The application of EGFP as a means o f fluorescently labelling proteins for use in live cell
imaging experiments requires that the toxicity o f the fluorophore be assessed for the cell line
in question. Deleterious effects on cell behaviour that result as a consequence o f the over
expression and/or photo-toxicity o f the protein may render the fluorophore unsuitable for a
particular cell system.
X I5 sarcoma cells expressing EGFP were assayed in the Dunn chemotaxis chamber to
determine any possible effects that expression o f the fluorophore alone may have on cell
behaviour. The speed and directionality o f 131 EGFP expressing cells from 10 independent
chemotaxis experiments were analysed. The non-expressing cells within the same
experimental group were used as a comparative control. Differences in speed and
directionality between the expressing and non-expressing cell populations were assessed
using ANOVA.
EGFP expression was not found to have a significant effect on either the speed or
directionality of T15 sarcoma cells. Cells from both the expressing and non-expressing
populations exhibited strong chemotactic responses in PDGF-BB/IGF-1 gradients (Figure
4 4 A & B , page 19). See also the representative filmstrip in Figure 45A, page 19. The
fluorescent cells can clearly be seen to migrate towards the outer well o f the chemotaxis
chamber. No significant difference existed between the chemotactic response o f the
It was hypothesized that any toxic effects associated with EGFP expression would most
likely be manifested as an impairment o f cell motility. However, EGFP expression did not
have a noticeable effect on the speed o f cell motility (see trajectory plots Figure 44 C & D).
The ANOVA comparison o f the speeds o f non-expressing and expressing cells confirmed
that EGFP expression did not confer a significant inhibitory effect on the speed o f sarcoma
cell motility Figure 44E. Observations o f individual films also indicated that there was no
obvious effect o f EGFP expression on cell viability although this was not assessed
statistically.
EGFP fusion provided a useful means o f assaying the effects o f various proteins on the long
term motile behaviour o f T15 sarcoma cells in live cell imaging experiments. The fluorescent
protein alone had no effect on cell directionality and speed and thus provided a useful base
line control when analysing the effects of other EGFP fusion proteins on these aspects of cell
Control EGFP cells = 117 0 = - 1 1 . 5 “ p < 0 . 0 0 1 cells = 98 e = - 1 0 . 3 ° p < 0.001 - 2 0 0 - 1 0 0 0 1 0 0 2 0 0 C ontrol cells = 173 average speed = 13 3 nm/h - 2 0 0 - 1 0 0 0 100 200 EGFP cells =131 average speed = 11.7 pm/h C/3 2 5 n.s. 20 15 10 5 N = 173 N = 131
Figure 44. Effects o f EGFP
expression on sarcoma cell motility
Circular histograms, trajectory plots, and box and whisker charts demonstrating the effects o f EGFP expression on the chemotaxis and motility o f sarcoma cells. Figures summarise data obtained from the interactive tracking o f cells from 10 independent chemotaxis experiments. (A & B) Circular histograms demonstrating the directional response o f non expressing and EGFP expressing cells respectively. Both control and EGFP expressing cells exhibited strong chemotactic responses in PDGF- BB/IGF-1 gradients. EGFP expression did not significantly alter the chemotaxis o f cells (ANOVA, n.s.). (C & D) Trajectory plots fo r non expressing and EGFP expressing cells respectively revealing the individual paths, shifted to a common origin, o f cells over the course o f chemotaxis experiments. The paths o f both non expressing and EGFP expressing cells tended towards the source o f the gradient (top o f plot) over the course o f experiments. EGFP expression did not visibly suppress cell movement. Axes units are in microns. Filled circles denote the final position o f cells. (E) Box and whisker plots summarising the mean speeds o f non expressing (control) and EGFP expressing sarcoma cells over the course o f chemotaxis experiments. EGFP expression did not significantly impair cell speed (ANOVA, n.s.). Circles represent median values while boxes span 50 %, and whiskers span 80 % o f the data within each group.
3,3.3 Cdc42 and T e l0 are implicated in the motility but not the chemotaxis o fT lS rat sarcoma cells
Cdc42 function has previously been shown to be essential for the chemotaxis o f certain cells
o f the mammalian immune system including macrophages and T lymphocytes (Allen et al.,
1998; Haddad et al., 2001). It was therefore hypothesized that this protein would be o f
similar importance in the chemotaxis o f the T15 rat sarcoma cell line. To determine the role
o f Cdc42 in the chemotaxis of T15 rat sarcoma cells an EGFP fusion o f the dominant
negative variant Cdc42(T17N) was microinjected into cells and cell behaviour subsequently
assessed using the Dunn chemotaxis chamber. Figure 45B, page 19 is an image sequence
from a representative film demonstrating the directional response o f EGFP-Cdc42(T17N)
expressing cells recorded in the chamber. The analysis o f 57 cells from 7 independent
cultures revealed that EGFP-Cdc42(T17N) had no significant effect on the chemotaxis of
T15 sarcoma cells, with cells exhibiting a strong directional response in gradients o f PDGF-
BB/IGF-1 (Figure 47B, page 19). No significant difference existed between the chemotactic
response o f EGFP-Cdc42(T 17N) expressing cell and control cells expressing EGFP alone as
confirmed by ANOVA (Table 4, page 19). Expression o f wild-type protein also had no effect
on chemotaxis (Figure 48B, page 19). EGFP-Cdc42(wt) expressing cells could clearly be
seen to migrate towards outer well o f the chemotaxis chamber (see representative film
sequence Figure 46B, page 19) and no significant difference existed between the directional
response o f these cells and controls (Table 4). In all cases the non-expressing cell populations
provided inbuilt controls for chemotaxis, confirming that effective chemotactic gradients had
established during individual experiments, see Figure 49, page 19 (dominant negative
The expression o f dominant negative Cdc42 did, however, affect the speed o f T15 rat
sarcoma cell motility. EGFP-Cdc42(T17N) expressing cells were capable o f detecting and
translocating towards the source of the chemotactic gradient but they did so more slowly than
the surrounding, non-expressing cells (this is clear from the image sequence in Figure 45B).
Cells exhibited a 25 % reduction in speed when compared to EGFP control cells. Trajectory
plots illustrating the individual paths taken by cells during the course o f experiments
demonstrate effectively the suppressive effects that Cdc42 inhibition had on the speed o f cell
motility (Figure 5 IB, page 19). Plots reveal that the paths o f EGFP-Cdc42(T17N) expressing
cells were visibly suppressed compared to those o f EGFP control cells (Figure 51, compare B
with A). The significance o f the speed reduction was confirmed by ANOVA (p < 0.01)
(Figure 53A, page 19 & Table 5, page 19). Expression o f the wild-type protein had no
significant effect on cell speed (Figure 53B & Table 5), confirming that the reduction in
speed was specific to the dominant negative mutation and not a consequence of toxicity
associated with an increased presence o f Cdc42 protein per se. Note how the trajectories of EGFP-Cdc42(wt) expressing cells were similar to those o f control cells expressing EGFP
alone (Figure 52, compare B with A. See also example film sequence. Figure 46B & A).
As Cdc42 was not required for the chemotaxis o f T15 rat sarcoma cells it was possible that
the closely related GTPase TclO was o f more importance in regulating chemotactic
behaviour in this particular cell line. TclO shares many down-stream signalling target with
EGFP-PAK1(83-149) EGFP-PAK1(1 -149) EGFP-N-WASP(AVCA) EGFP-Tc10(T31N) EGFP-Cdc42(T17N) EGFP
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m
Figure 45, (previous page) Film sequences o f migrating sarcoma cells expressing inhibitory constructs
Image sequences from chemotaxis experiments demonstrating the behaviour o f Tl 5 sarcoma cells in gradients o f PDGF-BB (80 ng/ml)/IGF-l(60 ng/ml) following microinjection and expression o f various inhibitory constructs fused to EGFP. Each image sequence is derived from a representative film fo r a given treatment group and represent 0, 8 and 16-hour time points. Images are composites o f overlaid phase contrast and EGFP fluorescence channels allowing the simultaneous observation o f both expressing and non-expressing cells. Cells were microinjected with expression constructs encoding either EGFP control (A), EGFP- Cdc42(TI7N) (B), EGFP-TcI0(T31N) (C), EGFP-N-WASP(AVCA) (D), EGFP-PAKI(1 - 149) (E), or EGFP-PAK1(83 - 149) (F). Yellow trajectories illustrate the individual paths o f cells expressing fluorescent proteins, while red trajectories represent those o f the non expressing cells. White filled circles illustrate the current position o f each cell fo r the image shown. In all experiments the camera was rotated so that the outer well was positioned at the top o f each image. Consequently the direction o f increasing growth factor concentration runs from the bottom to the top o f each image, as represented by the white arrow in the first image
o f the first sequence. Scale bar - 1 0 0 pm.
Figure 46, (overleaf) Film sequences o f migrating sarcoma cells expressing wild type constructs
Image sequences from chemotaxis experiments demonstrating the behaviour o f TI 5 sarcoma cells in gradients o f PDGF-BB (80 ng/ml)/IGF-l (60 ng/ml) following microinjection and expression o f various wild-type constructs fused to EGFP. Cells were microinjected with expression constructs encoding either EGFP control (A), EGFP-Cdc42(wt) (B), EGFP- TclO(wt) (C), EGFP-N-WASP(wt) (D), or EGFP-PAKl(wt) (E). Time intervals, image compositions, orientation o f the chemotactic gradient, and scales are the same as fo r Figure 45.
EGFP-PAK1 (wt) EGFP-N-WASP(wt) EGFP-Td 0(wt) EGFP-Cdc42(wt) EGFP
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O)
fibroblast cell lines, a phenotype often associated with Cdc42 expression (Neudauer et al.,
1998). Furthermore, TclO has been implicated in control o f axonal outgrowth in neurons
(Abe et al., 2003), and is therefore an important regulator o f cell polarity in other mammalian
systems.
Expression o f an EGFP fusion o f the dominant negative TclO variant, TclO(T31N), had no
effect on the chemotaxis o f T15 sarcoma cells. The analysis o f 35 cells from 5 independent
cultures revealed that EGFP-Tc 10(T31N) expressing cells could exhibit a strong chemotactic
response in PDGF-BB/IGF-1 gradients (see representative film sequence in Figure 45C,
page 19). A significant chemotactic response was confirmed for cells expressing both
dominant negative and wild-type proteins (Figure 47C, page 19 & Figure 48C page 19
respectively). EGFP-Tc 10(T31N) expression did, however, impair cell speed (Figure 53A,
page 19). Interestingly the degree o f speed inhibition was comparable to that associated with
the inhibition o f Cdc42 (25 % reduction). Again, the reduction in speed was specific to the
dominant negative variant o f the protein and was significant compared to the speed o f control
cells (Table 5, page 19). As with cells expressing EGFP-Cdc42(T17N), the trajectories of
EGFP-Tc 10(T31N) expressing cells appeared suppressed compared to those o f control cells
(Figure 51 compare C & A, page 19). Interestingly, the expression o f EGFP-TclO(wt)
significantly enhanced the speed o f cell motility (Figure 53B & Table 5). Endogenous TclO
expression levels may therefore present a limiting factor in the speed o f T15 rat sarcoma cell