The beautifully orchestrated regulation of cell shape and volume are central themes in cell biology and physiology. Though it is less well recognized, cell surface area regulation also constitutes a distinct task for cells. Maintaining an appropriate surface area is no automatic side effect of volume regulation or shape change. The issue of surface area regulation (SAR) would be moot if all cells resembled mammalian erythrocytes in being constrained to change shape and volume using existing surface membrane. But these enucleate cells are anomalies, possessing no endomembrane. Most cells use endomembrane to continually rework their plasma membrane, even while maintaining a given size or shape. This membrane traffic is intensively studied, generally with the emphasis on targeting and turnover of proteins and delivery of vesicle contents. But surface area (SA) homeostasis, including the controlled increase or decrease of SA, is another of the outcomes of trafficking. Our principal aims, then, are to highlight SAR as a discrete cellular task and to survey evidence for the idea that membrane tension is central to the task. Cells cannot directly "measure" their volume or SA, yet must regulate both. We posit that a homeostatic relationship exists between plasma membrane tension and plasma membrane area, which implies that cells detect and respond to deviations around a membrane tension set point. Maintenance of membrane strength during membrane turnover, a seldom-addressed aspect of SA dynamics, we examine in the context of SAR. SAR occurs in both animal and plant cells. The review shows the latter to be a continuing source of groundbreaking work on tension-sensitive SAR, but is principally slanted to animal cells.
SummaryThe relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis of energetic considerations. Here, we examine the dynamics of the plasma membrane (PM) in turgid guard cells of Vicia faba by monitoring with confocal microscopy the fate of¯uorescent styryl dyes (FM1-43, FM2-10 and FM4-64). As a second marker, we also observe the retrieval of a¯uorescent chimaera of the K -inward rectifying channel from Arabidopsis thaliana and the green¯uorescent protein (KAT1::GFP). Analysis of cytoplasmic structures, which became labelled by the different styryl dyes, revealed that only FM4-64, the most hydrophobic dye, was a reliable marker of endocytosis, whereas the two other styryl dyes resulted also in an unspeci®c labelling of different cytoplasmic structures including mitochondria. Over some minutes of incubation in continuous presence of these dyes, endocytic vesicles in the cortical cytoplasm beneath the PM were¯uorescently labelled. The identi®cation is based on the observation that the size distribution of these structures is very similar to that of endocytic vesicles obtained from patch-clamp capacitance recordings. Also, these structures are frequently co-labelled with KAT1::GFP. Taken together, the data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K -channel KAT1 from the PM via endocytic vesicles.
Edited by Michael R. Sussman Keywords:Fluorescent nano bead Clathrin-independent endocytosis Clathrin-dependent endocytosis IKA Wortmannin BY-2 a b s t r a c tTo follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20 nm in diameter were internalised rapidly and accumulated partially in compartments also labelled by the endocytic marker FM4-64. Studies in BY-2 cells and protoplasts revealed that larger beads (100 nm) were excluded from uptake into turgescent and plasmolysed cells while protoplasts were able to internalise beads with a diameter of up to 1000 nm. Endocytosis of beads was only partially inhibited by the clathrin-specific inhibitor Ikarugamycin and strongly blocked by wortmannin. These results imply that uptake of beads involves clathrin-dependent and clathrin-independent endocytic mechanisms and supports the hypothesis that clathrin-independent endocytosis plays a general role in plants.
Stomatal movement requires large and repetitive changes in cell volume and consequently changes in surface area. The patch-clamp technique was used to monitor changes in plasma-membrane surface area of individual guard-cell protoplasts (GCPs) by measuring membrane capacitance (C m ), a parameter proportional to the surface area. The membrane capacitance increased under hypoosmotic conditions and decreased after hypertonic treatment. As the speci®c capacitance remained constant, this demonstrates that osmotically induced changes in surface area are associated with incorporation and removal of membrane material. Osmotically induced fusion and ®ssion of plasma-membrane material was not aected by removal of extracellular Ca 2+ . Dialysing protoplasts with very low (<2 nM) or high (1 lM) Ca 2+ had no eect on changes in C m under hypo-and hyperosmotic conditions. However, the rate of change in surface area was dependent on the size of the dierence in osmotic potential applied. The larger the osmotic dierence and thus changes in membrane tension caused by water in¯ux or eux, the faster the change in C m . The results therefore demonstrate that osmotically induced fusion and ®ssion of plasma-membrane material in GCPs are Ca 2+ -independent and modulated by membrane tension.During stomatal movement, guard cells undergo large changes in cell volume and surface area over a period of minutes, as a result of changes in ion accumulation and consequently water in¯ux or eux. So far, little is known about the mechanism underlying osmotically induced changes in plasma-membrane area. Studies of the mechanical properties of the plant cell membrane have demonstrated that the maximum possible elastic stretching of membranes is only about 2% (Wolfe and Steponkus 1983). During stomatal movement, changes in surface area of about 40% occur (Raschke 1979). These large changes cannot be the result of stretching of the existing membrane, but require the insertion of new membrane material into the plasma membrane. Kell and Glaser (1993) postulated that water in¯ux causes an increase in membrane tension which results in an increase in exocytotic activity, a process which they proposed was regulated by membrane voltage and extracellular and intracellular Ca 2+ -activity.The formation of endocytotic vesicles in protoplasts from rye leaves after hyperosmotic treatment suggests that osmotic contraction of protoplasts is due to endocytosis and not membrane folding (Gordon-Kamm and Steponkus 1984). Similar results were found in guard-cell protoplasts (GCPs) after osmotically induced shrinkage (Diekmann et al. (1993). However, endocytotic vesicles were not observed in intact guard cells.To investigate the mechanisms involved in osmotically induced changes in the surface area in GCPs, I used the whole-cell patch-clamp technique. This technique allows the measurement of changes in surface area at a high time resolution by measuring the membrane capacitance (C m ), as well as controlling the cytosolic composition by dialysing the cell via the patch pip...
Stomata in the epidermis of photosynthetically active plant organs are formed by pairs of guard cells, which create a pore, to facilitate CO2 and water exchange with the environment. To control this gas exchange, guard cells actively change their volume and, consequently, surface area to alter the aperture of the stomatal pore. Due to the limited elasticity of the plasma membrane, such changes in surface area require an exocytic addition or endocytic retrieval of membrane during stomatal movement. Using confocal microscopic data, we have reconstructed detailed three-dimensional models of open and closed stomata to precisely quantify the necessary area to be exo- and endocytosed by the guard cells. Images were obtained under a strong emphasis on a precise calibration of the method and by avoiding unphysiological osmotical imbalance, and hence osmocytosis. The data reveal that guard cells of Vicia faba L., whose aperture increases by 111.89+/-22.39%, increase in volume and surface area by 24.82+/-6.26% and 14.99+/-2.62%, respectively. In addition, the precise volume to surface area relationship allows quantitative modeling of the three-dimensional changes. While the major volume change is caused by a slight increase in the cross section of the cells, an elongation of the guard cells achieves the main aperture change.
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