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PAR-1 Alters Morphology of Schwann Cells as Assessed by Cytoskeletal Staining
Samuel Hulbert, Victoria Turgeon, PhD, and Callie Van Koughnett
Furman University; Department of Biology, Greenville, SC 29613
ABSTRACT
METHODS
The purpose of this study was to investigate the morphological
changes associated with activation of PAR-1 in Schwann cells.
Previous work has shown that oligodendrocytes in the central
nervous system (CNS) show a morphological response to
thrombin/SFLLRNP and PAR-1 activation; however, less is
understood about the response of Schwann cells in the peripheral
nervous system (PNS). Cultured Schwann cells were treated with
SFLLRNP, a synthetic thrombin-like peptide chain, and stained at
12, 24, 48, and 96 hours. The number of cell processes (0, 1, 2, 3,
4+) was counted for each cell using fluorescent imaging as a
quantitative indicator of cell morphology. Relative percentages of
cells with 0, 1, 2, 3, and 4+ processes were significantly different
between the wells treated with SFLLRNP and controls for all time
points, with a p-value range of p<0.0001 and p=0.0195. As a
general trend treated cells are more likely to have fewer extensions
than untreated, especially at 2 and 3 uM concentrations.
Cells were cultured from a previously cryo-frozen line of Schwann cells using the standard Dulbecco’s modified
(DMEM + FBS) media. Schwann cells were placed in a 24 well plate at a 500 cell/ml concentration with 1 ml in
each well and given 24 hours for attachment. At 24 hours, cells were treated with either 1uM, 2uM, or 3uM or
0uM (control) SFLLRNP. Figure 2 shows the setup for each plate treatment. Immunocytochemisty was used to
stain cells, actin filaments, nuclei, and, ideally, focal adhesion points, at each plate’s respective time point: 12 hr,
24 hr, 48 hr, and 96 hr. Specific antibodies used were TRITC/Rhodamine-phalloidin. Cells were located and
imaged in 5 locations of each well, approximating the center, and four quadrants of the well for each picture.
The images were then analyzed by tallying the number of cells in each treatment and time point with 0, 1, 2, 3,
or 4+ cell processes.
DISCUSSION
As a general trend treated cells are more likely to have fewer
extensions than untreated, especially at 2 and 3uM
concentrations. The distribution of the 1uM treatment was slightly
more erratic and this may be attributed to the variety of PAR-1
responses that can occur. It has been shown that various body
cells respond differently to thrombin and its inhibitors especially in
reference to vascular damage in the nervous system [3].
Therefore, the 1uM treatment may have a compensatory effect on
Schwann cells, while increasing the amount of thrombin (or
damage) would be detrimental.
INTRODUCTION
Of note to Schwann cell behavior are the frequency of bipolar or 2
process category cells. Although higher in the control, they were
seen frequently in all treatments and are assumed to denote axon
“searching” behavior. These cells were most often found together
with other cells of the same category as shown in Figure 5 below.
Figure 2: Well Plate Treatment Map
24 Hour
0.6
0.4
1 uM
0.2
2 uM
3 uM
0
0
1
2
3
4+
control
Percentage of Cells
12 Hour
Figure 5: Bi-polar Schwann cell Behavior
0.5
0.4
0.3
1 uM
0.2
2 uM
0.1
3 uM
0
0
Number of Cell Processes
1
48 Hour
0.3
1 uM
0.2
2 uM
0.1
3 uM
0
1
2
3
3
4+
96 Hour
0.4
0
2
control
Number of Cell Processes
4+
Number of Cell Processes
control
Percentage of Cells
Schwann cells are neuroglial cells found in the peripheral nervous
system that myelinate the axons of motor neurons insulate nerve
fibers outside the central nervous system. Schwann cells function by
locating an axon by an extension of their plasma membrane and
then wrapping themselves around the numerous times, resulting in
an insulating cover around
the entire axon. The
membrane extension is
supported by a variety of
myelin proteins, creating a
myelin sheath that increases
the resistance of the axon
membrane to improve nerve
signaling. Figure 2 shows an
image of Schwann cells from
the 48 hour control group.
RESULTS
Percentage of Cells
PAR-1 is a large G-class protein that sits in the membrane of many
cells in the body and can be activated by thrombin. Thrombin
activation leads to a PAR-1 response that can induce multiple cell
activities, including cell shape changes, cell death, inhibited
repair/regeneration, and reduction in cell proliferation and growth
[2,3]. Nervous cells and their response to PAR-1 is of significant
interest due to the fact that vascular damage in areas of the nervous
system has been shown to impair the nervous system’s ability to
repair itself. Previous work has shown that oligodendrocytes in the
CNS show a morphological response to thrombin/SFLLRNP and
PAR-1 activation; however, less is understood about the response of
Schwann cells in the PNS [1].
Figure 1: Counting Cell Processes
Percentage of Cells
The purpose of this study was to investigate the morphological
changes associated with activation of PAR-1 in Schwann cells.
Previous studies have shown that PAR-1 is activated by SFLLRNP,
a synthetic peptide chain that mimics the serine protein Thrombin
[1,3]. Differences in morphologies were studied using fluorescent
imaging of the actin filaments in the cytoskeleton of the Schwann
cells. The number of cell processes or extensions was counted for
each cell as a quantitative indicator of cell morphology (Figure 1).
Figure 4 shows the percentage of cells with 0, 1, 2, 3 or 4+
processes for each treatment group and time point. A Pearson’s
Chi Square Test was run for each frequency distribution, using the
proportion of cells in each treatment or control group. Relative
percentages of cells with 0, 1, 2, 3, and 4+ processes were
significantly different between the wells treated with SFLLRNP
and controls for all time points, with a p-value range of p<0.0001
and p=0.0195.
RECOGNITIONS
0.4
0.3
1 uM
0.2
2 uM
0.1
3 uM
0
0
1
2
3
In order to assess the point at which thrombin or SFLLRNP
treatments become significant, further study should be completed
using smaller concentrations. As the 1uM treatments were
significant it would be informative to test concentrations in
between 0uM and 1uM in the future. In addition, using alternative
cell classifications (other than cell processes) to assess cell
morphology may be of benefit. For example, staining focal
adhesion points and excluding mitotically active cells would both
offer clarity into cell morphology and classification during study.
4+
control
1. Turgeon, VL et al. Thrombin perturbs neurite outgrowth and induces apoptotic cell death
in enriched chick spinal motorneuron cultures through caspase activation. J.
Neurosci. 1998; 18(17): 6882-6891
2. Turgeon, VL, Houenou, LJ. The Role of Thrombin-like proteases in the development,
plasticity and pathology of the nervous system. Brain Research Reviews 1997; 25:
85-95.
3. Turgeon, VL, Salman, N, Houenou, LJ. Thrombin: A Neuronal Cell Modulator.
Thrombosis Research 99 (2000): 417-427.
Number of Cell Processes
Furman University; Howard Hughes Medical Institution: RET Grant Program
Figure 3: Example of stained cell image
Figure 4: Percentage of cells with 0, 1, 2, 3 or 4+ processes for each treatment group and time point