Alternative Techniques for Determining Myosin
Density in a Standard Actin Myosin Motility Assay
Kevin M. Rice, M.S1, 2, Shinichi Asano M.S. 1, Hideyo Takatsuki Ph.D. 1, David Neff M.S. 3, and Eric R. Blough, Ph.D.1,2 :
1 Department of Biological Sciences, Marshall University, 2 Department of Physiology, Pharmacology, and Toxicology, Joan C. Edwards School of Medicine,
Marshall University, 3 Department of Chemistry, Marshall University,
Abstract
Background: Active transport in cells, utilizes molecular motors like kinesin and myosin. Such biological nano-machinery has provided the inspiration for integrating active transport into nano-synthetic devices. The first
prototypes of such molecular shuttles are hybrid devices employing motor proteins in a synthetic environment. However, working on the nano-scale has proven to require the development of new understanding
regarding the physics, chemistry and biology of such sub-microscopic systems. To date no man-made load-carrying motor smaller that 1 um bas been developed. With this in mind nanotechnologist has set out to
unravel the mysteries of nano-scale mechanics utilizing the cells predesigned machinery. Unraveling these mysteries has driven scientist to develop new and more advanced mechanisms for visualizing the activity in
the nano-world. Recent studies have demonstrated that in vitro actin motility is dependent on many factors including surface composition, PH, temperature, ionic concentration of solution and motor density. Many
techniques have been developed to determine motor density such as ATPase activity, heavy myromyosin (HMM) depletion fro solution, and total internal reflection fluorescence. These techniques have provided a
means of estimating motor density but have not been able to demonstrate the physical surface density. Here we investigate alternative techniques for determining myosin motor density in a standard actomyosin motility
assay. Through the use of antibody protein interaction and various microscopic techniques we have demonstrated variation in surface density, with the hope of determining the exact surface density of the HMM.
A.
Methods cont.
3
velocity ( μm / sec ).
Introduction
The in vitro motility assay developed in the 1980s has proven
to be a valuable experimental system for the study of actin
myosin function. Nanotechnologists are looking to these
biological nano-motor proteins as models for the construction
of synthetic nano-motors, or as key components in the
development of nano-devices. Recent studies have
demonstrated that in vitro actin motility is dependent on many
factors including surface composition, pH, temperature, ionic
concentration of solution and motor density. Many techniques
have been developed to determine motor density such as
ATPase activity, HMM depletion from solution, and total
internal reflection fluorescence microscopy (TIRF). These
techniques have provided a means of estimating motor density
but have not been able to demonstrate the physical surface
density.
Actual Velocity
2.5
3) HMM coated cover slips were incubated for 1 hr at room
temperature biotin labeled G-actin 8ug/ml. Cover slips were
then incubated with Rhodamine labeled streptavidin and
visualized with confocal microscopy. 4) HMM coated cover
slips were incubated for 1 hr at room temperature with 1:100
dilution of abcam™ myosin antibody. Cover slips were then
incubated with a Texas Red conjugated secondary and
visualized with confocal microscopy. 5) Cover slips were
incubated for 1 hr at room temperature with 1:100 dilution of
abcam ™ myosin antibody. Cover slips were then incubated
with a Q-dot ™ conjugated secondary and visualized with
confocal microscopy using ex. and em. parameters below.
2
1.5
1
0.5
0
60
90
120
150
180
[HMM] μg/ ml
B.
label
Texas red
Q-Dot ™
Purpose
Methods
The inverted, gliding motility assay was used to measure
changes in the velocity of Fascin bundled F-actin. In short,
flow cells were constructed from microscope slides, doublesided tape and a glass cover slip. Standard cover slips
preparation was prepared by coating cover slip with
nitrocellulose by placing 50ul of isoamyl acetate solution
containing 0.2 % nitrocellulose on the cover slip and
incubating at 80 C for 1 hour. Motility studies were conducted
using varying concentrations of heavy myromyosin (HMM) (60,
90, 120, 150, and 180 ug/ul). To determine the binding
efficiency of the HMM, standard cover slip preparations were
incubated with heavy myromyosin (HMM) (60, 90, 120, 150,
and 180 ug/ul) for 5 min. After incubation cover slips were
blocked with 0.1% or 5% BSA for 5 min. HMM coated cover
slips were then analyzed using 5 techniques. 1) HMM coated
cover slips were incubated for 1 hr at room temperature with
1:100 dilution of abcam™ myosin antibody. Cover slips were
then incubated with a 40 nm Gold conjugated secondary and
visualized using atomic force microscopy (AFM), scanning
electron microscopy (SEM), and SEM with energy dispersive
x-ray spectroscopy (EDS). 2) HMM coated cover slips were
incubated for 1 hr at room temperature with 1:100 dilution of
abcam ™ myosin antibody. Cover slips were then incubated
with 40 nm Gold conjugated secondary, sliver enhanced for
~20 min and visualized using atomic force microscopy (AFM),
scanning electron microscopy (SEM), and SEM with energy
dispersive x-ray spectroscopy (EDS).
D.
8
7
6
5
4
3
2
1
0
60 ug
2500
Frequency
.
C.
Surface RMS (nm)
To examine alternative techniques for measuring myosin
surface density in a standard actin myosin motility assay.
3000
90 ug
120 ug
150 ug
180 ug
2000
1500
1000
500
0
1
Dehydrated
19 37 55 73 91 109 127 145 163 181 199 217 235 253
8 Bit Pixel Value
Hydrated
120 ug
60 ug
90 ug
150 ug
180 ug
E.
Silver enhanced 40 nm
Gold bead
20nm Q-Dot labeled
2 ° Antibody
40 nm Gold bead
em.
598/40nm
705/40nm
Results
The effect of HMM concentration of the Fascin bundled F-actin
is shown in figure 1a. With increase in HMM concentration
velocity increases. Nitro-cellulose surface topography is
altered with addition of hydrating solutions (Figure 1b, c). This
increase in surface topography may play a role in various
components of motility but is not likely to be the causative
agent in HMM concentration dependent changes to velocity.
All techniques used to determine HMM density were
unsuccessful. Technique number 4 using the Texas Red
labeled secondary with confocal microscopy showed a trend of
increasing intensity (figure 1d). Figure 1e is a pictorial diagram
of the various techniques utilized in this study.
Conclusion
In the present study we show that HMM density alters Fascin
bundled F-actin motility. However we were unable to
definitively detect changes in surface density through the
techniques utilized in this study.
Rhodamine labeled
Streptavidin
Texas Red labeled 2
° Antibody
Biotin labeled GActin
ex .
568nm
488nm
Acknowledgements
HMM 1°
Antibody
1
2
HMM
3
4
5
Figure 1. A) Velocity curve of Fascin bundled F-actin motility. B), 3D representation of
dehydrated and re-hydrated Nitrocellulose coated cover slips (AFM). C) Root mean
square roughness of the dehydrated and re-hydrated Nitrocellulose coated cover
slips D), 8 bit histographic analysis of Texas Red labeled HMM cover slips utilizing
techniques number 4. And E) Pictorial diagrams of the various techniques utilized in
this study.
Grant support for this study was provided by
NSF Grant 0314742 and NIH AG027103 to Eric Blough.
Dr. M.L. Norton for maintaining the MBIC imaging facilities