1. Evaluation of cargo carrying efficiency of acto- myosin II motors in in-vitro motility assays Murali k. Gadde 1, Hideyo Takatsuki Ph.D. 1, David Neff 3, Madhukar kolli M.S. 1, Kevin M. Rice, M.S 1, 2, Siva K Nalabotu 1,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. Introduction Experimental details Acknowledgements 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. A biological living cell is a miniature factory that is equipped with protein machines to perform complex mechanical tasks such as intracellular transport or cell division. In particular, we study actomyosin bionanomotors in artificial environments, which have proven to be versatile building materials in construction of nano-mechanical devices. In previous studies, many researchers successfully demonstrated the assembly of these myosin motors on solid substrates like nitrocellulose for in-vitro motility assays (Refs. 2,3,5,6). Recently, Hideyo Takatsuki from Dr. Blough’s lab has successfully prepared actin bundles by using fascin protein (Ref. 1) and demonstrated that myosin II transports these actin bundles loaded with microparticles and E.coli in in-vitro motility assays (unpublished data). In support of this work, we are trying to image rhodamine labeled actin bundle coupled to genetically engineered, green fluorescent protein expressing, E.coli by biotin-streptavidin interactions (Fig.1B). To improve our ability to analyse time dependent activities in our assay, we attempt to image both carriers and cargo in the same field simultaneously. Myosin II was extracted from the back and leg muscles of a rabbit and heavy meromyosin (HMM) was prepared from it by using α- chymotrypsin (Margossian and Lowey,1982). G-actin was prepared from actin acetone powder of chicken breast muscle and then the polymerized actin filaments were labeled with 1:1 stoichiometric mixture of tetramethyl rhodamine-phalloidin (Molecular probes) and Biotin-phalloidin(Molecular probes). Human fascin-1 was expressed and collected from a genetically transformed E. coli strain and purified using column chromatography (thanks to Dr. Kohama). Purified fascin was mixed with F-actin in (1:3 mass ratio) in buffer and allowed to incubate overnight at 4 0 for preparing fascin bundled actin filaments (Fig.1C). We used artificially biotinylated E. coli that had been genetically engineered to express green fluorescent protein as the cargo to be carried. Materials : Methods : We prepared flow cells that had dimensions of approximately 6mm by 0.09 mm in cross section with nitrocellulose coating on the interior surface of coverslip (Fig. 1A). The flow cell was filled with 120 µg/ml HMM diluted in the assay buffer (25 mm KCl, 2.0 mm MgCl 2, 0.2 mm CaCl 2 and 25 mm imidazole at pH 7.0) and incubated for 5 minutes to allow immobilization of HMM onto nitrocellulose. Bovine serum albumin (0.1 % BSA in water) was used to prevent the actin bundles from nonspecifically binding to the surface. After 5 min incubation, the flow cell was washed with the assay buffer, and biotinylated bundles were introduced into the flow cell. After washing to remove unbound bundles, the flow cell was filled with neutravidin followed by biotin labeled GFP-E.coli. Flow cell was washed with assay buffer after addition of linker and cargo. We confirmed (microscopically) cargo linkage to bundles before adding ATP. 1.5mM ATP was added and system was observed under fluorescence microscope (Nikon te200, Japan) having Optosplit II (CAIRN) (Fig.1D) with a 60X objective (1.4 N.A., oil-immersion) with EMCCD camera. This setup allows us to image two wavelengths simultaneously and in near perfect registration on the same camera chip (Fig.1E, Fig. ). In addition we compared the actin filaments and actin bundles by taking high resolution images and tried to image E.coli loaded actin bundles in confocal laser scan microscopy(Bio- Rad MRC1024) with optical characteristics as seen in Fig.1F A necessary part of cell physiology involves the transport and localization of bio-molecules and subcellular structures by linear motion motor proteins using chemical energy as fuel. These transport mechanisms have inspired the integration of Bio-molecular motors into nano--engineered structures related to assembly and transport of nano or micro sized synthetic materials. We report the kinematic characteristics of myosin II in in-vitro motility assay with faster velocity of actin than kinesin-propelled microtubule and the potential ability of fascin bundled actin to control unidirectional movement without U-turn and to confine bundled actin movement in microchannels. Additionally it is demonstrated that bundled actin can be coupled with micro-particles as well as E-coli cells engineered to express green fluorescent proteins and they are transported over myosin II Bundled actin/myosin II system can be a new actuator for transport of molecular cargos in micro fluidic chip and NEMS (nano electro-mechanical systems). Abstract Neutravidin References 1. Ryoki Ishikawa, Takeshi Sakamoto, Toshio And, Sugie Higashi-Fujime and Kazuhiro Kohama Polarized actin bundles formed by human fascin-1; their sliding and dissembly on myosin II and myosin V in-vitro,J.Neurochem. 2003 87, 676-685 2. L. Jia, S. G. Moorjani, T. N. Jackson, W. O. Hancock, Microscale transport and sorting by kinesin molecular motors. Biomed. Microdevices 6, 67-74 (2004). 3. M. Sundberg, R. Bunk, N. Albet-Torres, A. Kvennefors, F. Persson, L. Montelius, I. A. Nicholls, S. Ghatnekar-Nilsson, P. Omling, S. Tagerud, A. Mansson, Actin filament guidance on a chip: toward high- throughput assays and lab-on-a-chip applications, Langmuir 22, 7286-95 (2006). 4. M. G, van den Heuvel, C. Dekker, Motor proteins at work for nanotechnology, Science 317, 333-6 (2007). 5. S. J. Kron, J. A. Spudich, Fluorescent actin filaments move on myosin fixed to a glass surface, Proc. Natl. Acad. Sci. U S A 83, 6272-6 (1986). 6. M. G. van den Heuvel, C. T. Butcher, R. M. Smeets, S. Diez, C. Dekker, High rectifying efficiencies of microtubule motility on kinesin-coated gold nanostructures, Nano Lett. 5, 1117-22 (2005). Figure 2: A & B Images of actin filaments and GFP-E.coli in long wavelength channel and short wavelength channel of optosplit respectively (see Fig. 1E&D). C. Image of GFP-E.coli (white arrows) and actin bundles in long wavelength channel of confocal laser scanning microscope (see Fig. 1F). Ideally, E.coli emissions would be filtered out of this detector channel. D. Emission spectrum of rhodamine phalloidin and EGFP showing broad EGFP emissions and the effect of overwhelming EGFP emissions over those of rhodamine. of the confocal laser scanning microscopic images (see Fig. 2E, white arrows indicate the E.coli that are not supposed to show up in this channel that is filtered by a 598nm / 40nm bandpass filter). We concluded that it is difficult to image both the rhodamine fluorophore labeled actin bundle and the EGFP simultaneously and so will continue work with cargo that is either less bright or cargo/carrier labels that are spectrally further separated from each other. We measured the length and widths of actin filaments and actin bundles from high resolution CSLM images (Fig. 3A-E) and these values are compared between the actin filament and fascin bundled actin filaments (see Fig. 3E). Data from electron micrographs (reference 1, Fig. 1C) indicate that fascin bundled actin (made similarly to ours, in a 1:3 fascin to actin ratio) have a mean width of 136nm with SD of 44nm. Two factors combine to explain our much larger diameters: 1 - Dr. Kohama ’ s fascin bundles (in Ref. 1) are not complexed with phalloidin as are ours, we are unsure as to how this actin binding molecule will change the dimensions of the actin bundles and; 2 – light microscopy has much lower resolving power than electron microscopy. Traditional definitions of diffraction limited resolution in light microscopy would predict a resolution value for our CSLM of ~200nm. This level of resolution is achieved when measuring small features on the surface of EGFP expressing E. coli (fig. 3F). As mentioned above, these bacteria fluoresce brightly thus allowing us to minimize the size of our confocal iris and maximize resolution (fig. 3G). However, we confirmed that the least width of both actin filaments and actin bundles (smallest diameter found in all samples of both groups), are the same at around 330 nm (Fig. 3 C&D). This implies that, due to resolution constraints, we are unable to measure features smaller than 330 nm. As mentioned above, rhodamine/actin emissions were relatively weak which necessitated that we use a large confocal iris to gather more light, this compromised our resolving power (fig. 3G). The fact that actin bundles averaged far above this 330nm value (754nm) implies that this broadening is not entirely due to limited resolving power. Finally, our attempts to link our E.coli cargo with fascin bundles showed a very low efficiency of linkage as measured by # of cargo-carrier complexes / # total actin bundles. Efficiency was measured at ~3% ( Fig. 4C). One example of this linkage is seen in figure 4B, there were 2 such linkages in the scanned sample field, these are shown in 4A inset and 4B. Notice in 4A the yellow appearance of the EGFP expressing E. coli, this is due to the intense emissions detected in both confocal channels (Fig. 1F). This merged image, 4A, shows our actin bundles only in red because their much lower emissions were well confined to this channel. Results and Discussion We detected signal from rhodamine-phalloidin and EGFP on the same camera chip with the optosplit setup (see Fig. 1D&E and 2A & 2B). Arrows in 2A&B indicate labeled actin filaments in the rhodamine channel and E.coli in GFP channel. However we are unabale image these actin filaments and E.coli simultaneously due to the dominance of EGFP emissions over those of rhodamine. As the emission spectra of EGFP is so broad and bright (Fig. 2C&D) that they are seen even in the red channel Results and Discussion Contd. HgXe lamp; peaks at 405, 436, 546, 577nm Sample Fixed mirror Cairn Optosplit 2 image splitter Fixed mirrors camera chip Fixed mirror D Blue Laser Yellow Laser Rd-Ph Emission filter GFP Emission filter F Biotin Figure 1: A. Basic setup of motility assay flow cell. B. Diagram of expected coupling of E.coli to Actin bundle by biotin-streptavidin linkage. C. TEM image of Fascin bundled actin (Dr. Kohoma). D. Optosplit setup. E. & F. Excitation/emission fluorescence spectra of Rhodamine phalloidin (dk. green/red) and EGFP (blue/lt. green) plotted with optical charachteristics of optosplit (E.) and Confocal microscope (F.). A Nitrocellulose coverslip Actin bundle E.coli B C 1145 E Figure 3: A & B Images of actin filaments and actin bundles from CSLM respectively, measurements were taken from images like these. C & D Images of actin filaments and actin bundles with lowest observed width. E. Graphs showing the comparision of length and width between actin filaments and actin bundles. F. Confocal Image of GFP-E.coli. G. Confocal iris adjustments and effect on resolution of images. Figure 4: A & B are confocal images of flow cell after loading of cargo (E.coli) to the actin bundles. Figure A shows many unlinked carriers and cargo and few that are linked. Inset from Fig. A and Fig. B are images from smaller regions in the original sample field specifically highlighting the loaded actin bundle (also visible in these are unloaded actin bundle). C is a table showing the values for determining coupling efficiency (entire field was analyzed). c When each samples contain only one fluorescent molecule (rhodamine in A and GFP in B), camera settings allow emission channel separation; in C this is not the case.