These experiments were conducted under the mentorship of Dr. Cecilia Arriagada in the Astrof Lab.
Our results have implications for treating cardiovascular development defects, with fibronectin (Fn1) playing a crucial role in morphogenesis and embryo development. Knockouts and mutants of Fn1 are shown to cause heart deformations in its developmental stage.
In the future, we hope to investigate ways integrin activation can be applied in vivo. The hope is that by targeting pathways that promote fibronectin rescue, we can save hearts and consequently lives in the earliest stages of human growth.
3. Methods - Generation of Fn1-KO cells
• LoxP (flox) sites recognized by Cre recombinase
• f/+ Cre+: homologous for Fn1, one allele gets cut, expresses Fn1 and
tdTomato
• f/- Cre+: no Fn1 expressed, GFP expressed instead of tdTomato
4. Experiment 1: Colocalization of
Fn1 in Coculture
Research Question: Does Fn1 assemble in a cell-autonomous manner?
Hypothesis: Fn1 assembles in a cell-autonomous manner. Fn1 will not assemble in
KO cells as readily as they do in WT cells, even in coculture.
5. Materials & Methods
• Three cocultures per trial: 90% WT + 10% KO, 75% WT + 25% KO, 25%
WT + 75% KO
• We prepared 4 trials (4x3 = 12 cocultures total, 10 pictures taken and
analyzed per coculture).
• To prepare coculture, used cell counting method. Optimal: 15000
cells per slide
• Immunofluorescence
• No permeabilization
• Primaries: Rabbit α -Fn1, Chicken α -GFP (1:500)
• Secondaries: DAPi (1:10000), rabbit 555 (1:300), chicken 488 (green color,
1:300)
6. Assembly of Fn1 is Cell-autonomous
Figure 1: Co-culture of WT and
Fn1-KO cells
Very little Fn1 colocalized in KO
cells.
Results imply that FN is assembled
in a cell-autonomous manner.
7. Results - Fn1 assembles more in WT cells than KO cells
W
T
9
0
%
K
O
1
0
%
W
T
7
5
%
K
O
2
5
%
W
T
2
5
%
K
O
7
5
%
0
2
4
6
8
10
Comparing Fn1 Coverage in WT Cells vs Fn1-KO Cells
in Cocultures with Diff. Concentrations
Cell Type &
Coculture Percentage
%
Cell
Area
Covered
by
Fn1
✱✱✱
*
*
WT KO WT KO WT KO
0
2
4
6
8
10
Comparing Fn1 Coverage in WT Cells vs Fn1-KO Cells
in Cocultures with Diff. Concentrations
Coculture Samples
%Cell
Area
Covered
by
Fn1
90% WT, 10% KO
75% WT, 25% KO
25% WT, 75% KO
Figure 2: Data used in the figures was analyzed using Fiji/ImageJ software and Prism.
A) Analysis of 4 trials. Only 3 trials were analyzed for 90%/10% and 25%/75%, while a 4th was used for 75%/25%
B) Similar graph, only using 2 trials. Error bars represent 1SEM.
A. B.
12. Fn1
depletion
reduces
Itga5 levels • Compares Itga5 fibril formation in WT vs KO cells
• Suggests that Fn1 activates Itga5 (likely in the secretion
process)
14. Experiment 2: Activation of Itga5
with Manganese
Research Question: How does Itga5 activation affect Fn1 assembly?
Hypothesis: Increasing Itga5 activation enhances Fn1 fibril assembly outside the
membrane, and vice versa.
15. Materials and Methods
• Need a way to culture WT cells in Mn without killing them
• Prepared WT and KO cultures (separately) in 0.5 mM and 1.0 mM
MnCl2 for varying durations
• 2h, 4h, 6h, 8h, overnight
• Immunofluorescence
• No permeabilization (Mn activates extracellular site of Itga5)
• Primaries: rabbit α-Fn1 287.1 (1:500), rat α-Itga5 (1:200), chicken α-GFP
(1:500)
• Secondaries: DAPi (1:2000), rabbit 555, rat 647, chicken 488 (all 1:300)
16. Materials and Methods
• Cocultures with/without Mn (15000 cells each)
• 100% WT
• 90% WT + 10% KO
• 75% WT + 10% KO
• 100% KO
• Immunofluorescence:
• No permeabilization
• Stained for Fn1
• Used auto-fluorescence for GFP and tdTomato cells
17. Testing Various Mn Concentrations/Durations
0.5
mM
for
6h
1.0
mM
for
6h
Fn1 Itga5 Merge Zoom Figure 3: WT cells
Plated in Different
Concentrations Of Mn
More integrin fibrils
visible in 1.0 mM, but
with greater cell death.
KO cells (not shown) all
survived.
We conclude that it’s
suboptimal to culture
WT cells in Mn.
Possibly because of
existing Fn1?
18. Cocultures w/w.o. Mn: Does it affect Fn1 assembly in KO cells?
No
Mn
Mn+
WT cells Fn1 in WT cells Merge Zoom (WT cells
shown)
KO cells Fn1 in KO cells
Figure 4: 75%/25%
coculture samples
with and without Mn
Pictures suggest that
Fn1 fibril assembly is
more successful in KO
cells with Mn than
without.
19. Problems Encountered
• WT tdTomato cells die in Mn if we increase the concentration
• At low concentrations of Mn, integrins don’t display sufficient
activation
Rather than using WT cells to produce Fn1 for the coculture, we can
use a Fn1-tdTomato medium.
20. Experiment 3: Itga5 and Fn1
Assembly in Fn1-tdTomato
Medium
Research Question: How does Itga5 activation affect Fn1 assembly?
Hypothesis: Increasing Itga5 activation enhances Fn1 fibril assembly outside the
membrane, and vice versa.
21. Materials and Methods
Mn- Mn+
Mn+ Mn-
Mn-
Mn+
Mn-
Mn+
With Fn1-tdTomato medium
No Fn1
All KO cells, permeabilized (to make
cytosolic Itga5 visible)
Primaries: rabbit anti-mCherry
(1:200), rat anti-Itga5 (1:200)
mCherry binds to Fn1-tdTomato
Secondaries: rat 647 (1:300), rabbit-
555 (1:300), DAPi (1:2000)
22. Materials and Methods
• With a high concentration of Fn1, Itga5
will activate even without Mn. We don’t
want this!
• Thus, the medium must be diluted to
mimic the concentration of a standard:
Our 90% WT 10% KO cultures
• Used ELISA to match Fn1 concentration
of the medium with the coculture
WT cells Medium
23. Plating in Fn-tdTomato Medium + Mn: Results
No
Mn
Mn+
GFP Fn1 Fn1 Itga5 Zoom Zoom
Figure 5: Comparing Fn1 and Itga5 fibril formation in Mn vs control
25. Fn1 Fibril Concentration - Results
M
n
+
N
o
M
n
M
n
+
N
o
M
n
0.0000
0.0005
0.0010
0.0015
0.0020
Fn1 Fibril Concentration in Cells
Treated with Manganese vs Without
With/without Mn
#
Fn1
Fibrils
/
Total
Area
of
Cells
Trial 1
Trial 2
Mn+ No Mn
0.0000
0.0005
0.0010
0.0015
0.0020
Fn1 Fibril Concentration in Cells
Treated with Manganese vs Without
Culture Type
#
Fn1
Fibrils
/
Total
Area
of
Cells
A. B.
Figure 6: Comparing Fn1 fibril concentration in Mn vs in control samples. Results suggest a significance difference between
the groups. In Mn solution, Fn1 forms more fibrils.
26. Conclusions
• Fn1 assembles cell-autonomously
• However, it can be rescued with integrin
activation
• Fn1 and Itga5 appear to have a “mutual
activation” relationship
For future investigation:
• How can we activate integrins in-vivo safely? How
much Fn1 restoration is too much?
• What’s the mechanism of integrin-assisted Fn1
assembly?
27. Thank you!
Special thanks to Dr. Cecilia Arriagada for
mentoring me through this project!
Also Dr. Astrof and the Astrof Lab’s members
for their support along the way!
If you’d like to contact me, email me at
elin0606@outlook.com.
