Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the me mbrane capacitance (C-m) to monitor membrane changes in protoplasts from Vi cia faba L. guard cells. Confocal images of protoplasts incubated with FM1- 43 (10 mu M) at constant ambient osmotic pressure (pi(o)) revealed in confo cal images a slow internalisation of FM1-43-labelled membrane into the cyto plasm. As a result of this process the relative fluorescence intensity of t he cell interior (f(FM,i)) increased with reference to the total fluorescen ce (f(FM,t)) by 7.4 x 10(-4)min(-1). This steady internalisation of dye sug gests the occurrence of constitutive endocytosis under constant osmotic pre ssure. Steady internalisation of FM1-43 labelled membrane caused a prominen t staining of a ring-like structure located beneath the plasma membrane. Ab rupt elevation of pi(o) by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cyt oplasm concomitant with a decrease in cell perimeter. Within the first 5 mi n the cell perimeter decreased by 7,9%. Over the same time f(FM,i)/f(FM,t) increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm, Combined measurements of C-m and total fluorescence of a protop last (f(FM.p)) showed that an increase in pi(o) evoked a decrease in C-m bu t 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 interna lisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalis ation of giant vesicles (median diameter 2.7 mu m) with FM1-43-labelled mem brane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we concl ude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membr ane surface area.