Abp1 is known to dimerize. Srv2 is known to hexamerize. We hypothesize that this polymerization leads to the cross-linking ability of Abp1 and Srv2, directly influencing actin dynamics. We utilized photobleaching and total internal reflection fluorescence (TIRF) microscopy to visualize actin dynamics in real time.

1. Effects of Actin Crosslinking Proteins on Actin Network Remodeling
Sumana Shashidhar, Alec Hoyland, and Sean Guo
Dept. of Quantitative Biology, Brandeis University, Waltham, MA 02454
Introduction Methods
Discussion
Our experiments conclusively determine that Abp1 and
Srv2 act as crosslinking proteins, consolidating actin
networks. We demonstrate that the actin filaments
coalesce into crosslinked fibers, producing a denser
structure. Our quantitative analysis demonstrated
significant effects of each protein individually and in
tandem. We found that the effect of Abp1 compounds with
Srv2 producing a multiplicative model of reduced returns.
At the final time point (t = 1200 s) the area is represented:
𝐴 𝐴𝑏𝑝1+𝑆𝑟𝑣2 1200
𝐴 𝐴𝑏𝑝1+𝑆𝑟𝑣2 0
= 1 −
𝐴 𝐴𝑏𝑝1 1200
𝐴 𝐴𝑏𝑝1(0)
1 −
𝐴 𝑆𝑟𝑣2 1200
𝐴 𝑆𝑟𝑣2(0)
This is the formula for calculating the fold-change given
two statistically independent operations, indicating that the
mechanisms of Abp1 and Srv2 are biochemically
independent, but that interaction with the same substrate
alters the crosslinking efficiency of each protein species.
Results
The protein actin forms many structures with various
functions that are central to cell functioning. Actin plays an
important role in biological processes such as movement, the
transportation of vesicles and organelles, endocytosis, cell
division, and sensing the environment [1]. These processes
are dependent upon the interactions between actin
filaments and other proteins that aid in crosslinking and
forming actin networks such as actin patches and the
contractile ring [1].
Actin-binding protein 1 (Abp1) and suppressor of Ras-Val19
(Srv2) are two of these crosslinking proteins that enable actin
to form networks or bundles and play a role in cellular
activities. This project studies these proteins in a system
constructed with actin, so that the networks that Abp1 and
Srv2 create can be analyzed in vitro. Better understanding
how these crosslinking proteins form actin networks allows
for the mechanisms of many biological processes to be better
defined. A basis for the development of hypotheses and
models is then developed.
Abp1 is known to dimerize [2]. Srv2 is known to hexamerize
[3]. We hypothesize that this polymerization leads to the
cross-linking ability of Abp1 and Srv2, directly influencing
actin dynamics. We utilized photobleaching and total internal
reflection fluorescence (TIRF) microscopy to visualize actin
dynamics in real time.
Sources
[1] Pollard, Thomas D. and John A. Cooper. "Actin, a Central Player in
Cell Shape and Movement." Science 326, no. 5957 (2009): 1208-
1212. Accessed March 10, 2016.
[2] Woo E-J, Marshall J, Bauly J, et al. Crystal structure of auxin-
binding protein 1 in complex with auxin. The EMBO Journal.
2002;21(12):2877-2885.
[3] Chaudhry F, Breitsprecher D, Little K, Sharov G, Sokolova O,
Goode BL. Srv2/cyclase-associated protein forms hexameric
shurikens that directly catalyze actin filament severing by cofilin.
Pollard TD, ed. Molecular Biology of the Cell. 2013;24(1):31-41.
Protein Analysis: Concentration
Question
We expressed a recombinant protein in E. coli through the
transfection of genes via antibiotic plasmids attached to
the lac operon. The proteins were purified from the cell
lysate by biochemical methods. Column chromatography;
His-tag affinity was used to purify Srv2. To determine
concentration, two methods were used.
Spectrophotometric methods analyzed the absorbance of
aromatic rings using known values and Beer’s Law. SDS-
PAGE was also performed on the proteins to assay purity.
Oligomeric state was assessed by single molecule
photobleaching using TIRF microscopy. Real-time analysis
of actin network remodeling in the presence of Abp1 and
Srv2 was then assessed using TIRF microscopy. 10% of actin
filaments were labeled with fluorophores in a microfluidic
chamber. Each protein was flowed in individually. In
addition, a trial with equal half concentrations of each
protein was performed.
Special Thanks
We would like to thank Avital Rodal and Bruce Goode and
their dogs.
Fig. 2
Fig. 1 Fig. 3
Fig. 4
Photobleaching
Fig. 1 and 2 indicate the photobleaching steps as
indicated by Heaviside drops in fluorescence over
time. Fig 3 shows the initial fluorescence of Abp1
and Fig. 4 the fluorescence at t = 190 s.
Due to incomplete binding of fluorophores to
proteins and Boltzmann dissociation of polymers,
we observed incomplete full polymerization. The
maximum polymeric Markov state accounting for
experimental error indicates the maximal
polymerization state for the protein: dimer for
Abp1 and hexamer for Srv2.
Over time, fluorophores “burn out” and cease
illumination. This process is temporally-correlated
and random, allowing us to observe quantized
drops in radiant flux of individual protein
polymers.
-10
0
10
20
30
40
0 1 2
NumberofAbp1Proteinswith
CertainNumberof
PhotobleachingSteps
Number of Photobleaching Steps
Photobleaching of Apb1 Protein
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200 1400
Area/InitialArea
Time (s)
Abp1: Normalized Area of Radiance over
Time
Fig. 8
Quantitative Analysis
Fig. 8 and 9 measure the area of the
viewing plate upon which actin
fluoresces above the noise
threshold. Actin is shown to
crosslink. Fig. 11 and 12 show the
mean intensity of the fluorescence.
The area of illumination decreases as
the crosslinking proteins aggregate
the actin into crosslinked polymeric
fibers. The radiant flux increases as
the area above threshold decreases
due to crosslinking. These
quantitative measures indicate the
crosslinking capabilities of Abp1 and
Srv2.
Fig. 10 measures the normalized
area of both proteins acting in
concert and Fig. 13 measures the
normalized mean intensity.
The normalized area and mean of
both proteins acting together obeys
probabilistic laws of independence.
0.8
0.9
1
1.1
1.2
1.3
1.4
0 200 400 600 800 1000 1200 1400
MeanIntensity/IntitialMean
Intensity
Time (s)
Abp1: Normalized Mean Radiant Flux above
Threshold
Fig. 10
Fig. 9
Fig. 11
Fig. 5, 6, and 7. Apb1, Srv2, and a 1:1 mixture acting on the actin network in silico
Actin Dynamics
Fig. 5 and 6 visualize a montage of
the actin network, with an image
taken every 20 seconds under TIRF
microscopy. Both proteins crosslink
actin filaments, producing thickened
fibers. Fig. 7 shows the action of
Abp1 and Srv2 acting together in a
1:1 mixture.
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000
Area/InitialArea
Time (s)
Abp1 and Srv2: Normalized Area of
Radiance over Time
0
0.5
1
1.5
2
0 200 400 600 800 1000
MeanIntensity/InitialMean
Intensity
Time (s)(
Abp1 and Srv2: Normalized Mean Radiant
Flux above Threshold
Fig. 12
Fig. 13
Method 1: SDS – PAGE Method 2: Spectrophotometry
After denaturing the protein and placing diluted
protein samples in the gel, the molecular weight can
be determined through comparison with the ladder as
seen in Fig. 14. Impurities are separated and purity of
the protein can be assessed.
Fig. 14
Determining the absorbance of 2uL samples of
the Abp1 and Srv2 at 280 nm allowed for
concentration to be calculated using the known
extinction coefficient and pathway length.
Impurities are not separated and counted within
the concentration.
0
0.2
0.4
0.6
0.8
1
1.2
1
6
11
16
21
26
31
36
41
46
51
56
61
66
71
76
81
86
91
Area/InitialArea
Time (s)
Srv2: Normalized Area of Radiance over
Time
0
0.5
1
1.5
1
6
11
16
21
26
31
36
41
46
51
56
61
66
71
76
81
86
91
MeanIntensity/InitialMean
Intensity
Time (s)
Srv2: Normalized Mean Radiant Flux above
Threshold