Download Endocytosis of GFP tagged potassium channel KAT1 against high

Endocytosis of GFP tagged potassium channel KAT1
against high turgor pressure in guard cells of Vicia faba
Tobias Meckel, Annette C. Hurst, Ulrike Homann, Gerhard Thiel
Darmstadt University of Technology, Schnittspahnstrasse 3, 64287 Darmstadt, Germany
E-mail: [email protected]
KEY WORDS:
endocytosis, single vesicles, living cells, high turgor, FM4-64, GFP quantification
ABSTRACT:
Pairs of guard cells form pores in the epidermis of photosynthetic active plant organs to facilitate
CO2 and water exchange with the environment. In order to control this gas exchange, guard cells
actively change their volume – roughly by a factor of 2 – to alter the aperture of the stomatal
pore. These volume changes are achieved by an uptake or release of K+ through K+-selective
channels followed by the respective osmotic water fluxes. However, since biological membranes
only have a limited elasticity of about 2-5 %, the surface area of the guard cell plasma membrane
(PM) needs to accomplish these volume and surface changes by addition or retrieval of
membrane to or from the PM. With patch-clamp capacitance measurements we demonstrated that
guard cell protoplasts achieve surface area changes by exo- and endocytosis of vesicles with a
mean diameter of around 300 nm. In parallel with the changes in membrane area also the number
of potassium channels is adjusted with the result that the K+-channel concentration is kept nearly
constant during excursions of the surface area. Collectively this suggests that these K+-channel
proteins are delivered or retrieved from the PM by vesicular transport.
These measurements have been carried out on cells with removed cell wall and consequently no
turgor pressure, however, the relevance of endocytosis in plants against high turgor pressure has
frequently been questioned on the basis of energetic considerations. To investigate this process
in highly turgescent guard cells, we examine the dynamics of the PM by monitoring with
confocal microscopy the fate of the fluorescent styryl dye FM4-64. As a second marker we also
observe the retrieval of a fluorescent chimera of the K +-channel KAT1 (KAT1::GFP).
With this approach we are able to show that turgescent guard
cells undergo vigorous constitutive endocytosis (Fig.1)
Moreover, a subset of single vesicles gets labeled with the
externally supplied membrane marker and the intracellular
produced KAT1::GFP. Consequently these vesicles carry K+channels, which had already been delivered to the PM and are
now retrieved via endocytosis. These data provide strong
evidence for an endocytic retrieval of PM located K+-channels
also in turgescent guard cells. Calculations based on patch10 µm
1 µm
clamp data show that each of the endocytosed vesicles
contains on average one potassium channel. However, because
Fig.1: Endocytosis of FM4-64
vesicles with a single GFP labeled protein are unlikely to be
detected with a conventional CLSM-setup, we hypothesize, that at least two different vesicle
pools exist in guard cells. One of which does not carry any K+-channels and is required to adjust
the surface area of the PM, the other carries clustered K+-channels with each vesicle to and from
the PM. Quantifications of the number of GFP-tagged KAT1-channels in single vesicles are
currently under way.