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,

AbstractBackground: 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.

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.

Introduction

MethodsThe 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, double-sided 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).

PurposeTo examine alternative techniques for measuring myosin surface density in a standard actin myosin motility assay.

Results

Conclusion

AcknowledgementsGrant support for this study was provided byNSF Grant 0314742 and NIH AG027103 to Eric Blough.Dr. M.L. Norton for maintaining the MBIC imaging facilities

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.

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.

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.

B.

D.

A. Actual Velocity

0

0.5

1

1.5

2

2.5

3

60 90 120 150 180

[HMM] μg/ ml

velo

city

( μm

/ se

c ).

0

500

1000

1500

2000

2500

3000

1 19 37 55 73 91 109 127 145 163 181 199 217 235 2538 Bit Pixel Value

Freq

uenc

y

120 ug 60 ug 90 ug 150 ug 180 ug

60 ug 90 ug 120 ug 150 ug 180 ugC.

012345678

Dehydrated Hydrated

Surfa

ce R

MS

(nm

)

.

40 nm Gold bead

Silver enhanced 40 nm Gold bead

Biotin labeled G-Actin

Rhodamine labeled Streptavidin

Texas Red labeled 2 ° Antibody

1 2 3 4 5

HMM 1° Antibody HMM

20nm Q-Dot labeled 2 ° Antibody

Methods cont.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.

label ex . em.Texas red 568nm 598/40nmQ-Dot ™ 488nm 705/40nm

E.