Lindegaardgonzales5217

Since the first moderate resolution, structural description of Taxol bound to tubulin by electron crystallography in 1998, several tubulin crystal systems have been developed and optimized for the high-resolution analysis of tubulin-ligand complexes by X-ray crystallography. Here we describe three tubulin crystal systems that have allowed investigating the molecular mechanisms of action of a large number of diverse anti-tubulin agents.High-speed atomic force microscopy (AFM) is a versatile method that can visualize proteins and protein systems on the nanometer scale and at a temporal resolution of 100 ms. The application to microtubules can not only reveal structural information with single-tubulin resolution but can also extract mechanical information and allows to study single motor proteins walking on microtubules, among others. This chapter provides a step-by-step guide from microtubule polymerization to successful observation with high-speed AFM.The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. How γTuRC nucleates microtubules, and how nucleation is regulated is not understood. To gain an understanding of γTuRC activity and regulation at the molecular level, it is important to measure quantitatively how γTuRC interacts with tubulin and potential regulators in space and time. Here, we describe a total internal reflection fluorescence microscopy-based assay on chemically functionalized glass slides for the in vitro study of surface immobilized purified γTuRC. The assay allows to measure microtubule nucleation by γTuRC in real time and at a single molecule level over a wide variety of assay conditions, in the absence and presence of potential regulators. This setup provides a previously unavailable opportunity for quantitative studies of the kinetics of microtubule nucleation by γTuRC.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.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.Since its discovery, several decades ago, microtubule dynamic instability has been the subject of countless studies that demonstrate its impact on cellular behavior in health and disease. Recent studies reveal a new dimension of microtubule dynamics. Microtubules are not only dynamic at their tips but also exhibit loss and incorporation of tubulin subunits along their lattice far from the tips. Although this phenomenon has been observed to occur under various conditions in vitro as well as in cells, many questions remain regarding the regulation of lattice dynamics and their contribution to overall microtubule network organization and function. Compared to microtubule tip dynamics, the dynamics of tubulin incorporation along the lattice are more challenging to investigate as they are hidden in classical experimental setups, which is likely the reason they were overlooked for a long time. In this chapter, we present a strategy to visualize and quantify the incorporation of tubulin subunits into the microtubule lattice in vitro. The proposed method does not require specialized equipment and can thus be carried out readily in most research laboratories.Fluorescence spectroscopy is routinely used for the determination of the interaction of a ligand with a protein. The quick detection of the interaction between the ligand and the protein is one of the most significant advantages of fluorescence spectroscopic methods. In this chapter, we have described assays to monitor drug -tubulin interactions using several fluorescence spectroscopic techniques. We have provided detailed protocols for different assays for investigating tubulin-drug interactions with key practical considerations for performing the experiments. We have also discussed how to deduce the binding parameters by fitting the fluorescence change data in different binding isotherms. Further, we have described detailed protocols to monitor the binding site of a ligand on tubulin by competitive inhibition. Though the methods are described for tubulin, these methods can also be used to monitor any drug -protein interactions.Microtubules (MTs) are tubular cytoskeletons, which are used for the various applications such as active matters and therapeutic targets. Although modification of the exterior surface of MTs is frequently used for functionalization of MTs, there was no approach to introduce molecules inside MTs. We previously developed a unique peptide binding to the inner surface of MT, which is derived from a MT-associated protein, Tau. The Tau-derived peptide (TP) can be used to introduce various nanomaterials inside MTs. Here we describe the TP-based encapsulation of fluorescent dye, gold nanoparticle, green fluorescent protein, and magnetic CoPt nanoparticles inside MTs.Fabrication of molecular devices using biomolecules through biomimetic approaches has witnessed a surge in interest in recent years. DNA a versatile programmable material offers an opportunity to realize complicated operations through the designing of various nanostructures such as DNA origami. Here we describe the methods to use DNA origami for the self-assembly of the biomolecular motor system, microtubule (MT)-kinesin. A rodlike DNA origami motif facilitates the self-assembly of MTs into asters. A smooth muscle like molecular contraction system could be realized following the method where DNA mediated self-assembly of MTs permits dynamic contraction in the presence of kinesins through an energy dissipative process.Swarm robotics has been attracting much attention in recent years in the field of robotics. This chapter describes a methodology for the construction of molecular swarm robots through precise control of active self-assembly of microtubules (MTs). Detailed protocols are presented for the construction of molecular robots through conjugation of DNA to MTs and demonstration of swarming of the MTs. The swarming is mediated by DNA-based interaction and photoirradiation which act as processors and sensors respectively for the robots. Furthermore, the required protocols to utilize the swarming of MTs for molecular computation is also described.The propulsion of motile cells such as sperms and the transport of fluids on cell surfaces rely on oscillatory bending of cellular appendages that can perform periodic oscillations. buy PF-04957325 These structures are flagella and cilia. Their beating is driven by the interaction between microtubules and motor proteins and the mechanism regulating this is still a puzzle. One approach to address this issue is the assembling of synthetic minimal systems by using natural building blocks, e.g., microtubules and kinesin motors, which undergo persistent oscillation in the presence of ATP. An example of an autonomous molecular system is reported in this chapter. It dynamically self-organizes through its elasticity and the interaction with the environment represented by the active forces exerted by motor proteins. The resulting motion resembles the beating of sperm flagella. Assembling such minimal systems able to mimic the behavior of complex biological structures might help to unveil basic mechanisms underlying the beating of natural cilia and flagella.In vitro gliding assay of the filamentous protein microtubule (MT) on a kinesin motor protein coated surface has appeared as a classic platform for studying active matters. At high densities, the gliding MTs spontaneously align and self-organize into fascinating large-scale patterns. Application of mechanical stimuli e.g., stretching stimuli to the MTs gliding on a kinesin-coated surface can modulate their self-organization and patterns according to the boundary conditions. Depending on the mode of stretching, MT at high densities change their moving direction and exhibit various kinds of patterns such as stream, zigzag and vortex pattern. In this chapter, we discuss detail procedures on how to apply mechanical stimuli to the moving MTs on a kinesin coated substrate.In this chapter, protocols for spontaneous alignment of microtubules (MTs), such as helices and spherulites, via tubulin polymerization in a narrow space and under a temperature gradient are presented for tubulin solutions and tubulin-polymer mixtures. These protocols provide an easy route for hierarchical MT assembly and may extend our current understanding of cytoskeletal protein self-assembly under dissipative conditions.Studied for more than a century, equilibrium liquid crystals provided insight into the properties of ordered materials, and led to commonplace applications such as display technology. Active nematics are a new class of liquid crystal materials that are driven out of equilibrium by continuous motion of the constituent anisotropic units. A versatile experimental realization of active nematic liquid crystals is based on rod-like cytoskeletal filaments that are driven out of equilibrium by molecular motors. We describe protocols for assembling microtubule-kinesin based active nematic liquid crystals and associated isotropic fluids. We describe the purification of each protein and the assembly process of a two-dimensional active nematic on a water-oil interface. Finally, we show examples of nematic formation and describe methods for quantifying their non-equilibrium dynamics.This chapter describes compiled methods for the formation and manipulation of microtubule-kinesin-carbon nanodots conjugates in user-defined synthetic environments. Specifically, by using inherited self-assembly and self-recognition properties of tubulin cytoskeletal protein and by interfacing this protein with lab synthesized carbon nanodots, bio-nano hybrid interfaces were formed. Further manipulation of such biohybrids under the mechanical cycle of kinesin 1 ATP-ase molecular motor led to their integration on user-controlled engineered surfaces. Presented methods are foreseen to lead to microtubule-molecular motor-hybrid based assemblies formation with applications ranging from biosensing, to nanoelectronics and single molecule printing, just to name a few.