Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm |
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| Authors: | Ulrich Kubitscheck Ulrike Homann Gerhard Thiel |
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| Institution: | Institut für Medizinische Physik und Biophysik, Westf?lische Wilhelms-Universit?t, Robert Koch Str. 31, 48149 Münster, Germany, DE
Albrecht-von-Haller Institut für Pflanzenwissenschaften, Biophysik der Pflanzen, Untere Karspüle 2, 37073 G?ttingen, Germany, DE
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| Abstract: | The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (πo) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process
the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 × 10−4 min−1. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure.
Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the
plasma membrane. Abrupt elevation of πo by 200 mosmol kg−1 caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant
with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in πo evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and
internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally
associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases,
osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude
that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically
driven decrease in membrane surface area.
Received: 4 May 1999 / Accepted: 19 August 1999 |
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| Keywords: | : Endocytosis – FM1-43 fluorescence internalisation – Guard cell – Membrane capacitance – Osmotic shrinking – Vicia (guard cell protoplast) |
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