Bioscience Reports, Vol. 6, No. 11, 1986
Electrorotation of Lymphocytes .........the
Influence of Membrane Events and Nucleus
Heiko Ziervogel, Roland Glaser 2, Dieter Schadow 1 and
Stephan Heymann t
Received December 9, 1986
KEY WORDS: electrorotation;lymphocytes;mitogenicstimulation;nuclear envelope.
Electrorotation--the spin of cells in rotating high frequency electric fields--has been
used to investigate properties of human peripheral blood lymphocytes. The rotation
spectra of lymphocytes deviate from those of single shell spheres. The deviations are
caused by the electrical properties of the nucleus in the cell interior.
Electrorotation allows the distinction between successfully stimulated
lymphocytes and unstimulated cells after application of concanavalin A.
Notwithstanding the fact that only a proportion of the cells will be mitogenically
stimulated we detected an enhanced cell membrane conductivity for the whole cell
population immediately after the addition of mitogen.
INTRODUCTION
If cells are placed in a rotating high frequency electric field they start to spin, although
at a rate much slower than the applied field. This so-called electrorotation allows the
determination of the dielectric properties of cellular constituents (Arnold and
Zimmermann, 1982a; Fuhr et al., 1985a; Gimsa et al., 1985; Glaser et al., 1985; Glaser
and Fuhr, 1986; F u h r e t al., 1986). At low frequencies the cells rotate against the spin
direction of the field, whereas at higher frequencies they spin with it. The
electrorotation spectrum (i.e. the spin of the cells as a function of the frequency of the
external field) shows two characteristic frequencies (fcl and fcz), where the rotation of
the cells is maximal. According to theoretical predictions (Glaser et al., 1983; Holzapfel
Department of Biology,Humboldt-Universityof Berlin, Berlin.
1 Central Institute of Molecular Biology,Academyof Science, Berlin-Buch.
2 Correspondence should be addressed to: Prof. R. Glaser, Humboldt-Universitatzn Berlin, Sektion
Biologie, Bereich Biophysik,1040 Berlin, Invalidenstr.42, GDR.
973
0144-8463/86/1100-0973505.09/09 1986 Plenum Publishing Corporation
974
Ziervogel,Glaser,Schadowand Heymann
et al., 1982; Fuhr, 1985, Sauer and Schloegel, 1985) the first characteristic frequency
mainly depends on membrane electrical properties, whereas the second reflects
parameters of the internal media.
Arnold and Zimmermann (1982a,b) first applied the method of electrorotation to
biological objects. The electrorotation of liquid filled glass spheres, erythrocytes,
thrombocytes and plant protoplasts generally corresponds with the theoretical
expectations of a single shell model. This model considers the cell as a homogeneous
sphere surrounded by a sihgle shell membrane of low conductivity (Fuhr et al., 1985a;
Glaser et al., 1983; Gimsa et al., 1985; Egger et al., 1986).
Many cellular subjects, however, have a more complicated structure. Recent
studies on protoplasts containing a large central vacuole indicate that such subjects
cannot be described by the single shell model; the rotation spectra deviate more or less
from the fitted curves of the single shell model. In such cases a three shell model has to
be applied (first shell--tonoplast, second shell--cytoplasm, third shell-plasmalemma) (Fuhret al., 1985b; Gimsa et al., 1985). This report addresses the
question of the influence of nuclei, in animal cells on electrorotation despite the fact
that the nucleus is separated from the cytoplasm by a porous membrane ("nuclear
envelope") (Paine et al., 1975; Reynolds and Tedeschi, 1984).
An intracellular organelle will produce an electrorotational signal if it is
surrounded by a low conductivity membrane. Our assumption was that the two main
factors influencing the conductivity of the nuclear envelope were the number of nuclear
pores per unit area, the nuclear pore frequency, and the portion of the nuclear envelope
which is free of pores.
We chose human lymphocytes for our investigations because their nuclear
envelope exhibits a ve~;ylow nuclear pore frequency (3 pores/#m 2) (Maul et al., 1971).
They offer a model for studying changes of the nuclear structure related to the
proliferative response induced by mitogenic stimulation (Johnson et al., 1974;
Pompidou et al., 1984; Setterfield et al., 1985) as well as to the properties of the cell
membrane (Kaplan, 1971; Owen and Kaplan, 1980; Hume and Weideman, 1980;
Owen and Crumpton, 1981; Szamel et al., 1985).
MATERIALS AND METHODS
Isolation and Culture of the Human Peripheral Blood Lymphocytes
Human peripheral blood lymphocytes were isolated from fresh buffy coat as
described by Boyum (1968). The Ficoll-Hypaque-technique with a dextran density
gradient was used (R. Grade, unpublished modification). After isolation the
lymphocytes were removed into RPMI-medium (Immunpraparate Weissensee)
containing 10 % v/v fetal calf serum (Immunpraparate Weissensee) and 200 units/ml
streptomycin and penicillin (Jenapharm).
The pH-value was adjusted to 7.4 with 5 mM HEPES (Serva) using NaOH for
titration. The final cell density was about 106/ml. For mitogenic stimulation 10 #g/ml
concanavalin A (ZIGOK Gatersleben) was added. Lymphocytes were incubated in
culture flasks in the complete medium at 37~ and continuously aerated (5 % 02, 21%
CO2, 74% N2).
Electrorotation of Lymphocytes
975
Electrorotation Measurements
A four electrode chamber was used driven by sinusoidal pulses as described by
Glaser et al. (1983). We applied a field strength of 5130 V/re. The rotation speed of the
cells was measured during free sedimentation in the centre of the square chamber. A
complete revolution of a cell needs more than one second. The revolution time was
measured by an electronic watch. The rotation speed per square of the applied field
strength is defined as "Rotation" (R) (in rad m 2 V-2 S-1). In the figures illustrating
electrorotation spectra (Figs. 1, 2, 5) rotation against the field spin is indicated as a
negative value.
At various times before and after mitogenic stimulation 3 ml of the cell suspension
was centrifuged at 500 x 9- The cell pellet was diluted (1:1000) in a sucrose solution
(300 mOsm) containing 1 m M phosphate (pH = 7.4). The conductivity of this solution
was 25 _+ 1 mS/m. In the case of application of gramicidin (SIGMA) 0.5 mM EDTA
was added to the sucrose solution to minimise blocking effects of external calcium
(Heitz and Gavach, 1983; Andersen, 1984). All experiments were carried out at 20~
Isolation of the Cell Nuclei
The nuclei from freshly drawn lymphocytes were prepared according to Fenske et
al. (1980). Finally, the isolated nuclei were suspended in a solution of 0.25 M sucrose
containing 1 mM MgC12. All steps in nuclear isolation were performed at 0-4~
RESULTS AND DISCUSSION
The rotation speed of the cells divided by the square of the field strength
("rotation") was measured for 11 fixed frequencies in the range from 0.01 to 10 MHz.
According to the equations for a single shell model (Fuhr, 1985; Glaser and Fuhr,
1986) a rotation spectrum was fitted to these measured points.
For a number of spectra the points could easily be fitted (cf. Fig. 2C). In some
cases, however, remarkable deviations occur from the curve predicted by the single
shell model (Fig. 1). In these cases the measured points do not fluctuate statistically but
rather indicate systematic deviations from the fitted curve. The character of these
deviations corresponds to that of protoplasts with large central vacuoles (Fuhr et al.,
1985b; Gimsa et al., 1985). This clearly suggests that the single shell model is not
applicable to these cells.
Fuhr, Gimsa and Glaser (1985) could show from theoretical considerations based
on the three shell model that in the case of an increased cell membrane conductivity,
which is reflected by a reduced Rmax-value,the rotation curve changes in different ways
from the curve of a single shell sphere. As shown in Fig. 1 it seems that the measured
spectra of lymphocytes containing a large nucleus (in relation to ceil size) are different
in every case from the fitted curves independently from the Rma~-value.Comparison of
these results with the investigations of Fuhr, Gimsa and Glaser (1985) allows us to
distinguish qualitatively between the spectra shown in Fig. 1A and 1C. The rotation
spectrum of a resting lymphocyte is determined mainly by the properties of the outer
976
Ziervogel, Glaser, Schadow and Heymann
5
A)
/
R
Log f
-5
5
B)
/
R
log
!
-5
c)
5
R
~ +
6 ~og f
-5
Fig. 1. Electrorotation spectra of stimulated human
peripheral blood lymphocytes with a constant mean
thickness (d) of cytoplasm (d = 0.8/zm) deviating in
rotation (Rmax) at the first characteristic frequency
(fcl)--the curve is a nonlinear fit according to the
equations of a single shell sphere (Glaser and Fuhr,
1986). f--frequency of the rotating field; R--cell
rotation in 10E - 8 [rad m z V-2 s 1]
Case
Cell
radius
r [Izm]
(A)
(B)
(C)
3.1
3.1
3. I
R ( x 10)
fcl of the
[rad
fitted curve
m 2 V -z s -1]
[kHz]
- 8.07
-6.62
- 5.32
121.4
171.3
232.7
cell m e m b r a n e w h e n its c o n d u c t i v i t y is low (i.e. Rmax is high) (see Fig. 1A). T h e h i g h e r
the cell m e m b r a n e c o n d u c t i v i t y (i.e. the lower Rmax is), t h e g r e a t e r will be the i n f l u e n c e
of a n i n n e r m e m b r a n e s y s t e m o n the r e s u l t i n g r o t a t i o n s p e c t r u m (Fig. 1C).
F u r t h e r m o r e F u h r , G i m s a a n d G l a s e r (1985) p o i n t e d o u t t h a t the i n f l u e n c e of the
i n n e r m e m b r a n e o n t h e first p e a k of the r o t a t i o n c u r v e declines if t h e t h i c k n e s s of the
c y t o p l a s m i c layer b e t w e e n the cell m e m b r a n e a n d the i n n e r m e m b r a n e increases. T h e
Electrorotation of Lymphocytes
977
curves in Fig. 2 support this prediction. Curve A (Fig. 2) shows the maximal deviation
of the measured rotation from the fitted curve; this is the cell with the thinnest
cytoplasmic layer. Thus the nuclear envelope influences the cell rotation mainly in the
range between the first characteristic frequency (fcl) and the frequency, where the
rotation reverses (f0)- The deviations of the measured rotation disappear when the
nucleus is surrounded by a relatively thick cytoplasmic layer. In addition, a decrease in
the thickness of the cytoplasm shifts the first characteristic frequency of the spectrum to
a higher value. This frequency shift is greater than expected because for a single shell
sphere the product of the cell radius and the frequency fcl remains constant.
A] 5
R
0
"' ~
~
8
-5
B)
5
R
0
-5
c) 5
R
g f
-5
Fig. 2. Electrorotation spectra of unstimnlated
lymphocytes,which exhibit a differentthickness (d) of
cytoplasm between the celt membrane and the nuclear
envelope--R, f see legend to Fig. 1
R
Case
Ceil thick- ( x 10-8)
radius ness [tad m2
r [#m] d [/lm] V -2 s-']
fcl
fcl * r
[kHz] [Hzm]
- 6.23
- 6.62
- 7.27
225.6 0.699
175.4 0.677
135.2 0.568
(A)
3.1
0.8
(B)
(C)
3.9
4.2
1.6
1.9
978
Ziervogel, Glaser, Schadowand Heymann
Another influence of the nucleus on the rotation curve of an intact lymphocyte can
be inferred from the data presented in Fig. 3A. A drastic increase of the cell membrane
conductivity induced by an ionophore strongly reduces the rotation of a single-shell
object. The concentration of the ionophore applied here stops the rotation of human
erythrocytes immediately in the range of the first characteristic frequency (the
membrane conductivity increasing to 400-600 pS/m from about 0.1 pS/m).
e
R max
7
$
.5
4
0
3
9
O0
9
eO
1
i
I
i
I
1
0
i
I
2
d
5
i
I
3
i
4
5
t~ml
R max
0
O0 o
q~oOo
0
I~0
"...
i
0
,
*
!
|
50
i
i
i
I
100
i
i
,-~
150
fcl
[kHz]
Fig. 3. Evidence of the influence of the nucleus on the electrorotation of
lymphocytes: (A) Electrorotation of ionophore-treated lymphocytes. Relation
between the Rrnax-values (absolute amounts) of the rotation spectra after drastic
increase of the cell membrane conductivity by application of 5 #g/ml gramicidin
and the cytoplasmic thickness (d) of the lymphocytes [f-l-l-mean value of the
untreated lymphocytes. (B) Electrorotation of nuclei isolated from unstimulated
lymphocytes. Relation between the Rmax-values (absolute amounts) and the
characteristic frequency f. I-O-[-isolated nuclei of unstimulated lymphocytes at
20~ [HI-isolated nuclei at 37~ (external medium conductivity--19 mS/m).
Electrorotation of Lymphocytes
979
In case of the lymphocytes we could not completely stop cell rotation with
ionophore. This fact as well as the dependency of the residual rotation on the thickness
of the cytoplasmic layer supports the assumption that the nuclear envelope influences
the cell rotation more strongly after a drastic increase of cell membrane conductivity. If
the ionophore also acted on the nuclear envelope it should eventually diminish the
resting rotational signal of the ionophore-treated lymphocytes. Such a decrease was
not found after an extended incubation with ionophore.
Further evidence for the influence of the nucleus on the electrorotation of
nucleated cells is given by the measured rotation curves of isolated nuclei. Figure 3B
shows the relation between the Rmax-value and the characteristic frequency fcl for
isolated nuclei. A direct comparison of these values with the frequency range where we
detected the influence of the nuclear envelope in the entire cell is not possible because
tile conductivity of the measuring solution differs from that of cytoplasm. We do not
give a numerical interpretation of the spectra of the nuclei because we do not know if
the single shell model is applicable.
We also investigated the mitogenic stimulation of resting lymphocytes, which
involves an enlargement of both the cell and the nucleus. Structural changes also occur,
for example the remodelling of large heterochromatic masses present in the inactive
nucleus (Pompidou et al., 1984; Setterfield et al., 1985), an increase of the nuclear
envelope, and a doubling of the nuclear pore frequency (Maul et al., 1972; Scott and
Marchesi, 1972). Mitogenic stimulation is accompanied by increased transport rates of
several metabolites and ions (Kaplan, 1977; Owens and Kaplan, 1980; Owen and
Crumpton, 1981); both the potassium influx and ettlux increase whereas the potassium
content of the growing cells remains constant (Segel et al., 1976a,b). One of the earliest
changes detectable after mitogen addition is an enhanced Ca 2 § uptake (Gelfand, 1984;
Hesketh, 1985). At the same time the turnover of membrane phospholipids increases,
predominantly their fatty acid moieties (Szamel, 1985).
After a preincubation in the RPMI-medium for 24 hr the lymphocytes were
mitogenically stimulated by addition of concanavalin A. During the preincubation
period we found a continuous increase of Rmax which reached a constant value 10 hr
after adding the lymphocytes to the culture medium (data not shown). This indicates
that cell membrane conductivity shifts slowly to a !ower value during the
preincubation period.
Figure 4 shows the change of the cell rotation and of the resonance frequency (fcl)
at different times after the addition of mitogen. Cells were classified, after microscopic
inspection, as "unstimulated" (i.e. cells which do not grow after stimulation) or
"stimulated" depending on the ratio of the radius of the cell (r2) to the radius of the
nucleus (q). Stimulated cells had a ratio (r2/q) in excess of 1.46; unstimulated cells,
before the addition of mitogen, had a ratio of 1.44 _+ 0.1.
Figure 4B indicates that immediately after mitogen addition the Rm~x-value of the
whole population of lymphocytes decreased. About 20 hr later unstimulated cells had
a rotation comparable to that of cells during preincubation period. On the other hand
the stimulated lymphocytes exhibited a diminished rotation over the whole time. This
indicates that the conductivity of the cell membrane of stimulated cells exceeds that of
unstimulated cells.
In such a case we should expect an increased characteristic frequency as well as
980
Ziervogel, Glaser, Schadow and Heymann
10
mQx
+
wJl
i
I
0
i
I
20
i
40
t
i
i
I
&O
I~0
[h]
250
fc 1
[kHz]
2OO
150
100
#
SO
jpl
r
0
I
.
20
i
40
i
I
I~
.
i
I10
t Ihl
Fig. 4. Electrorotation of lymphocytes after mitogenic stimulation with
concanavalin A. (A) Change of the Rmax-values (absolute amounts) of successfully
stimulated lymphocytes (R) and unstimulated lymphocytes (O) after addition of
concanavalin A (B) Change of the first characteristic frequency fcl after mitogenic
stimulation: (1)-successfully stimulated cells and (C))-unstimulated cells--each
point represents the data of 15-20 individual cells with exception of the first point
on the time scale (O), which summarises the data of the lymphocytes in the
stationary phase (i.e. with a constant value for Rmax and fct) before mitogenic
stimulation.
diminished rotation. But this is neither the case for the whole cell population
immediately after mitogen addition nor for the successfully stimulated cells.
As shown in Fig. 5, the spectrum of the stimulated lymphocyte is different, even
qualitatively, from that of an unstimulated cell, deviations of the measured rotation
from the fitted curve occurring only near the freqnency f0- The enlargement of the
Electrorotation of Lymphocytes
98t
AI
R
~s
/tog
-5
BI
2,5
Fig. 5. Comparison of a typical spectrum of
an unstimulated lymphocyte (18 hours after
stimulation--case A) with that of a
successfully stimulated cell (case B). (A) Rmax:
- 5 . 2 x 10 -s [rad m 2 V -2 s - l ] ; d = 0 . 8 #m;
r = 3 . 1 #m. (B)Rmax.: - 2 . 6 x 1 0 -~ [rad
m 2 V -2
s-lJ;
d=3A
pro;
(d-thickness of cytoplasm between the nucleus
and the cell membrane; r--cell radius). The
numerical interpretation of both spectra
according to the equations of the single shell
model (Glaser and Fuhr, 1986) leads to the
following results:
R
J
to9 f
-2,5
Case
(A)
(B)
(B)
Fixed
Calculated
parameter:
parameters:
Membrane
Inside
Membrane
conductivity conductivity dielectric
[~S/m]
IS/m]
constant
0.1
0.1
20,0
0.3
0.65
0.92
6.2
22.6
31.6
cytoplasm separates the signal due to the nuclear envelope from that of the cell
membrane, because fc~ shifts to a lower frequency if the cell radius increases. On the
one hand this should diminish the signal of the nuclear envelope, but on the other hand
the enhanced cell membrane conductivity provides conditions for a better
manifestation of the inner membrane system in the whole rotation curve (Fuhret a!.,
1985b). We could also see that the calculated membrane dielectric constant for the
stimulated cells was drastically enhanced.
Two explanations of these effects are possible:
Firstly, the membrane dielectric constant calculated from the measured points
using the single shell model is not correct because the single shell approach is not valid
for lymphocytes. The change of this value during stimulation, therefore, is apparent
rather than real.
Secondly, there is a real change of the dielectric constant which reflects the binding
of concanavalin A to the cell membrane.
As we were not able to measure the second peak of the rotation curve, an exact
determination of the internal conductivity was not possible, but there were indications
that the stimulated cells possess a lower internal conductivity than the unstimulated
cells. This would also explain the low fcl-value of the stimulated cells.
We conclude that the well-known increase of several transport rates immediately
after mitogen binding (Segel et al., 1976a,b; Gelfand, 1984), which is detected by our
method as an enhanced membrane conductivity, takes place in the whole cell
population. It seems that the first membrane events do not determine whether there
will be a successful growth stimulation of the lymphocytes. We found enhanced cell
membrane conductivity persisted for several hours only in successfully stimulated
lymphocytes whereas the unstimulated lymphocytes reverted to the low conductivity
state observed before the addition of mitogen.
982
Ziervogel, Glaser, Schadow and Heymann
ACKNOWLEDGEMENTS
F o r c o n s t r u c t i n g the e l e c t r o n i c devices for e l e c t r o r o t a t i o n we a r e very grateful to
J a n G i m s a . W e t h a n k D r R. G r a d e for the excellent p r e p a r a t i o n of the nuclei. F o r
helpful d i s c u s s i o n of the m a n u s c r i p t we t h a n k M a r c e l Egger a n d J a n G i m s a .
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