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Measuring the galvanotaxic response of Amoeba proteus to a changing DC electrical field
J. Bryce Sealander, Joseph Merriman, James K. Brown, Francis X. Hart PhD, John R. Palisano PhD
Departments of Biology and Physics, The University of the South, 735 University Avenue, Sewanee, Tennessee 37383
Abstract
Galvanotaxis is the change in cellular movement created by an
electrical field. Different types of motile cells have been shown to
exhibit galvanotaxis on a time frame of an hour in previous
investigations. Amoeba typically migrate towards the negative pole.
In contrast, we have video recorded the directional movement of
Amoeba proteus on a time frame of minutes in response to a DC
electrical field (dcEF) for which the polarity is occasionally
reversed. In addition to observing directional changes, the video
recording makes it possible to continuously measure the velocity
and acceleration of Amoeba proteus and the cytoplasmic
streaming as the polarity and strength of the field change. We have
confirmed that even on a time scale of a few minutes the amoeba
migrates toward the negative pole in a field of about 300 V/m. We
have also observed that the amoeba can respond to changes in
the direction of the field within a minute.
In this investigation, we describe the effect of dcEF
on galvanotaxis on Amoeba proteus. One of our aims
was to examine the short-term (up to half an hour)
galvanotaxis of individual amoebae under precisely
controlled experimental conditions. Moreover, we are
unaware of investigations in which amoebae were video
recorded during the movement in dcEF. Using video
recording software, we were able to characterize the
directed cell movement and cell shape changes induced
by the electric stimulus. We were also able to examine
the effect on reversing the polarity of the electric field
that the amoebae were subject to. This allowed us to
follow the change in migration of amoeba towards the
negative pole.
Exposure of Cells to dcEFs
Platinum electrode wires were placed 3.1 cm apart in the trough.
The wires were connected in series to a resistor and a switching
apparatus. Voltage was continually monitored across the
resistor with a Digital Multimeter to insure current continuity. The
switching apparatus permitted rapid reversal of the applied
polarity. Voltages of 5V-20V, direct current, were used in
experiments. However, 10V was used to monitor migrational
changes in response to polarity reversal. Amoeba were
introduced into the apparatus, and the chamber was sealed with
silicone grease (Fig. 3).
Conclusions
•
It has been hypothesized that this directional change
is due to migration of electrically charged receptors
in the plasma membrane. However, receptor
migration cannot occur over the period of seconds in
which we were able to observe change in amoeba
movement towards the new cathode upon reversal of
the field. This leads us to believe that changes in
cellular migration in response to an electrical field is
due to some other factor.
•
Future studies will examine the movement
of
individual amoeba exposed to different field
strengths. We will also analyze the kinetics of
cytoplasmic flow which controls the movement and
the cell shape after the application of an electric field
or its reversal.
Literature cited
Fig. 2 Diagram of slide preparation
Fig. 4 Amoeba Culture
Cells
Cultures of Amoeba proteus were maintained in spring water
purchased from Carolina Biological Supply. The amoeba were fed
protozoan that were cultured in Petri dishes containing boiled wheat
seeds (Fig. 4). The cells were taken out of the culture and placed in
pure spring water to wash any excess materials debris off them.
Amoeba were then transferred to microscope slide where they
attached to the surface of the slide. Their migrations in response to
dcEF were observed with video recording software.
Fig. 1 Amoeba proteus
Introduction
The importance of the electrical control of cell physiology has
been stressed since the famous frog leg experiments of Luigi
Galvani in the late 1700s (Adler and Shi 1988). Galvanotaxis, one
aspect of this control, is the change in cellular movement created by
an electrical field and has been an object of study for many years.
This migration occurs in the direction of either the cathode (negative
pole) or the anode (positive pole).
In recent years, research on the effects of dcEF on growth of
plants and animal tissues, organs and entire organisms have been
carried out. Galvanotaxis experiments usually last several hours.
However, our experiments with Amoeba proteus (Fig. 1)
investigated a response that occurred on a time scale of minutes.
Amoebae are particularly suitable subjects of galvanotactic
experiments studying the mechanism of movement because the
experiments can be done under simple conditions. Also, amoebae
are easier to work with for other reasons – no plating on a substrate
is required, for example.
Fig. 3 Top View of Apparatus for Amoeba
Materials and methods
Slides
The slides were prepared by cutting one cover slip in
half and gluing it to a glass slide with silicone sealant.
The cover slips were placed approximately three to
four millimeters apart creating a trough for the amoeba
(Fig. 2). A cover slip was then placed on top of the
apparatus to seal the trough.
Results
Our studies have confirmed previous investigations that in an
isotropic environment and in the absence of any electric stimulus,
the amoebae randomly migrated in all directions but towards the
negative pole under dcEF (Korohod et al. 1997). Moreover, our
preliminary evidence suggests that amoeba detect the field and
start to change direction within approximately 45 seconds to one
minute. For an applied electric field of 300 V/m, the amoeba
migrated toward the negative pole and often exhibited elongation
into a leech-like structure (Fig. 5).
Adler, J. and W. Shi. Galvanotaxis in Bactera. 1988.
Quantitative Biology 3: 23-25.
Korohoda, Wlodzimierz, Mycielska, M., Janda, E. and
Zbigniew Madeja. 2000. Immediate and Longterm Galvanotactic Responses of Amoeba proteus
to dc
Electric Fields. Cell Motility and the
Cytoskeleton 45: 10-26.
Nishimura, K.Y., Isseroff, R.R., and Richard Nuccitelli.
1996. Human keratinocytes migrate to the
negative pole in direct current electric field
comparable to those measured in mammalian
wounds. Journal of Cell Science 109: 199-207.
Acknowledgments
We would like to thank the biology and physics
departments of the University of the South for providing
us with all of the equipment used in the study. We also
want to acknowledge the support of a Faculty
Development Fund Grant to JRP.