Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum

Biochimica et Biophysica Acta, 720 (1982) 87-95 Elsevier Biomedical Press

87

BBA 11006

S P I N LABEL S T U D I E S ON O S M O T I C A L L Y - I N D U C E D C H A N G E S IN T H E A Q U E O U S C Y T O P L A S M OF PHAEODA C T Y L U M T R I C O R N U T U M BRIGITI'E SCHOBERT a,* and DEREK MARSH b

" lnstitut fi~r Botanik und Mikrohiologie, l,ehrstuhl fiir Botanik, Techmsche Unit;ersitiit, Arcis.~'tra.vse 10, D-S000 Miinchen 2 and /" A hteilung Spek trosk opw. Max-Plain'k- lnstitut fiir hiophvsikalische Chemic, D 3400 Gi)uingen- Ntk olausherg ( F. R. G. ) (Received June 15th, 1981) ( Revised manuscript received November 2nd, 1981 )

K
The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2 M external Ni z ~ to obtain spectra solely from labels within the cells. From partitioning of the T E M P O spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50-60% in media of high osmotic strength (1.9 osmoi/I). During the accumulation of proline in the cells (8.8 m g / m l packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further additi6n of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 m g / m l packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the T E M P O N E spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3 - 8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5-2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.

Introduction Hyperosmotic stress, imposed on several unicellular algal species can be divided into two phases: (1) During the onset of osmotic strain the cells shrink. The water activity in the suspension medium of the algal cells is reduced by the increased salt concentration, which primarily affects m

* Present address: Department of Physiology and Biophysics, California College of Medicine, University of California, Irxinc, CA 927[7, U.S.A. Abbreviations: TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl: TEMPONE, 2,2,6,6-tetramethylpiperidone-N-oxyl. 0167-4889/82/0000-0000/$02.75 " 1982 Elsevier Biomedical Press

the activity coefficient. Inside the cells the reduced water activity is balanced by a lowered water concentration. Since such changes in the water content are believed to exert an unfavorable influence on the stability of proteins in the cytoplasm, it was of interest to measure the extent of water removal from these cells. (2) The response to osmotic stress in the algal species investigated is synthesis and accumulation of proline. The function of proline during these water stress conditions is not yet proven. Formerly, its role was believed to restore the original cell volume [1], whereas in a new hypothesis its main function is interpreted as increasing the amount of ordered water structures, thus stabilizing the na-

8~

tive conformation of proteins (B. Schobert and R. Hartlieb, unpublished data). Therefore, the relationship between proline concentration and water content during the adaption phase of the cells was a further object of this investigation. Spin label studies on the aqueous cytoplasm of intact cells were introduced by Keith et al. [2], who mostly used T E M P O N E . Due to the narrow lines, the spectra of T E M P O N E are suitable to calculate rotational correlation times of this spin label in the aqueous phase of the cells. We additionally used T E M P O for a second series of experiments. This radical is more hydrophobic than T E M P O N E and partitions between the aqueous and the lipid phase (membranes). It is presumed that the partition coefficient is unchanged under the different conditions investigated. Thus, the amount of spin label in the lipid phase is constant and yields an internal reference to calculate the relative changes in the water content. Both radicals penetrated the cells passively and very rapidly. In order to measure solely the signal from the cell interior, the suspension medium contained NiCI e. Nickel is a paramagnetic ion, yet gives no visible signal in the temperature range investigated [3]. Due to spin-spin interaction the signal of the spin label is broadened. The signal, resulting from a 2 mM concentration of T E M P O or T E M P O N E , was completely quenched in the presence of 0.2 M NiC1 ~. Materials and Methods

Phaeodactylum tricornutum, strain 1090-1a, was obtained from the culture collection of algae, G6ttingen, F.R.G. The cells were cultivated and kept under conditions as described elsewhere [4], The different osmolarities, indicated in the experiments, were obtained by adding NaC1 to the culture solution. The respective osmolar concentrations of the solutions were measured by freezing point depression with an osmometer (Knauer, Berlin, F.R.G,). To determine the proline concentration in the cells, 5 ml aliquots of the different cell suspensions were centrifuged at 2000 × g for 5 rain. The cells containing 0.05M proline in their suspension medium were washed several times with an isoosmolar medium from which proline was omitted.

The pellets were extracted twice with 5 ml 50% ethanol. The supernatants were evaporated to dryness and the proline concentration was determined by the acid ninhydrin method as described earlier [51. For the ESR experiments, glass capillaries ( 1 mm outer diameter, 0.1 mm wall thickness) were sealed at one end and filled with 50 #1 of a solution of 0.4M NiC12 and 4 r a M spin label, In the salt stress experiments NaCI was added to the NiC1, solution to make it isoosmolar with the respective suspension medium. The algal cells were centrifuged at 2000 X g, for 5 rain, resuspended in a small amount of the respective suspension medium and again centrifuged. 90% of the supernatant was removed and the pellet was suspended as a thick slurry. By means of a syringe 50 #1 of the algal paste was filled into theglass capillaries, on top of the NiCI 2 solution. Centrifugation at 2000 X g for 2 min both sedimented the algal suspension and mixed it with the NiCI, solution to result in a final concentration of 0.2 M NiCI ~ and 2 mM spin label. The wet packed cells filled the bottom of the glass capillary to a height of approx. 2 cm. ESR spectra were recorded on a Varian E-12 9 G H z spectrometer with temperature regulation via a nitrogen gas flow system with double-wall quartz dewar. To reduce the reduction rate of the spin label in the cells, centrifugation and ESR measurements were performed at 2°C. The sample capillaries were placed into standard 4 mm quartz ESR tubes containing silicon oil for thermal stability. The temperature was measured using a thermocouple placed in the silicon oil just above the ESR cavity. Spectra were recorded at a microwave power of 10 mW and a modulation amplitude of 0.25 G. Field scan widths of 50 G were used when measuring lineheights and of 2 G when measuring linewidths. Scan times of 0.5 or 1 min were used with minimum filtering. When measuring relative lineheights, the measurements from successive scans were extrapolated to zero time in order to correct for the rate of spin label reduction. When required, spectral digitization and signal averaging was carried out using a PDP 11/10 dedicated computer and Digital Equipment Corp. LPS system with VT-11 display. Further details of the ESR spin label techniques are given by Marsh [7].

89

The T E M P O spin label was synthesized according to Rozantzev and Neiman [8] and the TEMP O N E spin label was obtained from Eastman Kodak, Rochester, NY. From the measurements with the T E M P O spin label, the internal volume, VW, of the cells is given by V~ = (Kn, Vm)" ( h ~ / h m )

(1)

where ( h w / h m ) is the ratio of the peak heights in the spectrum from T E M P O in the internal water phase and in the membrane, and K m, Vm are the partition coefficient of T E M P O into the membrane and the membrane volume, respectively. Thus the ratio, h , , / h m, (see Fig. 1, below), obtained in the presence of external Ni 2+ , was used as a measure of the internal aqueous volume of the cells. Comparison of digitized T E M P O spectra from cells under various conditions of osmotic stress indicated that there was no change in the linewidths, but only in the relative lineheights on osmotic stress. This point was also checked by intersubtractions of the digitized spectra. Rotational correlation times, ~-, for the TEMP O N E spin label were measured from the linewidth coefficients, B and C: B

(AHo/2) (¢ho/h.,-(ho/h

C = ( A H o / 2 ) (¢ho/h+ , + V ~ o / h

~)

(2)

,-2)

(3)

r 2 = a'I [C + a'2B ]

(7)

where the coefficients a~ depend on the magnetic parameters of the spin label and the axis of rotation. Fory-axis rotation the hyperfine'and g-tensors from Jost et al. [10] were used yielding: a~ = 4.95 • 10 9s, a 2 = 0 . 7 7 5 ; a ' 1 = - - 0 . 1 5 6 . 1 0 9s, a ~ = 8.85. By y-axis rotation is implied a preferential enhancement, or de-enhancement, in the rate of rotation around the nitroxide y-axis, relative to that about the other two axes. The allowed values for the I C / B I linewidth ratio depend on the axis of anisotropic rotation. For z-axis rotation: t C / B [ < 1 ; for y-axis rotation: 0.8~1C/B1<~8.8; and for x-axis rotation: 0.5 <~l C/Bt<~ 1.5 (Polnaszek, unpublished data). The correlation time for rotation perpendicular to the axis was obtained simply from ~'± = % and that for rotation around the axis from the expression [9]: = 2

/ (3% -

(8)

For the electron microscopic studies the algae were kept under the conditions described before. The cells were centrifuged for 5rain at 2000 × g and the pelleted cells were fixed with 4% glutaraldehyde and after that with 2% OsO 4. The fixed cells were embedded in a mixture of epon-araldid. The electron micrographs were obtained with a Zeiss EM-9. Results

where h + ~, h o, h _ ~ are the heights of the low-field, central and high-field lines, respectively, and A H 0 is the linewidth of the central line (c.f. Fig. 2. below). For isotropic rotation of the label I C/BI 1, the correlation time was calculated from (Polnaszek, unpublished data and Ref. 9): rff°(s) -- -- 1.22. 10 9. B

(4)

or

•

= 1.19.10 -9. c

(5)

For axial, anisotropic rotation the correlation times were calculated from expressions of the type (Polnaszek, unpublished data and Ref. 9):

%=al[C+a2B

]

(6)

Experiments with TEMPO The relationship between intensity of osmotic stress, water content and proline concentration in the algal cells was investigated in these experiments. As has been described earlier, proline accumulation in these algae is not only dependent on the intensity of osmotic stress, but also on the nitrogen source of the medium [6]. In their natural environment, nitrate is predominantly used as a nitrogen source for proline synthesis. However, the internal proline level can be increased by adding proline or several other amino acids to the suspension medium of the algae. On the opposite side, the water stress-induced proline accumulation is reduced in a nitrogen-depleted medium [6].

90

The spectra of TEMPO from cells of P. tricornutum under different conditions of osmotic

concentration but without proline was less con> plete, 80% only (lIc), than for cells adapted in the presence of proline, approx. 90% (Id). Cells, kept in a medium of high osmolarity, but without nitrogen supply, were able to accumulate only little proline. Consequently, the water content remained low after adaption, at approx. 60% (lle) compared to their controls (lid). Referred to the control cells of the complete medium, the control cells in the nitrate-depleted medium were already under water stress (IId; number given in parenthesis). Compared to the high stress condition lib, the water content in the adapted cells with the nitratedepleted medium (IIe; number in parenthesis) is e v e n lower.

stress are shown in Fig. 1 and the corresponding conditions are described in Table I. The first experiment, referring to Fig. 1, shows that a sudden increase in the osmolarity of the medium by 0.65 osmol/l was accompanied by a water removal of 50% (Ib). An increase by 1.1 osmol/l resulted in a 60% reduction of the cellular water content, which is extremely high (It). Cells which were kept for 3 days in a medium of high osmolarity, containing in addition 0.05 M proline to guarantee a high internal proline level, recovered almost entirely (Id). A second experiment was performed to investigate the effects of different nitrogen sources in the media. In this experiment (which was done after an interval of 4 weeks) the water removal from the cells during the onset of high osmotic strain was 46% (IIb), which is lower than for the cells in the first experiment (lc). The recovery of the adapted cells kept in a medium of high salt

Experiments with T E M P O N E

T E M P O N E gave a signal from the aqueous phase only. The spectrum from control cells of P. tricornutum is shown in Fig. 2A. The spectra of the algae, kept under the conditions described in Table I, differed in their relative

TABLE I RELATIONSHIP BETWEEN THE OSMOLAR CONCENTRATION IN T H E S U S P E N S I O N M E I ) I U M O F P. T R I ( ' O R \ ( TUM, T H E A C C U M U L A T E D PROLINE CONCENTRATION A N D T H E R A T I O O F T I f E L I N E H E I ( ; H T S IN T I l E H I ( } f t F I E L D R E G I O N F R O M T H E M E M B R A N E S I G N A L (h,,,) A N D T H E W A T E R S I G N A l . ( h , , ) O F T H E T E M P O S P I N L A B E L Expt. No.

Condition

Osmolarip, of the m e d i u m (osmoI ' 1 )

(a) N o r m a l m e d i u m (b) N o r m a l m e d i u m + N a C I (t 10 rain) (c) N o r m a l r n e d i m n + NaC1 " ( l - - 10 rain) (d) N o r m a l m e d i u m + N a C I 4 0.05 M p r o l i n c (t=3 days)

0.8

(a) (b) (t (c)

Prolinc content (rag ml p a c k e d celb.)

h , , . ti,,,

~;-

(I.85

2.8(I , 0.14

100

1.45

0.85

1.4

1.90

I).85

1.12 " (I.07

4O

1.90

12.16

2.59 " (I.0,";

')2

0.8

0.98

2.92 • O. 13

100

1.9

0.9:,';

1.58 • 0.14

54

' 0.1

5O

Normal medium N o r m a l m e d i u m 4 NaC1 10 rain) Normal medium ~ NaCI tt 3days) (d) M e d i u m N O a (t-3 days)

1.9

8.79

2.33 : 0. IV

8O

0.g

0. IV

1.94 • (/.28

100 (66)

(e) M e d i u m - N O 3 + N a C 1 (t-- 3 days)

1.9

3.07

1.13 ~ 0.06

58 t391

91 +1

A

o)

control ,,,-~

S b)

15 osm

L__] 5gauss

B -

\.

\

\

c) lg osm

d)

~ ~ ' \ \ \

/

~ adapted

buffer (+cells)

~

stress d

~

odopted '-

!

AH-

L__I

02 gauss

l__l

5 gauss Fig. 1. ESR spectra of TEMPO in cells of P. tricornutum under different conditions of hyperosmotic stress. T-2°C. All sampies contained 0.2 M Ni 2+ in the external medium, which quenched lhc signal from TEMPO outside the cells. (a) Norreal. 0.8 osmol/l: (b) low stress, 1.45 osmol/l: (c) high stress, 1.9 osmol/l: (d) high stress adapted, 1.9 osmol/l+(l./)5M prolinc See Table I and text for further details of the different media and incubations.

l i n e w i d t h s i n d i c a t i n g d i f f e r e n t rates of r o t a t i o n o f the TEMPONE in the c e l l u l a r c y t o p l a s m . T h e h i g h - f i e l d lines o f the v a r i o u s s a m p l e s are g i v e n in Fig. 2B. T h e l i n e w i d t h in the c y t o p l a s m of n o r m a l cells is seen to b e c o n s i d e r a b l y g r e a t e r t h a n t h a t in t h e e x t e r n a l m e d i u m , a n d is i n c r e a s e d f u r t h e r in t h e cells s u b j e c t e d to o s m o t i c stress. In the a d a p t e d cells the l i n e w i d t h is a l m o s t , b u t n o t c o m p l e t e l y ,

Fig. 2. ESR spectra of TEMPONE in cells of P. tricornutum, T 2°C. Samples contained 0.2M Ni 2+ in the external medium, which quenched the signal from TEMPONE outside the cells. A. Whole spectrum from cells in normal medium. B. High-field line from cells under different conditions of hyperosmotic stress. Normal, 0.9 osmol/1; stressed, 1.9 osmol/h adapted, 1.9 osmol/l+0.05 M proline. The spectrum: buffer (+ cells) is in the absence of Ni 2+. See Table I and text for further details of the different media and incubations.

r e s t o r e d to t h a t of the n o r m a l cells. T h e r a t i o s of t h e l i n e w i d t h c o e f f i c i e n t s , I C / B I, a n d the r o t a t i o n a l c o r r e l a t i o n t i m e s d e d u c e d f r o m t h e s p e c t r a o f T E M P O N E in cells u n d e r the vario u s i n c u b a t i o n c o n d i t i o n s are g i v e n in T a b l e II. T h e v a l u e s for n o r m a l cells in t h e a b s e n c e of N i 2 + (first line), w h i c h e s s e n t i a l l y c o r r e s p o n d s to the e x t e r n a l m e d i u m , are v e r y s i m i l a r to t h o s e obt a i n e d in b u f f e r alone. T h e o n l y d i f f e r e n c e was t h a t in the p r e s e n c e of cells, b e c a u s e of the dec r e a s e in s p i n l a b e l c o n c e n t r a t i o n d u e to c h e m i c a l

92

reduction, there was no detectable spin-spin broadening. The JC/BJ ratio is in all cases greater than one, and there appears to be a progressive increase in the ratio from buffer to normal cells, to stressed cells, although the error limits on this measurement are rather large. The values for the effective correlation time for isotropic rotation, "r~!~'~, are given in Table II. The only anisotropic axial rotation with which the data are consistent (assuming the spin tensors of Ref. 10) is rotation around the nitroxide v-axis. The JC/B] ratios lie at the extreme uppermost limit of the range of validity for x-axis rotation. Thus it is possible that the data could just be consistent with .Y-axis rotation with a very high degree of anisotropy. The correlation times for y-axis rotation are also given in Table II. The correlation times are about 3-times greater in the cytoplasm of the cells than in the external medium. They show up to a 2-fold increase on

high osmotic stress, which is only partially alleviated on adaptation for 3 days, the recovery being considerably less than that observed in the cell volume. Cells grown on a medium without nitrogen source display lesser correlation times for T E M P O N E than those grown on normal medium, but still have much greater T E M P O N E correlation times under osmotic stress, even after adaptation for 3 days. Cultivation in a nitrogen-deficient medium probably results in a reduction of cell substance (protein content and also proline concentration, cf. Table I). This could be the reason for the reduction in effective internal viscosity relative to that of non-depleted cells.

Electron microscopy Electron micrographs provided insight, whether there were visible structural alterations in the cells related to the osmotically-induced changes demonstrated in the previous experiments. The condi-

,¢

..... %

7

~

Fig. 3. Electron nlicrograph of a cell of P. tricornutum under normal conditions. Magnification 1:9500.

Fig. 4. Electron micrograph of P. trtcornutum during the onset of high osmotic stress (condition IIb). Magnification 1:9500.

4~ ¸

93

T A B L E II INFLUENCE OF DIFFERENT CONDITIONS OF HYPEROSMOTIC STRESS ON THE ROTATIONAL T I M E S O F T E M P O N E I N T H E A Q U E O U S P H A S E O F P. T R I C O R N U T U M Expt. No.

II

Condition

osmol/l

]C / B[

r(i~° (10 Lt s)

CORRELATION

( $1,/~ L ) '

r[ (10

JLs)

B u f f e r + c e l l s (no NiCI 2)

0.8

1.27

2.9

(/.39

4.6

(a) N o r m a l m e d i u m (b) N o r m a l m e d i u m + NaC1 ( t = 10 min) (c) N o r m a l m e d i u m + NaC1 + 0.05 M proline (t-3days)

0.8

1.58-+0.16

7.3 ~ 0.7

0.22 ~ (/.(15

1S ~: 2

1.9

1.67+0.07

12.1 m0.8

0.18 ~ 0.01

27 + 2

1,9

1.93+0.25

9.6+0.4

(I.14 ~ 0.04

24 ! 2

(a) N o r m a l m e d i u m (b) N o r m a l m e d i u m + NaC1 (t 10 rain) (c) N o r m a l m e d i u m + NaC1 (t 3 days) (d) M e d i u m - N O 3 (t 3 d a y s ) (e) M e d i u m - - N O 3 + N a C 1 ( t = 3 days)

0.8

1.56 ~0.03

8.2+0,6

0.22 ~ 0.01

17 ~ 2

1.9

1.81~0.14

12.0 * 0.9

0.16"0.03

28~3

1.9

1.88-+0.14

I().0 ~ 1.1

0.14 ~ 0.02

24 * 4

0.8

1.76+0.13

6.2~0.1

0.17 - 0.03

14 t I

1.9

1.78+0.06

10.0~ 0.5

0.16":().01

23 + 1

b Fig. 5. Electron m i c r o g r a p h of P. trwornutum, a d a p t e d to high o s m o t i c stress ( c o n d i t i o n IIc). M a g n i f i c a t i o n 1:18000.

Fig. 6. Electron m i c r o g r a p h of P. tricornutum, kept in a nitrate deficient m e d i u m for 3 days ( c o n d i t i o n IId). M a g n i f i c a t i o n 1:9500.

94

tions are the same as those described in Table I, Exp. II. Fig. 3 shows a control cell in normal medium (condition IIa). It is obvious that this algal species does not possess a central vacuole (the hole, shown in this picture, is an artefact). The cell interior is completely filled with cytoplasm and cell organelles. The onset of osmotic strain, corresponding to a water loss of approx. 50% (condition IIb), had clearly visible consequences on the structures of the cells (Fig. 4). Only outlines of the chloroplast were to be seen, the remaining cell interior appeared as a uniformly dense mass. Adapted cells, which accumulated proline (IIc) and regained a water content of approx. 80% of their controls, showed completely intact cell structures. No difference between these cells and their controls was detectable (Fig. 5). The cell structures of the nitrogen-depleted control cells (condition IId) appeared as faint and compact (Fig. 6), simi-

Fig. 7. Electron micrograph of P. trtcornutum, kept in a nitrate deficient medium of high osmolarity for 3 days (condition IIe). Magnification 1:9500.

lar to those of the cells under high osmotic stress (Fig. 4). Adapted cells under high osmotic stress, which contained only little proline (condition IIe) looked completely destroyed and no cellular structures at all were detectable (Fig. 7). On the other hand this low proline accumulation points to the fact that the cell was still alive, yet in a very diseased condition. Discussion The spin label studies showed that during the onset of hyperosmotic stress (by 1.1 osmol/l) approx. half of the water content from cells of P, tricornutum was lost. The electron micrographs revealed that the water loss was exclusively from the cytoplasm and the cell organelles, resulting in a doubled concentration of all dissolved solutes. From these results it seems probable that this water loss from the cells is followed by changes in the cellular water structure. During the accumulation of proline, the water content in the cells increased up to 80% of the controls only. In spite of this incomplete recovery no unfavorable influence on the structural intactness of the cell constituents was detectable. Addition of proline to the concentrated suspension medium resulted in a higher proline concentration and in a further increase in the water content. This artificial condition probably does not further optimize the functional state in the cells. In contrast, prevention of proline accumulation influenced not only the water content, but also the integrity of the cell structures. From the electron micrographs it was obvious that the cell content deteriorated under these conditions. Although it was evident from these investigations that proline accumulation and water content in the cells were related, it is not yet clear whether these volume changes are the primary or rather a secondary event. As regards water structure there are basically two types of solute only. Solutes increasing the structural organization of water exert a stabilizing influence on the native conformations of proteins. In contrast, water-structure breaking solutes lead to a destabilization and unfolding of protein molecules. The effect of both solute species is depen-

95

dent on the molecular structure and on their concentration. The effect of one solute species can be antagonized by a corresponding concentration of solutes belonging to the other group (Schobert and Hartlieb, unpublished data), Investigations with model systems have also revealed a water-structure breaking tendency of several proteins. Therefore, the first result of osmotic stress in the cells is believed to be a change in the state of water, favoring the denaturation of proteins due to the water-structure breaking effect of the enhanced protein concentration. This condition is antagonized by the accumulation of proline, a waterstructure forming solute, and followed by a water influx into the cells. From this point of view the proposed stabilization of proteins is the primary process, whereas the observed volume changes are the secondary process (Schobert and Hartlieb, unpublished data). However, this question cannot be decided with certainty. Even in a complete recovery of the water content the non-stress state of the cells is not restored, because the water activity is lower and corresponds to the water activity of the suspension medium. Furthermore it is obvious from the correlation times (Table II) that the aqueous phase of the stressed or adapted cells differed conisderably from the non-stress condition. The isotropic correlation times may be related to an effective microviscosity, either via the Stokes-Einstein equation "r i~'' = 4 w r l e f f a ' /~3 k T where a 3 A is the molecular radius of T E M P O N E [3], or via calibrations from glycerol-water mixtures given by Keith and Snipes [3]. Using the Stokes-Einstein equation the effective viscosity for the buffer is 1 cP as expected, and for normal stressed and adapted cells is approx. 2.5 cP, 4.0 cP and 3.5 cP, respectively. Using the calibration mixtures, the buffer is again 1 cP but the effective microviscosities of the cyto-

plasm are much larger: approx. 8 cP, 16 cP and 12 cP for normal, stressed and adapted cells. Clearly the effective cytoplasmic microviscosity is considerably higher than that of the external medium, and also appreciably greater than found for example in yeast cells [3]. High osmotic stress induces a 1.5 2-fold increase in effective cytoplasmic viscosity and there is only an approx. 50% recovery on adaption, much less than the recovery in the cellular volume. These latter experiments clearly show that the state of the stressed and adapted cells is different from the non-stressed state.

Acknowledgement We would like to thank Professor Dr. H. Ziegler, Mtinchen, and Mrs. K. Blase for the electron micrographs. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to B.S.

References 1 Kauss, H. (1977) in Int. Rev. of l~iochemistry, Plant Biochemistry II, Vol. 13, pp. 119-140 (Northcote, D.H., ed.), University Park Press, Baltimore 2 Keith, A.D., Arruda, D.H., Ruhlig, L., Snipes, W. and Verbalis, A. (1979) in The Aqueous Cytoplasm (Keith, A.D., ed.), pp. 179-211, Marcel Dekker, New York 3 Keith, A.D. and Snipes, W. (1974) Science 183, 666-668 4 Schobert, B. (1980) Physiol. Plant. 50, 37-42 5 Singh, T.N., Aspinall, D., Paleg, L.G. and Boggess, S.F. (t973) Aust. J. Biol. Sci. 26, 57-63 6 Schobert, B. (1977) Z. Pftanzenphysiol. 85,451-461 7 Marsh, D. (1981) in Membrane Spectroscopy (Grell. E., ed.), pp. 51-142, Springer-Verlag, Berlin 8 Rozantzev, E.G. and Neiman, M.B. (1964) Tetrahedron 20, 131-137 9 Goldman, S.A,, Bruno, G.V., Polnaszek, C.F. and Freed, J.H. (1972) J. Chem. Phys. 56, 716 735 10 Jost, P.C., Libertini, L.J., Hebert, V. and Griffith, O.H. (1971) J. Mol. Biol. 59, 77-98