The Effect of Mercury on Macropinocytosis in Glial Cells
KarryAnne
Belanger
Neurobiology
Short Report
Bio324
/ Neurobiology
Wheaton
College, Norton, Massachusetts, USA
April
24, 2013
Introduction:
Endocytosis
is used by cells to replace plasma membrane lost during exocytosis, aid in cell
surface repair, and
bring in nutrients. (Kandel, 2013 Pg. 272 and 87) The
endocytic pathway consists of the formation of an enodocytic
vesicle that fuses
with an endosome. (Atkinson, et al. 2002) This early endosome is transported
through the cell and
then undergoes endosome maturation. During endosome
maturation the late endosome binds with a degradative
compartment known as the
lysosome. The lysosome pumps out the useful material present in the endosome
such as
amino acids and sugars while leaving everything else in it. (Luzio,
et. al, 2000) (Wikipedia-endosome) (Morris, 2013)
There are numerous types
of endocytosis, of which the most common is receptor-mediated.
Receptor-mediated
endocytosis makes uses of vesicles coated in clathrin.
(Kandel, 2013 Pg. 87) The use of receptors allows specific
material to be
brought into the cell. In order for these clathrin-coated vesicles and
endosomes to fuse together and
proceed with the endocytic pathway, the
clathrin-coat must be lost. (Kandel, 2013 Pg. 87)
Another
form of endocytosis is macropinocytosis. Pinocytosis is “cell drinking”.
(Morris, 2013) Pinocytosis
requires ATP as energy to drive the process. (Feo,
et al, 1977) (Wikipedia-pinocytosis) Pinocytosis differs from
receptor-mediated
endocytosis in that it takes up everything rather than incorporating specific
material. (Swanson, et al
1995) (Wikipedia-pinocytosis) There are two types of
pinocytosis, macropinocytosis and micropinocytosis. (Swanson,
et al 1995) As
their prefixes indicate, micropinocytosis includes smaller pinosomes that can
either be coated or
uncoated. Macropinocytosis include large pinosomes that
form at areas of cell surface ruffling. Macropinosomes vary
in their size and
incorporate large amounts of non-specific material into the cell. (Swanson, et
al 1995)
Macropinosomes feed into the endocytic pathway in order for the cell
to access the contents within. (Morris, 2013)
Mercury
has been shown to have neurotoxic effects on the central nervous system and be
able to cross the bloodbrain barrier. The neurotoxicity is different between
compounds containing mercury and dependent on the redox
potential of a specific
compound. (Aschner, 1990) McHgCL and HgCl2 are two compounds that have been
used to test
the toxicity of mercury. Both have shown toxic effects on cells at
10^-6 M. With smaller concentrations than 10^-6 M
cells begin preparations to
handle the damage that will occur once the toxic mercury level is reached. For
example these
preparations include the formation of proteins used for gliosis
and microglial interactions. (Monnet-Tschudi, 1996)
When the central nervous
system experiences damage gliosis occurs and involves the proliferation of
glial cells such as
microglia and astrocytes. (Fawcett, et al, 1999)
(Wikipedia-gliosis) Additionally when the central nervous system
experiences
damage microglial aid in the immune response. (Gehrmann, et al, 1995) (Wikipedia-microglia)
Continuing
on, mercury has been shown to effect functions of the cell such as
mitochondria, synthesis of RNA and protein, and
concentration of ATP. One study
found that mercury directly effected the machinery used for protein synthesis.
(Sarafian, 1984)
In
this study, we tested the hypothesis that cultured chick (Gallus gallus) glial cells exposed to mercury will
exhibit
decreased macropinosome formation as observed through video phase microscopy.
In order to test this
hypothesis we designed an experiment where we exposed
cultured chick glial cells to a mercury dose and observed
their behavior
through video phase microscopy. From this data I focused on the cellular
behavior of macropinocytosis
in order to test my hypothesis. Whether or not
cultured chick glial cells exposed to mercury will exhibit decreased
macropinosome formation is an interesting hypothesis. This hypothesis will look
further into what the neurotoxic
concentration of mercury is for glial cells,
and what behaviors the cell may participate in expectation of damage and
post-damage. As mentioned previously at 10^-6 M of mercury toxic effects were
shown. (Monnet-Tschudi, 1996) In
addition inhibition of protein synthesis has
been seen at 2 to 4 micromolar mercury. (Gopal, 2003) In order to look
farther
at what concentration of mercury neurotoxic effects first occur a lower
concentration will be used. I believe that
cells exposed to mercury will
exhibit decreased macropinosome formation because cell processes will start
being
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]
inhibited such as protein synthesis and the cell will not have a need for
the nutrients nor the energy to get the nutrients
it needs.
Materials and Methods:
Dissection
of chick embryonic peripheral glial cells was completed following the procedure
“Primary culture of
chick embryonic peripheral neurons 1: DISSECTION”. (Morris,
2013a) Following this procedure coverslips were
cleaned in etOH, dried with a
kimwipe and inspected for any remnants. Once clean the coverslips were placed
in a petri
dish and stored for later use. These coverslips were later treated
by placing them on poly-lysine for 20 minutes and then
rinsing them off with
water and allowing them to dry. Finally the coverslips were placed on laminin
with the previously
treated side down for 20 more minutes.
Sympathetic
nerve chains and dorsal root ganglia were collected through the dissection of
10 day chick embryos.
The chick embryos were contained in a petri dish filled
with DMEM and observed under a dissection microscope.
Using forceps all body
components except for the spinal cord were carefully removed taking precaution
to not remove
the neurons and glial cells and discard them. Then the
sympathetic nerve chains and dorsal root ganglia were collected
and placed in a
35 mm petri dish with DMEM. The ganglia were then trimmed of their tails,
washed with DMEM,
followed by replacement with trypsin. They were then
incubated for 20 minutes at 37 degrees Celsius and then
resuspended in DMEM by
titration. This suspension was then added to growth medium and plated out.
(Morris, 2013a)
Isolated
cells that were fed two weeks later were used to construct a flow chamber
following the instructions in
“Primary Culture of Chick Embryonic Peripheral
Neurons 2: OBSERVATIONS of LIVE UNLABELED CELLS”.
(Morris, 2013b) Following
this procedure cover slip chips were placed in two lines on a clean microscope
slide while
wearing safety glasses. A few drops of growth medium were placed in
the center of the coverslip chips using a Pasteur
pipette and then the coverslip
with the isolated cells was placed cell side down on top. If the growth medium
didn’t
completely fill the space below the coverslip more was added. Once
completely filled the top and bottom of the
coverslip were sealed with VALAP
and the top of the coverslip was carefully cleaned with water. (Morris, 2013b)
Next
observations were conducted in the ICUC at Wheaton College following the
instructions in “Primary
Culture of Chick Embryonic Peripheral Neurons 3: DYING
and OBSERVING LIVE CELLS”. (Morris, 2013c) The
specifications of how these
observations were conducted came from (Laliberte C, 2013; Laliberte M, 2013;
McCloskey,
2013; and Superson, 2013) and occurred as follows. The flow chamber
was placed on the microscope stand and
warmed up to 37 degrees Celsius. The
microscope was aligned for Koehler illumination. The cells were then observed
in
the Imaging Center for Undergraduate Collaboration (ICUC) at Wheaton College,
Norton MA in phase optics at
400X magnification on a Nikon EFD-3 microscope
using a Sony DFW-x700 camera with a 1.0x C-mount and BTV
6.0b1 software running
on a Macintosh computer running OS 10.5.8 and then analyzed using ImageJ 1.46r.
Glial cells
were located and pictures were taken every 15 seconds for 10
minutes. The image was re-focused as necessary.
Following this, 100nm mercury
was flowed through the chamber to completely replace the growth medium and
create
the experimental slide. (Laliberte M, 2013) On the 40x objective the
same glial cell was located and pictures were taken
every 15 seconds for 10
minutes.
The pictures of the control glial cell were then compiled, as were the pictures of the experimental slide. These
were then used to create phase microscopy videos. For quantification purposes the pictures starting at time 0 and every
minute up till 10 minutes for the control were compiled together as were they for the experimental. These 11 pictures
from the control and 11 pictures from the experimental were then opened with ImageJ. The same portion of every
picture was cropped out by going to edit, selection, specify and typing in 200 for the width, height, x-coordinate, and ycoordinate. This area was selected because of its location next to the surface of the glial cell where most of the
macropinocytosis in the cell was observed. It was kept constant to allow for comparable data. Then under image, crop
was selected. These images were saved
and compiled in a new folder for control cropped and experimental cropped
which
then could be used to create another phase microscopy video of the area of
interest. Each image was then
observed and the number of macropinosomes were
counted. A macropinosome was defined as a circular green area
greater than 20
pixels that had a mean brightness between 120 and 150. Due to the fact that macropinosomes
are big
they include a large volume with solute in it. (Swanson, et al 1995) This large volume takes up most of the area
between the cell membrane and glass
used in the flow chamber. Due to this, the area of a macropinosome appears
brighter because it is not obscured by other objects. (Morris, 2013) Specifying
an average brightness allows other
endosomes to be excluded from the data
analysis. To determine if they met these criteria they were encircled with the
oval tool in ImageJ. Then while selected from the analyze menu the measure tool
was clicked. This tool gave the area
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]
of the selection and the color mean. Then
using the phase microscopy video it was noted the number of new
macropinosomes
that appeared in each image. This was done by tracking their location through
out the video. All of the
macropinosomes at time 0 were used as a foundation to
compare to. At time 1 minute any macropinosomes that
appeared in the image that
were not in the image at 0 minutes were considered new. At time 2 minutes any
macropinosomes that appeared in the image that were not in the image at 1
minute were considered new, and so on.
The average number of macropinosomes and
the average number of new macropinosomes was calculated for the
control and
experimental data. Microsoft Excel was used to create all graphs. The percent
decrease between the control
and mercury trials was calculated by finding their
difference and dividing by the control number.
Results:
Figure 1 shows the visual difference between the control and
mercury group at time 0 and ten minutes. The total number
of macropinosomes and
new macropinosomes were counted every minute starting at time 0 for 10 minutes
for both the
control and mercury trials. This information is provided in Figure
2 and Figure 4. It can be seen in Figure 3 that the
control trial has a higher
average of total macropinosomes per minute than did the mercury trial. It can
be seen in
Figure 5 that the control trial has a higher average number of new
macropinosomes per minute than did the mercury
trial.
Figure
1: A is the control at 0 minutes. B is the experimental mercury
at 0 minutes. C is the control at 10 minutes. D is
the experimental mercury at
10 minutes. Notice the visible difference in the number of macropinosomes in
the control
on the left as compared to the experimental mercury on the right.
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]
Figure
2: Comparison of the total number of macropinosomes for every
minute between the control and experimental
mercury data over time starting at
0 minutes. The control data is shown in blue and the mercury data is shown in
red. A
total of 96 macropinosomes were counted in one cell during the control
trial. A total of 47 macropinosomes were
counted in one cell during the mercury
trial.
Figure
3: The average number of total macropinosomes formed in
mercury dropped by 51% compared to the average
number of total macropinosomes
formed in the control. A total of 96 macropinosomes were counted in one cell
during
the control trial including a count at time 0 to give the average. A
total of 47 macropinosomes were counted in one cell
during the mercury trial
including a count at time 0 to give the average.
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]
Figure
4: Comparison of the number of new macropinosomes for every
minute between the control and experimental
mercury data over time starting at
0 minutes. The control data are shown in blue and the experimental mercury data
are
shown in red. Note that the control had no new macropinosomes at 0, 2, and
6 minutes and that mercury had no new
macropinosomes at 0, 1, 3, 5, 7, and 10
minutes. 26 new macropinosomes were counted in one cell during the control
trial. 11 new macropinosomes were counted in one cell during the mercury trial.
Figure
5: The average number of new macropinosomes formed in mercury
dropped by 58% compared to the average
number of new macropinosomes formed in
the control.
26 new macropinosomes were counted in one cell during the
control trial, including a count at time zero to get the
average. 11 new
macropinosomes were counted in one cell during the mercury trial including a
count at time zero to get
this average.
Discussion and Conclusions:
The
hypothesis that cultured chick (Gallus
gallus) glial cells exposed to mercury would exhibit decreased
macropinosome formation was supported by the current study. There was a clear
decrease in the number of
macropinosomes per minute between the control and the
mercury. The average number of total macropinosomes
formed in mercury dropped
by 51% compared to the average number of total macropinosomes formed in the
control.
The average number of new macropinosomes formed in mercury dropped by
58% compared to the average number of
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]
new macropinosomes formed in the control.
From this data we can draw that the control group had a higher rate of
formation of the macropinosomes and thus a higher concentration of the
macropinosomes. The mercury had toxic effect
on the glial cells and decreased
the rate of formation of the macropinosomes and thus the concentration of the
macropinosomes in the cell at a given time.
As
mentioned previously mercury is known to have an effect on mitochondria and the
concentration of ATP in a
cell. (Sarafian, 1984) It is also known that
pinocytosis requires ATP as energy to drive the process. (Feo, et al, 1977)
(Wikipedia-pinocytosis)
The observed result that mercury decreased macropinosome formation in chick
(Gallus gallus)
glial cells may be explained on a cellular level. It is known
that mercury directly effects mitochondria and ATP
concentration. (Sarafian,
1984) If mercury inhibited the function of mitochondria and thus the ATP
concentration in the
cell there would be less energy available for
macropinocytosis to occur. With decreased energy, the process of
macropinocytosis may be expected to slow down, and consequentially the number
of macropinosomes formed may
well be lowered.
To
fix errors and refine the experiment it could be done slightly differently next
time. The specifications of how
the observations were conducted came from
(Laliberte C, 2013; Laliberte M, 2013; McCloskey, 2013; and Superson,
2013) as
explained in the materials and methods section. This procedure did not wash the
cells or use Tyrode’s, but
rather observed the control
directly in the growth medium, and the mercury trial in mercury. The
application of the
mercury and the control buffer should be done as specified
by R. L. Morris in the next experiment. This would be done
by washing the
growth medium in the flow chamber out with DMEM, then replacing the DMEM with Tyrode’s
for the
control. The Tyrode’s would stay in the flow chamber during the
incubation time and be replaced with growth medium
after the incubation period.
This process would be repeated for mercury but instead we would use Tyrode’s
and
mercury. (Morris, 2013)
Future
experiments to extend the results obtained from this data would include
different concentrations of
mercury and different compounds of mercury. This
would allow for the observation of exactly what level mercury
becomes lethal in
glial cells and whether it has staggered level of effects with increasing
concentration up to this lethal
level. This would be helpful knowledge in
understanding neurotoxic effects and the role glial cells play in these
effects.
Additionally it would allow for the identification of which compounds
with mercury have a more toxic affect on the
glial cells and thus what levels
of mercury must absolutely be avoided.
References:
My collaborators:
1) Cailin McCloskey (2013), personal communication.
2) Corey Laliberte (2013), personal communication.
3) Magan Laliberte (2013), personal communication.
4) Michaela Superson (2013), personal communication.
R. L. Morris (2013), personal communication.
R. L. Morris (2013a) Neurobiology Bio324 Primary Culture of Chick Embryonic Peripheral Neurons 1: Dissection.
Accessed at: (http://icuc.wheatoncollege.edu/bio324/2013/morris_robert/BIO324_Lab_Proc_1_Dissection.htm)
R. L. Morris (2013b) Neurobiology Bio324 Primary Culture of
Chick Embryonic Peripheral Neurons 2:
OBSERVATION of LIVE UNLABELED CELLS
Accessed at :
(http://icuc.wheatoncollege.edu/bio324/2013/morris_robert/BIO24_Lab_Proc_2_ObserveLiveUnstained.htm)
R.L. Morris (2013c) Neurobiology Bio324 Primary Culture of
Chick Embryonic Peripheral Neurons 3: DYING and
OBSERVING LIVE CELLS Accessed
at:
(http://icuc.wheatoncollege.edu/bio324/2013/morris_robert/BIO324_Lab_Proc_3_StainAndObserv.htm)
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]
Joel A. Swanson, Colin Watts,
Macropinocytosis, Trends in Cell Biology, Volume 5, Issue 11, November 1995,
Pages
424-428, ISSN 0962-8924, 10.1016/S0962-8924(00)89101-1.
(http://www.sciencedirect.com/science/article/pii/S0962892400891011)
Kandel, Eric R, et al. Principles of Neural Science:
Fifth Edition. N.p.: Mcgraw Hill, 2013. Print.
Helen A Atkinson, Alison Daniels, Nick
D Read, Live-cell imaging of endocytosis during conidial germination in the
rice blast fungus, Magnaporthe grisea, Fungal Genetics and Biology, Volume 37,
Issue 3, December 2002, Pages 233244, ISSN 1087-1845,
10.1016/S1087-1845(02)00535-2.
(http://www.sciencedirect.com/science/article/pii/S1087184502005352)
Michael Aschner, Judy Lynn Aschner,
Mercury neurotoxicity: Mechanisms of blood-brain barrier transport,
Neuroscience & Biobehavioral Reviews, Volume 14, Issue 2, Summer 1990,
Pages 169-176, ISSN 0149-7634,
10.1016/S0149-7634(05)80217-9.
(http://www.sciencedirect.com/science/article/pii/S0149763405802179)
Florianne Monnet-Tschudi,
Marie-Gabrielle Zurich, Paul Honegger, Comparison of the developmental effects
of two
mercury compounds on glial cells and neurons in aggregate cultures of
rat telencephalon, Brain Research, Volume 741,
Issues 1–2, 25 November
1996, Pages 52-59, ISSN 0006-8993, 10.1016/S0006-8993(96)00895-5.
(http://www.sciencedirect.com/science/article/pii/S0006899396008955) Kamakshi V Gopal, Neurotoxic effects of
mercury on auditory cortex networks growing on microelectrode arrays: a
preliminary analysis, Neurotoxicology and Teratology, Volume 25, Issue 1,
January–February 2003, Pages 69-76,
ISSN 0892-0362, 10.1016/S0892-0362(02)00321-5.
(http://www.sciencedirect.com/science/article/pii/S0892036202003215)
Sarafian, T. A., M. K. Cheung, and M.
A. Verity. "In Vitro Methyl Mercury Inhibition Of Protein Synthesis In
Neonatal
Cerebellar Perikarya." Neuropathology and Applied Neurobiology
10.2 (1984): 85-100. Print. Accessed at
(http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2990.1984.tb00342.x/abstract)
"Endosome." Wikipedia. Wikimedia
Foundation, 04 Feb. 2013. Web. 10 Apr. 2013.
<http://en.wikipedia.org/wiki/Endosome>.
Luzio, J. P., Brian A. Rous, and Nicholas A. Bright.
"Lysosome-endosome Fusion and Lysosome Biogenesis." Journal
of
Cell Science 113.9 (2000): 1515-524. Journal of Cell Science. Apr.
2000. Web. 23 Apr. 2013.
<http://jcs.biologists.org/content/113/9/1515.long>.
"Pinocytosis." Wikipedia. Wikimedia
Foundation, 04 Jan. 2013. Web. 10 Apr. 2013.
<http://en.wikipedia.org/wiki/pinocytosis>.
Feo, C., and N. Mohandas. "Clarification of Role of ATP
in Red-cell Morphology and Function." Nature 265.5590
(1977): 166-68.
Nature. Web. 24 Apr. 2013.
<http://www.nature.com/nature/journal/v265/n5590/pdf/265166a0.pdf>.
"Gliosis." Wikipedia. Wikimedia Foundation,
29 Mar. 2013. Web. 10 Apr. 2013. <http://en.wikipedia.org/wiki/Gliosis>.
James W Fawcett, Richard.A Asher, The
glial scar and central nervous system repair, Brain Research Bulletin, Volume
49, Issue 6, August 1999, Pages 377-391, ISSN 0361-9230,
10.1016/S0361-9230(99)00072-6.
(http://www.sciencedirect.com/science/article/pii/S0361923099000726)
"Microglia." Wikipedia. Wikimedia
Foundation, 04 Sept. 2013. Web. 10 Apr. 2013.
<http://en.wikipedia.org/wiki/Microglia>.
Jochen Gehrmann, Yoh Matsumoto, Georg
W. Kreutzberg, Microglia: Intrinsic immuneffector cell of the brain, Brain
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]
Research Reviews, Volume 20, Issue 3, March 1995, Pages 269-287, ISSN
0165-0173, 10.1016/0165-0173(94)00015H.
(http://www.sciencedirect.com/science/article/pii/016501739400015H)
http://icuc.wheatoncollege.edu/bio324/2013/belanger_karryanne/index.htm[7/1/2015 1:49:11 PM]