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Induction of cell processes by local force
L. B. MAKGOLIS
A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow, 119899, USSR
and S. V. POPOV*
Laboratory of Bioelectrochemistry, A. N. Frumkin Institute of Electrochemistry of the USSR Academy of Sciences, Leninsky prospect,
31, Moscow, 117071, USSR
* Author for correspondence. Present address: Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Summary
A method of applying local mechanical force to the
plasma membrane of mouse embryo fibroblasts is
described. The force is generated by local treatment
of cells with an alternating current (a.c.) electrical
field. The phenomenon of cell process formation
under the action of this force was investigated.
Inhibitors of actin polymerization did not prevent
generation of processes in the electrical field. At the
early stages of cell spreading, the processes could be
induced at any part of the cell membrane. After cell
polarization was completed, protrusions could be
Introduction
Many cell phenomena are accompanied by morphological
changes in the cells, including global (rounding of the cells
during cell division and spreading) and local changes
(formation of cell outgrowths) (Vasiliev, 1985; Trinkaus,
1985). It has been established that in all cases the
cytoskeleton plays a key role in changing cell shape.
However, the mechanical properties of the cell membrane
and of the cortical layer might contribute to the cell
deformability.
To investigate the mechanical properties of cell membranes several experimental methods have been developed
(Evans and Skalak, 1979; Pasternak and Elson, 1985; Bray
et al. 1986; Bo and Waugh, 1989). Using these methods, the
mechanical properties of liposomes, sea urchin eggs,
erythrocytes and lymphocytes have been investigated, but
they are applicable mainly to suspended cells; the
mechanical properties are expressed in terms of elastisity
and viscosity, and are thought to be homogeneous over the
cell surface. However, it is important to understand
whether different areas of the cells with complex morphology differ locally in their mechanical properties. The
existing methods are inapplicable to cells with complex
cell surface structure (e.g. spread fibroblasts).
Previously (Popov and Margolis, 1988) we described a
new experimental system based on the phenomenon of
dielectrophoresis for applying a force directly to the
plasma membrane of suspended cells. The force was
sufficient to generate cell-specific membrane protrusions
of various types. Inhibitors of cytoskeletal activity were
not able to prevent generation of processes by an external
force.
Journal of Cell Science 98, 369-373 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
formed at the active edge of the cell, but not at its
stable edge. Pre-existing protrusions (but not retraction fibers) could be elongated by the external force.
The results of these experiments demonstrate that
different areas of the cell membrane differ in their
ability to form processes under the action of a
membrane-applied force. The significance of these
data to the structure of the cortical layer is discussed.
Key words: cell processes, cortical layer, local mechanical force.
In this study we investigated the potential for different
regions of mouse embryo fibroblasts (MEF) to form
processes under the action of a membrane-applied force.
The force, applied locally to the plasma membrane, was
generated during the treatment of cells with an a.c.
electric field.
Materials and methods
Cells
Primary mouse embryo fibroblasts (MEF) were prepared according to the method of Domnina et al. (1982). Cells were maintained
in Dulbecco's modified Eagle's medium containing 10 % foetal calf
serum and used after 1-3 passages.
After trypsinization, 100 jA of cell suspension containing
10 3 -10 4 cells was transferred to the surface of a 25 mm x 25 mm
coverslip. The cells were incubated at 37 °C in a humidified
atmosphere.
Cytochalasin D (Sigma) dissolved in dimethylsulphoxide
(lmgml" 1 ) was added to MEF immediately before incubation on
the coverslip. The final concentration of cytochalasin D was
1/igml"1. Carbonyl-m-chlorphenylhydrazon (Sigma) was dissolved in dimethylsulphoxide (lmgrnl" 1 ); sodium azide was
dissolved in water (200mgml~1). The final concentration of these
drugs in the medium were l/igml" 1 and 2mgml~ 1 , respectively.
In both cases 20 OM 2-deoxy-D-glucose (Sigma) was added to the
medium. Carbonyl-m-chlorphenylhydrazon and sodium azide
were added to MEF after a l h incubation of the cells on a
coverslip at 37 °C.
Cell treatment by electrical field
Immediately before electrical field treatment, cells were washed
three to four times with buffered sucrose (290raMsucrose, 1 HIM
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for different cells. When the electrodes were positioned so
that they were not equidistant from the cell the processes
were generated mainly at the membrane surface facing
the nearest electrode.
7-8 V, 1MHz
Fig. 1. An a.c. electrical field was applied to cells spread on
the surface of a coverslip by 2 tungsten microelectrodes (tip
diameter of 2-6 /an).
Hepes-NaOH, pH7.4) and placed on the stage of inverted
Tluovert' microscope (Leitz). Tungsten microneedles with 2-5 /an
diameter tips were used as electrodes. Electrodes were placed by
using Narishigi MO 303 micromanipulators 10-20/on from the
cell surface at an angle of 10° to the horizontal (Fig. 1). An a.c.
electrical field was applied (voltage 7-8 V, frequency 1 MHz).
Cells were treated by the field no later than lOmin after
replacement of the medium with buffered sucrose.
Previously it was shown that during the treatment of cells with
an a.c. electrical field in a low-conducting sucrose solution a force
is generated; it is applied to the plasma membrane and to a thin
(10-20 A; 1 A=0.1 nm) cytoplasmic layer and is directed outwards
(Popov and Margolis, 1988).
Force applied to the surface of MEF was used to investigate:
(1) cell process formation during cell spreading; (2) the role of the
cytoskeleton in this phenomenon; and (3) the potential of
morphologically different parts of the cell surface to generate
processes.
Results
Cell process formation under the action of membrane^
applied force
After a 1 h incubation on the surface of a coverslip, cells
adhered to the surface and spread, acquiring the characteristic shape with a circular lamella. A few seconds after
application of the electrical field, processes were generated
on that part of the membrane closest to the electrode
(Fig. 2). Processes were cylindrical (~0.5 /an in diameter),
with a thickening at the distal edge of the protrusion
(~-l /an in diameter). The processes were directed towards
the tip of the electrode and they reached it in approximately 30 s. The number of processes varied from 1 to 20
Influence of cytoskeleton inhibitors on cell process
formation in electrical field
Control cells incubated in cytochalasin D-containing
medium and not treated with an electrical field attached to
the coverslip after a 1 h incubation, However, practically
no lamellae were observed. The processes were ~1 /an in
diameter, which was approximately twice that observed
during spreading in cytochalasin-free medium.
Application of an electrical field to cytochalasin-treated
cells resulted in the formation of processes directed
towards the tip of the electrode like those formed by
untreated cells in an electrical field (Fig. 3). The diameters
of the processes did not exceed 0.5 /an.
Cells treated with sodium azide+2-deoxy-D-glucose for
l h also form processes after electrical field application
(Fig. 4). Again, the processes are formed only at the
electrode-facing part of the cell and reach the electrodes in
a few dozen seconds. The diameter of the processes is
—0.5 fan. Similar results were obtained when the time of
incubation was increased to 3h and when another
inhibitor of ATP synthesis, carbonyl-m-chlorphenylhydrazon, was added instead of sodium azide.
Cell process formation at different parts of the cell
membrane
During the early stages of spreading in normal physiological conditions the surface of some MEF were covered with
microblebs (located mainly at the lamella). These cells
easily formed processes (usually 10-20/cell) after electrical field treatment (Fig. 5) that quickly reached the
electrode.
After a few hours of incubation on the surface of a
coverslip cells polarized with the formation of active and
stable edges. By placing the electrodes close to certain
parts of the membrane, we were able to apply the force
locally to the lamella region of the cell and to the stable
edge. Under electrical field treatment the processes were
formed only at the active edge. No processes were formed
at the stable edge (Figs 6, 7).
Some cells in normal physiological conditions have
processes —0.5 /an in diameter. Usually they are located at
the lamella region of the cell. These pre-existing processes
easily elongated after electrical field application (Fig. 8).
\
\
2A
B
Fig. 2. Phase-contrast microscopy of MEF before (A) and 30 s after (B) application of the electrical field. Processes are formed at
the electrode-facing part of the cell. They are directed from the cell body to the tips of the electrodes. Bar, 20 /an, for all
photographs.
370
L. B. Margolis and S. V. Popov
i
Fig. 3. Prior to electrical field treatment, MEF were incubated with cytochalasin D (1/igml ') for l h . The cell before (A) and 30 s
after (B) electrical field application.
B
Fig. 4. (A) MEF were incubated with sodium azide (2mgml *) and 2-deoxy-D-glucose (20 mM). (B) 20 s after electrical field
application processes directed towards the tip of the electrode had formed.
i
5A
B
Fig. 5. (A) MEF after 30 min incubation on the surface of the coverslip. The cell surface was covered with microblebs —1—3/an in
diameter. (B) After electrical field application processes (10-20 per cell) were formed, which usually reached the tip of the electrode
in less than 10 s.
Their diameter (~0.5 /sm) did not change after electrical
field application.
Migrating fibroblasts in normal physiological conditions
as a rule have retraction fibers at the rear of the cell,
morphologically similar to the processes at the leading
edge. Retraction fibers could not be elongated by electrical
field treatment.
Discussion
Previously we developed an experimental system for
applying force to spherical cells brought in contact with a
planar electrode (Popov and Margolis, 1988). We have
found that the surface of the cells is deformed due to the
membrane-applied force that is generated during electriInduction of cell processes
371
Fig. 6. A cell after 3 h incubation on the surface of a coverslip (A). The cell is polarized: active and stable regions are formed.
(B) 30 s after electrical field application.
*
>
• . . *^%*'^*ty
*/
Fig. 7. A polarized cell 30 s after electrical field application.
No processes were formed at the stable edges.
cal field treatment. In this paper the method is modified to
investigate the mechanical potential of different parts of
cells with surfaces of complex morphology.
The principle of force generation is based on the
different conductivities of the cytoplasm and the extracellular solution (Zimmermann, 1982; Pohl, 1978; Engelhardt et al. 1984). After application of the electrical field
an undermembrane diffuse layer 10-20 A (lA=0.1nm)
thick is formed to compensate the field inside the cell
(Landau and Lifshits, 1960). This phenomenon is equivalent to the polarization of a dielectric in a vacuum in an
electrical field. A force applied to the surface of the
dielectric and directed outwards is generated.
In our experiments the medium outside is conducting
the current and a diffuse layer on the outer surface of the
membrane tends to form. This outer layer would balance
the inner diffuse layer and no force would be generated.
But owing to the low conductivity of the extracellular
solution the time of formation of the outer diffuse layer
(10~4s) is much longer than the period of the applied field
(10~6 s). So an outer diffuse layer does not form and a force
applied to the thin membrane layer and directed outwards
is generated. For more details see Margolis and Popov
(1988) and Pastushenko et al. (1985).
The method presented in this article permits application
of a local force to different parts of the membrane and
investigation of morphological changes in cells, spread on
a glass surface, induced by these forces.
After electrical field application processes were formed
at the electrode-facing parts of the cells. Light microscopic
data demonstrated that these processes were morphologically normal (~0.5 f.an in diameter, uniform in thickness
with a thickening at the end of the process).
Previously (Popov and Margolis, 1988) we have shown
that it is a membrane-applied force that generates the
processes. Other possible effects of an electrical field
(heating, electrical breakdown, electrophoresis etc.) do not
contribute to the observed phenomenon.
In normal physiological conditions cell process formation is substantially inhibited by cytochalasin D (inhibitor
of polymerization of G-actin into microfilaments) and by
metabolic poisons. In the experimental conditions used in
B
8A
Fig. 8. The processes at the active edge of a polarized fibroblast (A) elongate easily after electrical field application (B).
372
L. B. Margolis and S. V. Popov
this work these substances did not prevent protrusion
formation in the electrical field. Therefore, membraneapplied force per se is sufficient to generate processes with
normal morphology (see also Popov and Margolis, 1988).
The method of application of local force to the cell
surface used in this work permits investigation of the
potential of different regions of the cell surface to form
processes under the action of this force.
MEF were studied at the stage of radial spreading and
after cell polarization was completed. At the stage of radial
spreading MEF formed symmetrical lamella. Cell processes at this stage could be formed at any region of the
lamella.
The surface of parts of the cells at the stage of radial
spreading was covered with microblebs. These cells were
very 'deformable': under the action of membrane-applied
force numerous cell processes were formed, reaching the
tip of the electrode within a few seconds. This result is in
agreement with the previous suggestion about the higher
'deformability' of blebing cells (Trinkaus, 1985).
During polarization, the cell surface is divided into
active and stable domains (Vasiliev, 1985), with processes
forming only at the active region. Vasiliev et al. (1970)
showed that the treatment of cells with colcemide or other
microtubule-disrupting agents leads to the loss of cell
polarization: all of the cell edge becomes active. It is
proposed that the polarized form of the cell is determined
by preferential orientation of microtubules along the
stable edge (Goldman, 1971). Material transported along
the microtubules is inserted at the active edge (Hollenbeck, 1989). It is here that the protrusive activity of the
cell surface is localized. This model, however, does not
explain the independence of polarization of fibroblasts in
early primary cultures on microtubule integrity (Middleton et al. 1989).
The results of this paper suggest that, at least in part,
the absence of protrusive activity at the stable edge can be
explained by its 'rigidity': membrane-applied force does
not generate processes. This rigidity is most probably
determined by the structure of the cell membrane and/or
by the underlying cortical layer (Zand and AlbrechtBuehler, 1989).
The experimental system used in this study provides
information about the organization of the cortical layer at
different regions of the cell surface. Protrusions at the
active edge and retraction fibers react differently to the
applied force. The processes at the active edge are easily
elongated while retraction fibers are not.
The poorly understood internal organization of the blebs
also can be studied by the method described here. The
commonly held point of view about the absence of a
cortical layer beneath the membrane of the bleb is based
on three observations: blebs are spherical; the coefficient
of lateral diffusion of proteins in the bleb membrane,
measured by fluorescence recovery after photobleaching,
is higher than in the cell body (Tank et al. 1982); electron
microscopy does not reveal actin beneath the bleb
membrane. According to the results presented here it can
be proposed that the membrane of tbe bleb does not have
uniform mechanical properties. The nonuniformities have
the same diameter as that of the process (~0.5 (jm) and are
determined by the cortical structures beneath the bleb
membrane. The results are in agreement with the
hypothesis of Yechiel and Edidin (1987) about the
existence of micrometer-scale domains in the fibroblast
plasma membrane.
Thus the method of local application of mechanical force
to the membrane of substratum-attached cells gives an
insight into the fine organization of the cell membrane and
the underlying cortical layer.
We thank Drs Y. A. Chizmadzev and L. V. Chernomordik for
useful discussions.
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(Received 1 June 1990 - Accepted, in revised form, 12 October 1990)
Induction of cell processes
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