http://orcid.org/0000-0002-6719-2735
authors.initials: J
authors.lastname: Petrášek
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D018613:Microscopy, Confocal / Q000379:methods*; D059828:Plant Cells / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods*; D014026:Nicotiana / Q000737:chemistry
chemical_list: D008565:Membrane Proteins
keywords: auxin efflux; auxin influx; fluorescence recovery after photobleaching; plasma membrane; raster image correlation spectroscopy