Prolonged function and optimization of actomyosin motility for upscaled network-based biocomputation

Abstract

Significant advancements have been made towards exploitation of naturally available molecular motors and their associated cytoskeletal filaments in nanotechnological applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biological system. Here, we systematically investigated potentially critical factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay solution composition and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from <20 min to >60 min. In addition, we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chemical oxygen scavenger system (glucose oxidase–glucose–catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted commercially available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at −80 °C for more than 10 years before use for nanotechnological purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.

Through the Eyes of Creators: Observing Artificial Molecular Motors

“…One can get an idea of what these may be from the already emerging applications of biological motors, reviewed in refs and . These emerging applications include biocomputation, , biosensing, − diagnostic devices, and self-assembling structures . All these devices are based on cytoskeletal microtubules or actin filaments driven by kinesin and myosin.…”

“…In the future, microfabricated or similar patterned structures can be used for guiding molecular motors for a targeted delivery of cargos such as liposome vesicles and microstructures . There is a development of nanodevices with nanofabricated structures used for biocomputation when explored by the guided movement of cytoskeletal filaments propelled by molecular motors. , Directional movement of bead-based motors could be an alternative to the filaments in performing the computation. Channels can also serve as “testing grounds” for artificial motors, where effects of confinement are studied, for example, for bead-based motors of varying size, for which narrower channels can facilitate the directional bias but also may decrease the binding footprint.…”

“…The motors implemented so far have often been designed for a specific function, such as molecular switches and rotors, ,− transport of cargoes, , recording digital information, employing quantum tunnelling for directional motion, phototherapy of cancer, controlled supramolecular aggregation, governing cellular processes, and energy-efficient performance at low temperatures . In addition, artificial motors could be an advantageous alternative to their natural counterparts that are currently employed in emerging applications such as highly energy-efficient biocomputing devices. , It is also possible to modify natural motors by adding functional groups to the molecules or by using synthetic scaffolds for the motion of the natural motors. , …”

Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and Cells

“…Additionally, dysfunctional HMM may be blocked by unlabeled actin filaments increasing the overall actin filament velocity. 29 Next, we test if we can pull lipid nanotubes from GUVs using the directional force of gliding actin filaments. In order to bind actin filaments to the lipid membrane of GUVs as illustrated in Figure 2a, we prepare filaments with 10% biotinylated actin monomers and verify the successful functionalization with SDS-PAGE (SI Figure S1).…”

“…This might be due to the existence of defined tracks for aligned actin that allow for a higher motor processivity compared to when actin filaments are randomly distributed. Additionally, dysfunctional HMM may be blocked by unlabeled actin filaments increasing the overall actin filament velocity …”