Osmotically induced Exocytosis and Endocytosis in Guard Cell Protoplasts

Investigation of Exocytosis and Endocytosis by Patch-clamp Capacitance Measurements

Osmotically driven and pressure-driven changes in surface area of guard cell protoplasts have been investigated extensively by patch-clamp capacitance measurements. This technique allows the examination of exocytosis and endocytosis in single living protoplasts. Recordings can be performed with a resolution that is high enough to detect fusion and fission of vesicles with a diameter as low as 60 nm and a temporal resolution on the order of some 10 ms (Neher and Marty 1982; Kreft and Zorec 1997). Measurements of exo-cytosis and endocytosis via capacitance recordings are based on the fact that a biological membrane can be viewed as a capacitor. The capacitance of this ca-pacitordepends on its surface area. Fora numberofplant protoplasts, including guard cells, such a linear relationship between membrane capacitance and surface area has been demonstrated, and a specific capacitance (capacitance per unit surface area of membrane) between 7.5 and 8.1 mFm-2 has been calculated (Zorec and Tester 1992;Thieletal. 1994; Carroll etal. 1998; Homann 1998). Under the valid assumption that the specific capacitance remains constant during the time of observation, the changes in plasma membrane surface area resulting from exocytic and endocytic activity can be monitored by measuring membrane capacitance. In principle, the membrane capacitance of a cell is determined from the current measured in response to a voltage command which is applied to the cell (Gillis 1995; Homann and Tester 1998; Thiel et al. 2001). When measurements are carried out in the so-called whole-cell configuration, the cytoplasm is rapidly dialysed by the pipette solution. This allows control of cytoplasmic composition and the introduction of potential regulators of exocytosis and endocytosis.

The high temporal resolution and the potential of manipulating the cy-tosolic composition via the patch pipette make patch-clamp capacitance measurements a powerful tool for studying exocytosis and endocytosis. The main limitations of patch-clamp capacitance measurements are the general limitations of patch-clamp measurements: the requirement of an accessible membrane (measurements are generally carried out on protoplasts) and possible loss of endogenous substances that affect exocytosis and endocytosis during cell dialysis.

Membrane Tension as a Stimulus for Exocytosis and Endocytosis in Guard Cell Protoplasts

Patch-clamp capacitance measurements have been used to study osmotically induced surface area changes of guard cell protoplasts. Results from these measurements demonstrated that osmotically induced swelling and shrinking of guard cell protoplasts are associated with incorporation and removal of membrane material into and out of the plasma membrane, respectively (Homann 1998). High-resolution capacitance measurements which allow the detection of single exocytic and endocytic events revealed fusion and fission of single vesicles with a median diameter of 300 nm during osmotically induced changes in surface area (Homann and Thiel 1999). However, for most of the recordings the change in surface area occurred without resolvable ex-ocytic or endocytic events. This was most likely due to fusion and fission of vesicles below the resolution limit (less than 200 nm). Hence, vesicles with a diameter below 300 nm almost certainly also contribute significantly to the increase and reduction of the surface area. The vesicular retrieval of plasma membrane material during osmotically induced shrinking of guard cell protoplasts was confirmed by imaging of guard cell protoplasts stained with the fluorescent membrane probe FM1-43 (Kubitscheck et al. 2000). Confocal images of protoplasts incubated with FM1-43 at constant ambient osmotic pressure revealed a slow internalisation of FM1-43 labelled membrane into the cytoplasm without changes in cell perimeter. This indicated the occurrence of constitutive endocytosis (Samaj, this volume). Hyperosmotic treatment of protoplasts led to a rapid internalisation of FM1-43 fluorescence into the cytoplasm and a corresponding decrease in cell perimeter. Only occasionally was the shrinking of protoplasts associated with the internalisation of large vesicles (median diameter 2.7 ^m). Most hyperosmotically treated protoplasts showed a diffuse distribution of the FM1-43 label throughout the cytoplasm without any resolvable vesicular structures. This led to the conclusion that endocytosis of small vesicles below the resolution limit accommodates for the osmotically induced decrease in surface area (Kubitscheck et al. 2000).

Osmotically induced fusion and fission of plasma membrane material was not affected by changes in intracellular or extracellular Ca2+ concentration (Homann 1998); however, the rate of change in surface area was dependent on the size of the difference in the osmotic potential applied. The larger the osmotic difference the faster the change in surface area (Homann 1998). This strongly indicated that changes in membrane tension resulting from osmotically induced water influx or efflux can modulate exocytosis and endocytosis in guard cell protoplasts. Exocytic and endocytic activity of guard cell protoplasts could also be modulated by application of hydrostatic pressure, confirming the hypothesis of tension-sensitive surface area regulation (Fig. 1; Bick et al. 2001).

Fig. 1 Insertion and retrieval of K+ channels during pressure-stimulated exocytosis and endocytosis in a guard cell protoplast A Application of a hydrostatic pressure (AP) via the patch-pipette resulted in an increase and a decrease in membrane capacitance (Cm), which corresponds to a change in surface area via exocytosis and endocytosis. At the time points indicated by different symbols a voltage pulse of - 140 mV was applied and the resulting current passing through the K+ inward rectifier channel was measured. B Linear correlation between changes in the current passing through the K+ inward rectifier channel (I) and the membrane capacitance (Cm). The current was measured at the time points indicated in A

Fig. 1 Insertion and retrieval of K+ channels during pressure-stimulated exocytosis and endocytosis in a guard cell protoplast A Application of a hydrostatic pressure (AP) via the patch-pipette resulted in an increase and a decrease in membrane capacitance (Cm), which corresponds to a change in surface area via exocytosis and endocytosis. At the time points indicated by different symbols a voltage pulse of - 140 mV was applied and the resulting current passing through the K+ inward rectifier channel was measured. B Linear correlation between changes in the current passing through the K+ inward rectifier channel (I) and the membrane capacitance (Cm). The current was measured at the time points indicated in A

Tension-sensitive exocytosis and endocytosis have been implicated to be important components of surface area regulation not only in plant cells but also in animal cells (Morris and Homann 2001). Cells seem to detect and respond to deviations from a membrane tension set point. An increase in membrane tension above this set point results in addition of membrane material to the plasma membrane until the membrane tension set point is restored. Upon a decrease in membrane tension, excess plasma membrane material is retrieved to reestablish the resting tension. The mechanisms by which cells sense changes in membrane tension are not yet known. Neither have the signal transduction pathways been identified which link changes in membrane tension to changes in the rate of exocytosis or endocytosis.

Other important but yet unresolved questions in tension-modulated surface area changes are the origin and quality of the membrane material which is added and removed in the course of this process. In guard cells the addition of membrane material could often be detected immediately after application of hydrostatic pressure (Fig. 1 in Bick et al. 2001). This indicates the existence of an intracellular reservoir of membrane material which is instantly available for incorporation into the plasma membrane. Guard cell protoplasts can undergo several cycles of swelling and shrinking. It is therefore most likely that the membrane material that is retrieved from the plasma membrane during surface area decrease is reused in subsequent cell swelling.

Insertion and Retrieval of Plasma Membrane K+ Channels

Two types of plasma membrane K+ channels play a central role in the accumulation and loss of K+ during opening and closing of the stomatal pore. One, a K+ inward rectifier, conducts K+ uptake, the other, a K+ outward rectifier, mediates K+ discharge from guard cells. The density of these channels in the plasma membrane is an important factor for determining the transport rate across the membrane and thus for the cell function. The channel density can be modulated by exocytotic insertion and endocytic retrieval of ion channels, which in turn alters the membrane conductance.

Parallel measurements of membrane conductance and membrane capacitance provide a valuable tool to study the insertion and retrieval of ion channels. While the membrane capacitance is proportional to the cell surface area and monitors excursions in the plasma membrane area the membrane conductance provides information on the activity of plasma membrane ion channels.

When guard cell protoplasts are subject to swelling the membrane capacitance and the current passing through the K+ inward rectifier increase nearly in parallel (Fig. 1). A decrease in surface area is accompanied by a corresponding reduction in K+ inward current (Fig. 1). This implies that the vesicular membrane which is inserted and retrieved during pressure-driven changes in surface area carries active K+ channels. Detailed measurements of membrane capacitance and conductance in guard cell protoplasts demonstrated that osmotically driven and pressure-driven changes in surface area of guard cell protoplasts are associated with insertion and removal of K+ inward and K+ outward rectifiers (Homann and Thiel 2002; Hurst et al. 2004).

From the parallel measurements of changes in conductance and capacitance the number of K+ channels added for a given increase in surface area can be estimated. This led to the conclusion that only about one of nine vesicles, which fuse with the plasma membrane, contains a K+ channel (Homann and Thiel 2002). Similar results were obtained for endocytic vesicles.

The observation that the increase in surface area and the incorporation of K+ channels occurs immediately (i.e. within seconds) upon pressure stimulation (Fig. 1; Homann and Thiel 2002) means that the vesicles containing the active K+ channels are already present in the cell, probably in a pool close to the plasma membrane. Incorporation and retrieval of K+ channels can be observed even after several cycles of swelling and shrinking. It therefore seems likely that endocytosed K+ channels are retrieved back into the pool of "ready-to-fuse" vesicles.

During swelling of guard cell protoplasts an increase in plasma membrane channel density occurred (Homann and Thiel 2002). This increase was reversed during subsequent shrinking of protoplasts (Hurst et al. 2004). The results implied that the density of the K+ channels in the membrane of ex-ocytic and endocytic vesicles is higher (by a factor of about 10) compared with the channel density of the plasma membrane. (Homann and Thiel 2002; Hurst et al. 2004). Since it is unlikely that channels are first concentrated in small areas before they are retrieved by endocytosis, this strongly suggests that channels form stable clusters in the plasma membrane and remain in clusters during exocytosis and endocytosis. The formation of channel clusters is also supported by analysis of fluorescence images of turgid guard cells expressing the K+ inward rectifier KAT1 fused to green fluorescent protein (GFP) (see later; Meckel et al. 2004).

The available data also have some implications for the physiology of guard cell movement. Modulators of ion channel activity in guard cells typically have opposite effects on the K+ outward and K+ inward rectifiers owing to their different role in the regulation of the stomatal pore. They promote either uptake (K+ inward rectifier) or release (K+ outward rectifier) of K+, resulting in opening or closing of the pore, respectively (Blatt 2000). In the case of vesicle-mediated insertion or retrieval of K+ channels, however, the inward and outward rectifiers change in parallel. This implies that this process has nothing to do with the physiological regulation of K+ channel activity in the context of guard cell function. The observation that the channel density changes during swelling and shrinking of guard cell protoplasts suggests that osmotically induced insertion and retrieval of ion channels act as a mechanism for a reversible increase of the ion channel density without discriminating between K+ inward and outward rectifiers. However, in the physiological situation of stomatal movement the process of vesicle delivery and retrieval to and from the plasma membrane may be more complex, involving distinct mechanisms of vesicle sorting. In this context it is feasible to speculate that a more balanced insertion of K+ channels into the plasma membrane may occur. This can then serve as a mechanism for homeostasis of channel density during variations in surface area.

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