Chapter 3. Results
3.2 Caveolin-1 signaling pathway
We further investigated whether or not caveolin-1 played a role in α2β1 integrin
and lipid raft with electric field stimulation. Caveolin-1 polarized while DC electric
field stimulation (Figure 9). Knockdown of caveolin-1 expression by RNAi impair
cell directionality but not impact on cell motility (Figure 10 and Figure 12).
Knockdown caveolin-1 show no effect on caveolin-1 polarization but impair integrin
distribution; however, integrin functional block show no effect on lipid raft
polarization but impair caveolin-1 distribution (Figure 9 and Figure 11). RhoA
polarization induced by electric field also disrupt while RNAi treatment and lipid raft
deconstruction (Figure 13). It seems that the polarization of caveolin-1 and α2β1
integrin coincide with quantity of caveolin-1 and function of α2β1 integrin. The data
13
imply that caveolin-1 on the lipid raft could control galvanotaxis though RhoA and
cooperation with α2β1 integrin.
14
Chapter 4.
Discussion
It is evident from the results that overall, lipid rafts polarization induced by
electric field guide cell migration though caveolin-1. That electric field causes the
lipid rafts polarization correspond with that lipid raft can merge into large and stable
structure under stimulation. The polarization of lipid raft shares a similar pattern with
these of α2β1 integrin. These consist with previous finding, which show that α2β1
integrin polarize under electric field stimulation [9]. As previous research by Leitinger
indicated, lipid raft localization are relevant to integrin cluster [22]. The α2β1 integrin
colocalize with lipid raft. The deconstruction of lipid raft impairs the redistribution of
α2β1 integrin; however, the function inhibition of α2β1 integrin shows no change on
the polarization of lipid raft. It is indicated that lipid raft regulate the distribution of
α2β1 integrin with electric field stimulation.
According to Hart’s formula, the force from membrane, environment viscosity
can affect the electric force exerting on the membrane molecules [31]. The drag force
and viscous force could influence the lipid raft polarization. The larger size of lipid
raft causes the higher drag force from membrane. Furthermore, the huger lipid raft
can bear more plasma membrane proteins. The possible mechanism on AC sin wave
15
electric field is that the merge of lipid raft induced by electric field could reduce their
diffusion rate. Lipid rafts gather with each other at beginning then stay behind at
reverse electric field. It is reasonable that the larger lipid rafts become huger due to
the possibility of their contacting with small one. As hypothesized, lipid rafts polarize
at the cell area to which the initial 16ms current orient under AC sin wave electric
field stimulation. The opposite initial currents compared to DC electric field
determine the orientation of lipid raft redistribution. This finding reveals that lipid raft
can polarize at briefly electric field exposure time.
As expected, the increase of membrane viscosity by cholestryl hemisuccinate can
inhibit the lipid raft polarization not only on AC but also on DC. Consequently, the
results show that the lipid rafts size and the membrane viscosity affect lipid raft
diffusion rate. Lipid raft clustering is dependent on the initial orientation of current
due to the change of diffusivity.
Our results show that knockdown of caveolin-1 can impair galvanotaxis. How
caveolin-1 affect cell migration is that Rey-Barroso delineates that caveolin-1 control
focal adhesion and migration though activating FAK and β1 integrin [25]. Moreover,
the knockdown of caveolin-1 affect integrin polarization and functional block of α2β1
integrin influence caveolin-1 distribution. It imply that integrin and caveolin
cooperate with each other [24].
16
It also indicated that RhoA polarization induced by electric field is impaired by
caveolin-1 knockdown and lipid raft disruption. RhoA cause actin-based contraction
and lead to cell migration. RhoA is located on fibroblasts podosomes, which rich in
F-actin related to cell adhesion [32]. Therefore, the RhoA location affects the cell
motility. Our results show that lipid raft can affect directional migration though
controlling RhoA polarization.
We find that lipid raft polarize under electric field stimulation. The polarization
guides cell directional migration though caveolin-1. The initial orientation of electric
current determines the area at which lipid raft distribute and the direction to which
cell migrate. In future study, we would investigate the signaling mechanism of
caveolin-1 and RhoA. For instance, previous research indicates that
integrin/caveolin-1 complex could activate RhoA though p190 RhoGAP [24].
17
17
Table 1. Primers used in this study (Primer-BLAST, NCBI)
Pr im er An ne al in g Te m pe ra tu re (℃ ) P ro duc t si ze (bp) F or w ar d T CC CT G CT T CT A CCG G C G CT R ev er se G CC A G C CCC A G C A T C A A A G G T F or w ar d A CA T C T CT A CA C CG T CCC CA R ev er se A C G TC G TC G TTG A G A TG C TT
GAP D H Ca ve ol in -1
S eque nc e 5 ' t o 3 ' 59 281 54 181
18
18
Table 2. siRNA for caveolin-1 (Qiagen)
N am e S eq ue nc e 5' t o 3' Co nc . (n m ol /tu be ) T ar ge t C T GGAAT A AAT T C A AAT T C T T S en se s tr an d GGAAUAAAUUC A AAUUC UUUU An tis en se s tr an d AAGAAUUUGAAUUUAUUC C A G 20
Figu
20
10 mm
0.4 mm
3 mm
Slide
Microfluidic Channel
2% Agarose salt bridge
Figure 2. Microfluidic Channel.
It is made of MEMS. The channel is 0.4 mm height, 10 mm length and 3 mm width.
Two pores are punctured as an inlet and an outlet. The channel combines with glass
slide and connects to power supply with 2% agarose salt bridge.
Figu
Figu
Figu
24
Figure 6. α2β1 integrin riding lipid raft
(a) Electric field induces the AI increase of α2β1 integrin and MβCD impair that. (b)
Anti-α2β1 integrin antibody blocks the integrin function but does not affect the AI of
lipid raft. (* P < 0.05 DC vs. 0V, $ P < 0.05 Groups vs. Control, n = 21-50)
25
26
Figure 7. Lipid raft polarization disrupt by Ch and MβCD
The asymmetric distribution (AI) of lipid raft results from DC or AC. Significantly,
the AI of DC drop while treated with Ch and the AI of AC approximate to 0 while
treated with MβCD (* P < 0.05 Groups vs. 0V , $ P < 0.05 Groups vs. Control , &
P<0.05 Groups vs. DC, n = 17-118).
27
Figure 8. Ch and MβCD impair galvanotaxis
(a) DC EF stimulation enhances the fibroblasts’ migration speed but AC shows not
difference with 0V. No significant difference between control and the groups treated
with MβCD or Ch when fibroblasts exposed on 6V/cm. However, MβCD or Ch
improve the velocity simulated by AC. It seems that MβCD or Ch could enhance cell
performance induced by AC. (b) The directionality simulated by DC is higher than 0V
(a)
28
and by AC is more negative than 0V. It represents that the majority of fibroblasts
migrate toward cathode while DC electric field stimulation and toward anode while
exposed on AC. The MβCD and Ch could reduce the DC directionality and increase
the AC directionality. Therefore, pharmaceutical treatments inhibit directional
migration induced by DC or AC. (* P < 0.05 Groups vs. 0V, $ P < 0.05 Groups vs.
Control, & P<0.05 Groups vs. DC, n = 42-171)
Figu
Figu
Figu
Fibr
(* P
ure 11 Cave
roblasts wit
P < 0.05 Gro
eolin-1 knoc
th
caveolin-oups vs. 0V,
ckdown disr
1 knockdow
V, $ P < 0.05
31
rupt α2β1 in
wn show low
Groups vs.
ntegrin distr
wer integrin
. Control, n
ribution
n AI than ne
= 20-26)
egative conttrol.
32
Figure 12. Cav1 knockdown reduce directionality.
(a) Fibroblasts increase their migration velocity whether or not the caveolin-1
knockdown. The expression level of caveolin-1 do not alters cell motility.
(b) Caveolin-1 knockdown inhibit the directionality. (* P < 0.05 Groups vs. 0V, $ P
< 0.05 Groups vs. Control, n = 40-95)
Figu
34
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