RESUMEN
Microtubule (MT)-motor systems show promise as nanoscale actuator platforms for performing molecular manipulations in nanobiotechnology and micro total analysis systems. These systems have been demonstrated to exert a variety of functions, including the concentration, transportation, and detection of molecular cargos. Although gliding direction control of MTs is necessary for these applications, most direction control methods are currently conducted using micro/nanofabricated guiding structures and/or flow, magnetic, and electric field forces. These control methods force all MTs to exhibit identical gliding behaviors and destinations. In this chapter, we describe an active multidirectional control method for MT without guiding tracks. The bottom-up molecular design allowed MTs to be guided in designated directions under an electric field in a microfluidic device. By designing the stiffness and surface charge density of MTs, three types of MT (Stiff-MT, Soft-MT, and Charged soft-MT) with different mechanical and electrical properties are prepared. The gliding directions within an electric field are predicted according to the measured stiffness and electrophoretic mobility. Finally, the Stiff-MTs are separated from Soft-MTs and Charged soft-MTs with a microfluidic sorter.
Asunto(s)
Fenómenos Mecánicos , Microtúbulos , Electricidad , Microtúbulos/químicaRESUMEN
BACKGROUND: Microtubules (MTs) are highly dynamic tubular cytoskeleton filaments that are essential for cellular morphology and intracellular transport. In vivo, the flexural rigidity of MTs can be dynamically regulated depending on their intracellular function. In the in vitro reconstructed MT-motor system, flexural rigidity affects MT gliding behaviors and trajectories. Despite the importance of flexural rigidity for both biological functions and in vitro applications, there is no clear interpretation of the regulation of MT flexural rigidity, and the results of many studies are contradictory. These discrepancies impede our understanding of the regulation of MT flexural rigidity, thereby challenging its precise manipulation. RESULTS: Here, plausible explanations for these discrepancies are provided and a new method to evaluate the MT rigidity is developed. Moreover, a new relationship of the dynamic and mechanic of MTs is revealed that MT flexural rigidity decreases through three phases with the growth rate increases, which offers a method of designing MT flexural rigidity by regulating its growth rate. To test the validity of this method, the gliding performances of MTs with different flexural rigidities polymerized at different growth rates are examined. The growth rate-dependent flexural rigidity of MTs is experimentally found to influence the pattern formation in collective motion using gliding motility assay, which is further validated using machine learning. CONCLUSION: Our study establishes a robust quantitative method for measurement and design of MT flexural rigidity to study its influences on MT gliding assays, collective motion, and other biological activities in vitro. The new relationship about the growth rate and rigidity of MTs updates current concepts on the dynamics and mechanics of MTs and provides comparable data for investigating the regulation mechanism of MT rigidity in vivo in the future.
Asunto(s)
Crecimiento , Microtúbulos/metabolismo , Transporte Biológico , Citoesqueleto , Aprendizaje Automático , Tubulina (Proteína)RESUMEN
Kinesin-driven microtubules have been focused on to serve as molecular transporters, called "molecular shuttles," to replace micro/nanoscale molecular manipulations necessitated in micro total analysis systems. Although transport, concentration, and detection of target molecules have been demonstrated, controllability of the transport directions is still a major challenge. Toward broad applications of molecular shuttles by defining multiple moving directions for selective molecular transport, we integrated a bottom-up molecular design of microtubules and a top-down design of a microfluidic device. The surface charge density and stiffness of microtubules were controlled, allowing us to create three different types of microtubules, each with different gliding directions corresponding to their electrical and mechanical properties. The measured curvature of the gliding microtubules enabled us to optimize the size and design of the device for molecular sorting in a top-down approach. The integrated bottom-up and top-down design achieved separation of stiff microtubules from negatively charged, soft microtubules under an electric field. Our method guides multiple microtubules by integrating molecular control and microfluidic device design; it is not only limited to molecular sorters but is also applicable to various molecular shuttles with the high controllability in their movement directions.
RESUMEN
One of challenges for using microtubules (MTs) driven by kinesin motors in microfluidic environments is to control their direction of movement. Although applying physical biases to rectify MTs is prevalent, it has not been established as a design methodology in conjunction with microfluidic devices. In the future, the methodology is expected to achieve functional motor-driven nanosystems. Here, we propose a method to guide kinesin-propelled MTs in multiple directions under an electric field by designing a charged surface of MT minus ends labeled with dsDNA via a streptavidin-biotin interaction. MTs labeled with 20-bp or 50-bp dsDNA molecules showed significantly different trajectories according to the DNA length, which were in good agreement with values predicted from electrophoretic mobilities measured for their minus ends. Since the effective charge of labeled DNA molecules was equal to that of freely dispersed DNA molecules in a buffer solution, MT trajectory could be estimated by selecting labeling molecules with known charges. Moreover, the estimated trajectory enables to define geometrical sizes of a microfluidic device. This rational molecular design and prediction methodology allows MTs to be guided in multiple directions, demonstrating the feasibility of using molecular sorters driven by motor proteins.