Photo: Ksergent527 CC BY-SA4.0
Figure: Images obtained using TIRF microscopy showing actin filaments (green) bound to surface adsorbed myosin motor fragments before (left) and 30 s after (right) addition of 5 nM Alexa647-labelled (red) gelsolin in the presence of micromolar Ca2+. Note presence of single, red, gelsolin molecules at several filament ends after the gelsolin induced fragmentation, consistent with gelsolin capping of one of the filament ends after fragmentation.
A new method to regenerate bionanodevices forms an important milestone to saving material and energy and thus, towards sustainable alternative chip technology. The devices do not need to be disassembled, but are treated with proteinase K and washed with mild detergent. Thereafter the surfaces can be functionalized with motility proteins again and used for subsequent gliding assays. The quality of the assays after regeneration is similar to before.
Regeneration of Assembled, Molecular-Motor-Based Bionanodevices
M.A. Rahman, et al. Nano Letters 2019
Two important techological developments form the cornerstones of Frida Lindberg's doctoral thesis:
Ups and downs of two standard ways to improve motility in the in vitro motility assay by removing the effects of "dead" non-functional heads.
Rahman, M.A., Salhotra, A. & Månsson, A. J. Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay. Muscle Res Cell Motil (2019).
High throughput nanofabrication: To enable large scale network-based computation using molecular motors, a key requirement is the ability to fabricate big networks with a high resolution at a high throughput. All these devices require loading zones to feed filaments into the channel networks, regardless of the computational problem encoded. Nanoimprint lithography is an excellent, large scale fabrication method, but becomes difficult for high aspect ratio structures such as loading zones. By introducing nanoscaled pillars, we are able to high aspect ratio structures and provide a possibility to tailor the emptying rate and directional guiding of actin filaments.
Lindberg, F. W. et al. Design and development of nanoimprint-enabled structures for molecular motor devices. Materials Research Express 6, (2019).
Development of miniaturized devices for the rapid and sensitive detection of analyte is crucial for various applications across healthcare, pharmaceutical, environmental, and other industries. Here, we report on the detection of unlabeled analyte by using fluorescently labeled, antibody-conjugated microtubules in a kinesin-1 gliding motility assay. The detection principle is based on the formation of fluorescent supramolecular assemblies of microtubule bundles and spools in the presence of multivalent analytes. We demonstrate the rapid, label-free detection of CD45+ microvesicles derived from leukemia cells.
Chaudhuri, S. et al. Label-Free Detection of Microvesicles and Proteins by the Bundling of Gliding Microtubules. Nano Letters 18, 117-123, 2018 doi:10.1021/acs.nanolett.7b03619
In order to facilitate rapid screening of compounds affecting kinesin motor activity as well as optimizing kinesin-based in vitro motility assays for nanotechnological applications, we developed an in vitro motility assay where sample preparation, imaging and data evaluation are fully automated, enabling the processing of a 384-well plate within less than three hours. We demonstrate the automated assay for the analysis of peptide inhibitors for kinesin-1 at a wide range of concentrations.
Korten, T., Tavkin, E., Scharrel, L., Kushwaha, V. S. & Diez, S. An automated in vitro motility assay for high-throughput studies of molecular motors. Lab on a Chip 18, 3196-3206, 2018.
Controlled surface chemistry: The guiding of actin filaments is strongly regulated by the surface chemistry. By regulating the surface hydrophobicity using only one type of silane, we are able to maintain the same, motility promoting chemical environment while tuning the actin filament velocity.
Lindberg, F. W. et al. Controlled Surface Silanization for Actin-Myosin and Biocompatibility of New Polymer Resists. Langmuir 34, 8777-8784, 2018.
This is a review-article about the extent by which results from studies of single molecules of myosin and actin directly predict mechanical and contractile properties of muscle cells. The paper generally finds good agreement between model behavior based on parameters from single molecule studies and experimental data from muscle. This suggests that the 3D order of the contractile machinery in muscle together with a range of accessory proteins in addition to actin and myosin only have a minor modulatory role in the mechanisms leading to development of force and motion. This knowledge about basic mechanisms relies on input from Bio4comp but is also important for the project as actin and myosin are molecular work-horses that drive our computational machines.
Mansson, A., Usaj, M., Moretto, L. & Rassier, D. E. Do Actomyosin Single-Molecule Mechanics Data Predict Mechanics of Contracting Muscle? International Journal of Molecular Sciences 19, (2018).
The small-molecular chemical compound blebbistatin can be used together with varied ATP concentration and varied ionic strength to fine-tune the gliding velocity of myosin propelled actin filaments in biocomputation devices. This is desirable e.g. to tune multiplication rate of actin filaments to velocity. In this study we characterize the effects of blebbistatin on velocity as well as its mechanism of action on the force- and motion-generating cycle of myosin with actin. In these studies we also unveil hidden secrets in this process in the absence of any modifying substance.
Rahman, M. A., Usaj, M., Rassier, D. E. & Mansson, A. Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin. Biophysical Journal 115, 386-397, (2018).
Engineering cargo-loading strategies is crucial to developing nanotechnological applications of microtubule-based biomolecular transport systems. Here, we report a highly efficient and robust bioconjugation scheme to load antibodies to microtubules.
Chaudhuri, S., Korten, T. & Diez, S. Tetrazine-trans-cyclooctene Mediated Conjugation of Antibodies to Microtubules Facilitates Subpicomolar Protein Detection. Bioconjugate Chemistry 28, 918-922, (2017).
This review article describes the biological properties of actin filaments and how these properties may be altered by chemical engineering of the filaments. Such engineering is of relevance for Bio4Comp both for efforts to multiply actin filaments, to make them less flexible and to tag the filaments for certain computation approaches.
Kumar, S. & Mansson, A. Covalent and non-covalent chemical engineering of actin for biotechnological applications. Biotechnology Advances 35, 867-888, (2017).
Highly-efficient guiding of motile microtubules on non-topographical motor patterns. A UV-laser-based ablation technique can be used to programmably generate highly localized patterns of functional kinesin-1 motors on PLL-g-PEG-coated, planar polystyrene surfaces. We demonstrated that straight and curved motor tracks with widths of less than 500 nm reliably guide gliding microtubules. Moreover, the performance of complex motor patterns, recently designed by evolutionary algorithms for controlling the global directionality of microtubule motion on large-area substrates, could be experimentally verified. The presented results may open up new routes toward controlling microtubule motion in self-organized hybrid-devices.
Reuther, C., Mittasch, M., Naganathan, S. R., Grill, S. W. & Diez, S. Highly-Efficient Guiding of Motile Microtubules on Non-Topographical Motor Patterns.
Nano Letters 17, 5699-5705, (2017).
This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under grant agreement No 732482.
Call: FETPROACT-2016; Type of Action: RIA (Research and Innovation Action)