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Contact Information
1505 S. Atlantic Blvd. #E
Alhambra, CA 91803
Email: francislin96@yahoo.com
Tel: (+011-626-281-2637)
SUMMARY OF KEY FINDINGS
The actin binding proteins, Bnr and Bud6, are necessary for normal regulation of actin. Deletion of Bnr and Bud6 results in misregulation of the mutant actins, N115T and R116Q, as well as
increased proliferation of cells expressing these mutations. These findings are depicted in the cell growth curves, doubling times, and growth under stress conditions.
BACKGROUND
• Thoracic Aortic Aneurysm and Dissection (TAAD) is
characterized by dilation and rupture of the aortic wall
• Responsible for 0.5% to 1% of deaths in the U.S. per year
• 20% of the patients inherit the disease
• ACTA2, a gene that codes for α-smooth muscle actin, is the
most common genetic cause of TAAD
• N115T and R116Q are of special interest because their
location on the actin monomer suggests involvement in
intermonomer interactions within the filament
• Formins are actin-binding proteins and are involved in actin
polymerization
OBJECTIVES
• Observe and analyze the regulation of N115T and R116Q
mutations upon deletion of Bnr and Bud6
• Observe mutations under stress conditions
• Hypothesis: Select mutations in actin differentially affect
regulation by a select group of actin-binding proteins that
facilitate polymerization.
VARIABLES
Controls: Wild Type (WT), N115T, and R116Q at 30°C
Manipulated Variables: Mutations – N115T and R116Q
ΔBnr vs. ΔBnrΔBud6
Stress Conditions – 24°C, 30°C, 37°C, YPG, 0.5M NaCl, 0.9M NaCl
Dependent Variables: Doubling Time
Growth Curve
Stress Condition Plates
RESULTS
DISCUSSION
• Cells expressing the mutant actins doubled in growth
approximately 30 minutes faster than WT cells
• ΔBnr strains had a slightly faster doubling time than
ΔBnrΔBud6
• Proposed hypothesis was proven to be correct.
• Deleting Bnr and Bud6 results in misregulation and
excessive proliferation of cells
• Select mutations in actin differentially affect
regulation by a select group of actin-binding proteins
that facilitate polymerization
STUDY IMPLICATIONS
• While both N115T and R116Q mutations cause
TAAD, N115T results in stroke and R116Q results in
coronary artery disease
• The effect of Bnr and Bud6 deletion on cells
expressing N115T and R116Q have not previously
been studied
• Results could provide further insight into the N115T
and R116Q clinical phenotypes
• Complete characterization of ACTA2 mutations could
lead to a better understanding of the clinical
phenotypes and potentially result in improved
treatments
FUTURE RESEARCH
• Repeat experiments to determine accuracy
• Assess effects of ΔBnr and ΔBnrΔBud6 on WT cell
growth and other TAAD mutations
• Further investigation into why N115T and R116Q
have faster doubling times than wild type
• Determine the mutation-specific cause for TAAD
development in ACTA2 mutations
ACKNOWLEDGEMENTS
Heather L. Bartlett
Peter A. Rubenstein
Alyson R. Pierick
Nicole D. Vanderpool
Elesa W. Wedemeyer
Alex Greiner
University of Iowa Biochemistry Department
Regulation of Mutant Actin by Actin-Binding Proteins
Francis Lin
Mentor: Heather Bartlett
Department of Biochemistry, University of Iowa Carver College of Medicine, 51 Newton Rd., Iowa City, Iowa, 52242-1109
Doubling Time
Cell Type Hours
WT 2.63
115* 2.68
N115T ΔBnr 2.15
N115T ΔBnrΔBud6 2.22
116* 2.65
R116Q ΔBnr 2.10
R116Q ΔBnrΔBud6 2.11
0
5
10
15
20
25
30
0 20 40 60 80
O.D.600
Hours
Cell Growth Curve
WT
115*
115 ΔBnr
115 ΔBnr ΔBud6
116*
116 ΔBnr
116 ΔBnr ΔBud6
24°C 37°C
0.5M NaCl 0.9M NaClYPG
*Extrapolated from previous data*Extrapolated from previous data
Legend
1- WT
2- N115T ΔBnr
3- N115T ΔBnrΔBud6
4- R115Q ΔBnr
5- R115Q ΔBnrΔBud6
30°C
1000x
1x
10x
100x
1000x
1x
10x
100x
1000x
1x
10x
100x
1000x
1x
10x
100x
1 2 3 4 53 4 51 2 3 4 51 2 3
4 51 2 3 4 51 2 3 4 51 2 3
1
2
3
4
C
N
N115T
R116Q
Figure 1. Actin Monomer
Figure 2. Actin Filament
Bni1p or
Bnr1p
Actin Filament
Actin Monomer
Pointed End
Barbed EndBni1p and Bnr1p are
yeast formins. They
facilitate the addition of
monomers to the barbed
end of the filament
during polymerization.
Bud6p enhances Bni1p.
SUMMARY OF KEY FINDINGS
• Saccharomyces cerevisiae, or budding
yeast, was used as a model since it is
easy to manipulate. Yeast actin shares
a 94% similarity with human actin.
• A spectrometer was used to determine
total cell density in order to obtain a
growth curve for cells expressing the
mutant actins.
• Spotting of cells was done using 1x,
10x, 100x, and 1000x dilutions. Stress
conditions included 24°C, 30°C, and
37°C. A restricted carbon source
(YPG), 0.5M NaCl, and 0.9M NaCl
were additional stress conditions.
METHODS
The results from the spotting indicates that there is misregulation
among the mutations when Bnr and/or Bud6 are deleted. The
mutations display increased cell density, suggesting that the cell
has lost control of its cell proliferation mechanism.
All other possible
ACTA2 mutations
BACKGROUND
OBJECTIVES
METHODS
VARIABLES
RESULTS
DISCUSSION
STUDY IMPLICATIONS
FUTURE RESEARCH
ACKNOWLEDGEMENTS