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Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy

Published online by Cambridge University Press:  03 March 2016

Martina Laňková ,
Jana Humpolíčková ,
Stanislav Vosolsobě ,
Zdeněk Cit ,
Jozef Lacek ,
Martin Čovan ,
Milada Čovanová ,
Martin Hof  and
Jan Petrášek Open the ORCID record for Jan Petrášek [Opens in a new window]
Martina Laňková
Affiliation:
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic
Jana Humpolíčková
Affiliation:
J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 2155/3, 182 23 Prague 8, Czech Republic
Stanislav Vosolsobě
Affiliation:
Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
Zdeněk Cit
Affiliation:
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
Jozef Lacek
Affiliation:
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
Martin Čovan
Affiliation:
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic
Milada Čovanová
Affiliation:
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic
Martin Hof
Affiliation:
J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 2155/3, 182 23 Prague 8, Czech Republic
Jan Petrášek*
Affiliation:
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
*
*Corresponding author.[email protected]
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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.

Keywords

raster image correlation spectroscopyfluorescence recovery after photobleachingauxin influxauxin effluxplasma membrane
Type
Special Issue on Imaging Plant Biology
Information
Microscopy and Microanalysis , Volume 22 , Supplement 2 , April 2016 , pp. 290 - 299
Copyright
© Microscopy Society of America 2016 

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