Role of tubulin C-terminal tail on mechanical properties of microtubule.

Tubulin C-terminal tail (CTT) is a disordered segment extended from each tubulin monomer of αß tubulin heterodimers, the building blocks of microtubules. The tubulin CTT contributes to the cellular function of microtubules such as intracellular transportation by regulating their interaction with other proteins and cell shape regulation by controlling microtubule polymerization dynamics. Although the mechanical integrity of microtubules is crucial for their functions, the role of tubulin CTT on microtubule mechanical properties has remained elusive. In this work, we investigate the role of tubulin CTTs in regulating the mechanical properties of microtubules by estimating the persistence lengths and investigating the buckling behavior of microtubules with and without CTT. We find that microtubules with intact CTTs exhibit twice the rigidity of microtubules lacking tubulin CTTs. Our study will widen the scope of altering microtubule mechanical properties for its application in nano bio-devices and lead to novel therapeutic approaches for neurodegenerative diseases with altered microtubule properties.

Purification of Tubulin from Porcine Brain and its Fluorescence Dye Modification.

Filamentous microtubules, polymers of the heterodimeric protein tubulins play one of the major roles in the emergent nano-biotechnological devices. To develop the feature of those devices, it is important to understand the function of microtubule in in vitro, hence, the availability of purified αß-tubulin is required. Additionally, fluorescently labeled tubulin has become a powerful approach for extensively studying the dynamics of these components. In this chapter, the process of purifying the heterodimeric αß-tubulin from porcine brain will be described, as well as the process of labeling of the purified tubulin with fluorescence dye.

Cargo Transport by Microtubule-Associated Motor Protein Along Mechanically Deformed Microtubules.

Mechanical forces play pivotal roles in regulating various cellular functions. Biomolecular motor protein-driven intracellular transportation is one example which is affected by mechanical forces, although the mechanism at molecular level is unknown. In this chapter, we describe deformation of microtubules under compressive stress and we show that such deformation of microtubules affects the kinetics of dynein-driven cargo transportation along the microtubules. The extent of alteration in the kinetics of dynein-driven transportation is found strongly dependent on the extent of deformation of microtubules under compressive stress.

Mechanical Deformation of Microtubules on a Two-Dimensional Elastic Medium.

Microtubule, the most rigid filamentous protein in cytoskeleton, plays significant roles in cellular mechano-transduction and mechano-regulation of cellular functions. In cells, the mechanical stress serves as a prevalent stimulus to frequently cause deformation of the microtubules participating in various cellular events. While the experimental and simulation-based approaches have confirmed the role of mechanical stress to tune mechanical properties of microtubule. Yet, the effect of mechanical force on the structural stability and the mechanism of microtubule deformation have remained obscure. Here, we describe the mechanical stress-induced deformation of microtubules using a custom-made mechanical device. We designed the device in a way which allows the microtubules to undergo deformation as response to the applied stress while attached on a two-dimensional elastic substrate through interaction with microtubule-associated motor protein, kinesin. We provide here the method to cause controlled bucking or fragmentation of microtubules by applying compressive or tensile stress on the microtubules, respectively. Such study is crucial to understand the mechanism of deformation in microtubules in cellular environment and their consequences in physiological activities.

Deformation of microtubules regulates translocation dynamics of kinesin.

Microtubules, the most rigid components of the cytoskeleton, can be key transduction elements between external forces and the cellular environment. Mechanical forces induce microtubule deformation, which is presumed to be critical for the mechanoregulation of cellular events. However, concrete evidence is lacking. In this work, with high-speed atomic force microscopy, we unravel how microtubule deformation regulates the translocation of the microtubule-associated motor protein kinesin-1, responsible for intracellular transport. Our results show that the microtubule deformation by bending impedes the translocation dynamics of kinesins along them. Molecular dynamics simulation shows that the hindered translocation of kinesins can be attributed to an enhanced affinity of kinesins to the microtubule structural units in microtubules deformed by bending. This study advances our understanding of the role of cytoskeletal components in mechanotransduction.

Regulation of Biomolecular-Motor-Driven Cargo Transport by Microtubules under Mechanical Stress.

Mechanical stress on cells has profound influences on biological processes, such as cell shape regulation, the formation of tissue patterns, and development. Recently, mechanosensing properties of the microtubule, an important cytoskeletal component, have drawn attention. In this work, we studied cargo transport by dynein, a microtubule-associated motor protein, along microtubules deformed under mechanical stress. We reveal that the microtubule deformation took place as a response to the applied stress and that the deformation of microtubules facilitated the transport of dynein-driven quantum dots. This finding will provide opportunities to explore the role of microtubules as molecular mechanotransducers in cellular processes.

Stabilization of microtubules by cevipabulin.

We report the utility of cevipabulin as a stabilizing agent for microtubules. Cevipabulin-stabilized microtubules were more flexible compared to the microtubules stabilized by paclitaxel, the most commonly used microtubule stabilizing agent. Similar to the paclitaxel-stabilized microtubules, cevipabulin-stabilized microtubules were driven by kinesins in an in vitro gliding assay. The velocity of cevipabulin-stabilized microtubules was significantly higher than that of paclitaxel-stabilized microtubules. These findings will enrich the variety of microtubules with difference in mechanical and dynamic properties and widen their applications in nanotechnology.