Search

Host microtubules and motor proteins are crucial to the intracellular transport of a number of viruses. Disruption of microtubules or suppression of motor functions can remarkably inhibit the movement of viruses in host cells. It is now known that incoming viruses use motor proteins to travel along microtubules from the plasma membrane to the nuclear or perinuclear replication site, whereas progeny viruses depend on microtubules and motors to move from the assembly site to the cell periphery. Here, we describe several major methods for analyzing microtubule-mediated intracellular viral transport, using adenovirus as an example. [ABSTRACT FROM AUTHOR]

In neurons, the molecular machinery for axonal growth and navigation is localized to the growth cone region, whereas tubulin is synthesized primarily in the cell body. Because diffusion serves as an efficient transport mechanism only for very short distances, tubulin has to be actively transported from the cell body down the axon. Two mechanistically distinct models for tubulin transport have been proposed. "Polymer model" postulates that tubulin moves in the form of microtubules preassembled in the cell body, whereas "subunit model" assumes that axonal microtubules are stationary, and that tubulin is delivered from the cell body in unassembled form. We used three independent quantitative approaches (photobleaching, fluorescence speckle microscopy, and microtubule plus end tracking) to demonstrate that axonal microtubules are stationary in rapidly growing axons produced by Xenopus spinal cord neurons in culture. These experiments strongly support subunit model for tubulin delivery. [ABSTRACT FROM AUTHOR]

Taxol, the first microtubule stabilizer identified, is one of the most important new anticancer drugs to be brought to the clinic in the past 20 yr. The clinical success of Taxol? led to the development of a second-generation taxane, docetaxel (Taxotere™), and multiple third-generation taxane derivatives are under development. Non-taxane microtubule-stabilizers of diverse chemical structures, including the epothilones and discodermolide, show promising preclinical activities and several epothilones are progressing through clinical trials. One important advantage of the new stabilizers is their ability to circumvent drug resistance mechanisms. The clinical development of these new classes of agents suggests that microtubule stabilizers will continue to be important drugs for the treatment of cancer. This chapter provides a brief history of Taxol and the discovery and development status of other classes of microtubule stabilizers. Although all microtubule-stabilizers share similar mechanisms of action, interesting subtle differences among the stabilizers are being detected. This chapter also provides some strategies for identifying the differences among microtubule stabilizers that may help prioritize them for development and clinical use. [ABSTRACT FROM AUTHOR]

Drugs that affect microtubule dynamics are among the most effective anticancer agents in routine clinical use. The standard assay for antimicrotubule agents observes the ability of a particular substance to affect in vitro microtubule assembly. We have modified these procedures so that they can be performed in 96-well plates using a standard fluorescence plate reader. Two different protocols are provided in this chapter. One of these protocols is for ligands that inhibit microtubule polymerization, such as colchicine and related molecules. The second is for ligands that promote in vitro microtubule assembly, such as Taxol. [ABSTRACT FROM AUTHOR]

Vinca alkaloids play a vital role in chemotherapy protocols for a wide range of hematological and solid tumors. Studies of drug interactions with the drug target, tubulin or microtubules, have helped us to understand the cytotoxic and toxic effects. We present here in vivo and in vitro methods for studying vinca alkaloid interactions with tubulin. In vivo methods for examining drug effects on cell proliferation and intracellular tubulin or microtubules and direct visualization of drug effects by fluorescence microscopy are presented. In vitro methods for measuring drug affinity for tubulin by analytical ultracentrifugation, kinetics of drug binding by light scattering and drug effects on microtubules by turbidity are also presented. [ABSTRACT FROM AUTHOR]

The knowledge of the thermodynamics and kinetics of drug-microtubule interaction is essential to understand the structure/affinity relationship of a given ligand family. When a ligand does not show an appropriate signal change (absorbance or fluorescence) upon binding, the extensive direct characterization of its binding affinities and kinetic rate constants of association and dissociation becomes a complex task. In those cases it is possible to obtain these parameters by competition of the ligand with a reference one of the same binding site that shows such change. Nevertheless, although the experimental setup of the competition measurements is easier, the treatment of the data is complex because simultaneous equilibrium/kinetic equations have to be solved. In this chapter, the taxoid-binding site of the microtubules will be used as an example to describe experimental competition and data analysis methods to determine the binding constants and kinetic rates of association and dissociation of ligands for microtubules. [ABSTRACT FROM AUTHOR]

Tubulin, the microtubule building-block, is the target of numerous small molecule compounds that interfere with microtubule dynamics. Several of these ligands are in clinical use as antitumor drugs. There have been numerous studies on these molecules, with two main objectives: to determine their mechanism of action and to find new compounds that would expand the arsenal available for cancer chemotherapy. Although these studies would undoubtedly benefit from structural data on tubulin, this protein has long resisted crystallization attempts. We have used stathmin-like domains (SLDs) of stathmin family proteins as a tool to crystallize tubulin and have obtained three-dimensional crystals of the tubulin:SLD complexes. As many tubulin ligands bind to these complexes, the crystals are valuable tools to study tubulin-drug interactions by X-ray crystallography. They open the way to a structure-based drug design approach. [ABSTRACT FROM AUTHOR]

The microtubule-targeting agents have made significant contributions to cancer therapy over the past 50 years. The vinca alkaloids and taxanes have been used to treat a broad range of malignancies, including leukemias and lymphomas and many types of solid tumors. The taxanes have been frequently used in the treatment of advanced ovarian, breast, lung, head and neck, and prostate cancer, and they are increasingly being used in early stage disease. This chapter reviews the pharmacology, clinical indications, and toxicities associated with the vinca alkaloids and taxanes. [ABSTRACT FROM AUTHOR]

The mitotic spindle is an important target for cancer chemotherapy. The main protein target for drugs in clinical use is tubulin, the building block of microtubules. In recent years, other proteins of the mitotic spindle have been identified as potential targets for the development of more specific drugs with the hope that these will have fewer side effects than known antimitotics (taxanes, vinca alkaloids). The human genome contains more than 40 members of the kinesin superfamily, with at least 12 of these involved in mitosis and cytokinesis. HsEg5 (also called KSP, kinesin spindle protein), a member of the kinesin-5 family, involved in the formation of the bipolar spindle, is a very promising target for cancer chemotherapy with specific inhibitors in Phase I and II clinical trails. Several successful approaches exist today to screen Eg5 for inhibitors, including phenotype-based assays and simple in vitro assays that explore the intrinsic enzymatic ATPase activity of Eg5. Here, we describe a robust and straightforward in vitro method to rapidly screen Eg5 for inhibitors. The assay can easily be adapted to other mitotic kinesins that may be identified in the future as potential drug targets, or simply to obtain specific kinesin inhibitors for use in "chemical genetics" to study the function of this important class of proteins. [ABSTRACT FROM AUTHOR]

Microtubule-actin interactions are fundamental to many cellular processes such as cytokinesis and cellular locomotion. Investigating the mechanism of microtubule-actin interactions is the key to understand the cellular morphogenesis and related pathological processes. The abundance and highly dynamic nature of microtubules and F-actin raise a serious challenge when trying to distinguish between the real and fortuitous interactions within a cell. Xenopus oocyte wound model represents an ideal system to study microtubule-actin interactions as well as microtubule-dependent control of the actin polymerization. Here, we describe a series of cytoskeleton specific treatments in Xenopus oocyte wound healing experiments and use confocal fluorescence microscopy to analyze fixed oocytes to examine microtubule-actin interactions. [ABSTRACT FROM AUTHOR]

Numerous comparisons have shown that confocal imaging of fluorescently labeled samples has superior image clarity compared to traditional epifluorescence microscopy, especially when imaging through thick specimens. Nevertheless, one limitation of confocal microscopy is the speed at which these systems acquire images. Newer confocal technologies, however, have overcome this drawback by rapidly scanning images while still maintaining the precision and resolution of a confocal microscope. One example is the spinning disk confocal, which can rapidly acquire images with minimal photobleaching. The following protocol provides methodology using spinning disk technology to visualize spindle microtubule behavior in live breast cancer cells expressing green fluorescent protein:α-tubulin. It begins with sample preparation, covers equipment setup and image acquisition, and ends with image processing and archiving. Image acquisition is subdivided into two categories-imaging the complete mitotic cycle and imaging rapid mitotic events, to address the varying parameters required for the each experiment. [ABSTRACT FROM AUTHOR]

Kinetochores are essential for the proper positioning, movement and segregation of chromosomes on spindle microtubules. Live cell analyses of kinetochore movements on the spindle provide an important tool for dissecting the molecular machinery underlying kinetochore-based chromosome motility. Here, we describe contemporary techniques for studying and manipulating kinetochore function in live Drosophila syncytial blastoderm-stage embryos and S2 cells. [ABSTRACT FROM AUTHOR]

In this article, the author describes the needed instrumentation and the methods to be followed for the observation and measurement of the birefringence of single and bundled microtubules and of their ordered arrays using a polarizing microscope. As instruments, the traditional polarizing microscope and the recently developed LC-PolScope are discussed. As methods, we describe qualitative and quantitative observations, including notes on specimen preparations, which optimize the sensitivity and accuracy of measuring specimen retardance. [ABSTRACT FROM AUTHOR]

Stathmin is an important phosphorylation-controlled regulator of microtubule dynamics and plays a crucial role in cell division and cell proliferation. In its non-phosphorylated form, stathmin is the protein that interacts the most tightly with tubulin, in a 2:1 tubulin-stathmin (T2S) complex that does not participate in microtubule assembly. The importance of stathmin at different levels of phosphorylation in different steps of mitosis This article is a short overview of the different methods that have been or could be used to monitor the kinetic and thermodynamic parameters of tubulin-stathmin interaction and to evaluate the effects of phosphorylation. The author has tried to emphasize how hydrodynamic and spectroscopic methods measuring direct binding of stathmin to tubulin can be complemented by methods that make use of linked functions, measuring how the change in a functional property of tubulin upon binding stathmin provides information on binding parameters. [ABSTRACT FROM AUTHOR]

Cytoplasmic microtubules are noncovalent polymers of the protein tubulin. In the cells, the main function of microtubules is to provide tracks for organelle transport. Two experimental approaches based on fluorescence microscopy are commonly used to examine organization of microtubules in mammalian tissue culture cells. The first experimental approach involves indirect immunofluorescence staining of chemically fixed cells with tubulin antibody. Fluorescence microscopy of immunostained specimens allows the examination of the distribution of microtubules in the cytoplasm at the moment of fixation. The second experimental approach involves introduction of tubulin subunits covalently labeled with a fluorochrome into the cytoplasm of living cells. Time-lapse fluorescence microscopy of cells containing labeled tubulin subunits allows to examine changes in the spatial organization of microtubules in the cytoplasm and also to directly observe their behavior. In this chapter, we describe preparation of samples for fluorescence microscopy of microtubules. [ABSTRACT FROM AUTHOR]

Tubulin heterogeneity within eukaryotic cells is generated by differential gene expression and posttranslational modification of α- and β-tubulin gene products, either as heterodimers or when polymerized into microtubules. The characterization of posttranslationally modified tubulins from the crustacean Artemia franciscana is presented, although tubulins from other sources can be studied with these procedures. Tubulin is prepared from cell free extracts by taxol-induced assembly and centrifugation of microtubules through sucrose cushions, which also yields microtubule-associated proteins, or it is purified to apparent homogeneity by relatively simple chromatographic procedures and assembly/disassembly steps. To detect posttranslationally modified tubulins protein samples are electrophoresed in sodium dodecyl sulfate (SDS) polyacrylamide gels, blotted to nitrocellulose membranes and probed with isoform-specific antibodies. Isotubulins, for which gene-encoded amino acid differences and post translational modifications generate charge variations, are resolved in two-dimensional gels using isoelectric focusing followed by SDS polyacrylamide gel electrophoresis, a procedure useful for resolution of microtubule-associated proteins. Isoforms patterns are visualized by Coomassie blue and/or silver staining and individual isoforms are identified by antibody reactivity on Western blots. Tubulin isoforms are localized in Artemia by immunofluorescent staining of larvae. The focus of this chapter is the purification of tubulin from a nonneural source and characterization of tyrosinated, detyrosinated, and nontyrosinatable α-tubulins using polyclonal antibodies made to carboxy-terminal peptides of each i [ABSTRACT FROM AUTHOR]

Although the structures of individual proteins and moderately sized complexes of proteins may be investigated by X-ray crystallography, the interaction between a long polymer, such as a microtubule, and other protein molecules, such as the motor domain of kinesin, need to be studied by electron microscopy. We have used electron cryo-microscopy and image analysis to study the structures of microtubules with and without bound kinesin motor domains and the changes that take place when the motor domains are in different nucleotide states. Among the microtubules that assemble from pure tubulin, we select a minor subpopulation that has perfect helical symmetry, which are the best for three-dimensional reconstruction. Gold labeling can be used to mark the positions of certain regions of protein sequence. [ABSTRACT FROM AUTHOR]

Microtubule-binding proteins are conveniently divided into two large groups: MAPs (microtubule-associated proteins), which can stabilize, anchor, and/or nucleate microtubules, and motors, which use the energy of ATP hydrolysis for a variety of functions, including microtubule network organization and cargo transportation along microtubules. Here, we describe the use of Taxol-stabilized microtubules for purification of MAPs, motors, and their complexes from Xenopus egg extracts. Isolated proteins are analysed using sodium dodecyl sulfate gel electrophoresis and identified by various mass spectrometry and database mining technologies. Found proteins can be grouped into three classes: (1) known MAPs and motors; (2) proteins previously reported as associated with the microtubule cytoskeleton, but without a clearly defined cytoskeletal function; (3) proteins not yet described as having microtubule localization. Sequence-similarity methods employed for protein identification allow efficient identification of MAPs and motors from species with yet unsequenced genomes. [ABSTRACT FROM AUTHOR]

This method is a modification of the initial procedure employed to purify tubulin from mammalian brain. It consists of tissue homogenization, elimination of cell membranes, ammonium sulfate fractionation, and batch anion exchange, followed by selective precipitation with magnesium chloride. Half gram of electrophoretically homogenous, active, concentrated calf brain tubulin is typically purified in 9 h, dialyzed overnight, and stored under liquid nitrogen. Prior to use the protein is equilibrated in the experimental buffer and its concentration measured. This tubulin preparation has been very extensively characterized. Frozen aliquots have been found to retain microtubule assembly activity after 10 yr of storage. [ABSTRACT FROM AUTHOR]

Microtubules are found in all eukaryotes and are built from αβ-tubulin heterodimers. The α-tubulins and β-tubulins are among the most highly conserved eukaryotic proteins. Other members of the tubulin family have come to light recently and, like γ-tubulin, appear to play roles in microtubule nucleation and assembly. Microtubule assembly is accompanied by hydrolysis of GTP associated with β-tubulin so that microtubules consist principally of "GDP-tubulin" stabilized by a short "GTP cap." Microtubules are polar, cylindrical structures some 25 nm in diameter. Protofilaments made from tubulin heterodimers run lengthwise along the microtubule wall with the β-tubulin subunit at the microtubule plus end. The crystallographic structures of tubulins are essential to understand in detail microtubule architecture and interactions with stabilizing and destabilizing drugs and proteins. [ABSTRACT FROM AUTHOR]

In this paper, we propose a fast and robust face detection method. We train a cascade-structured classifier with boosted haar-like features which uses intensity information only. To speed up the process, we integrate motion energy into the cascade-structured classifier. Motion energy can represent moving the extent of the candidate regions, which is used to reject most of the candidate windows and thus accelerates the evaluation procedure. According to the face presence situation, we divide the system state into three modes, and process input images with an intensity detector, or motion integrated dynamic detector, or else keep the pre-results. Since motion energy can be computed efficiently, processing speed is greatly accelerated. Furthermore, without depending on any supposed motion model, the system is very robust in real situations without the limitation of moving patterns including speed and direction. [ABSTRACT FROM AUTHOR]

This paper presents a novel dynamic face and fingerprint fusion system for identity authentication. To solve the face pose problem in dynamic authentication system, multi-route detection and parallel processing technology are used in this system. A multimodal part face recognition method based on principal component analysis (MMP-PCA) algorithm is adopted to perform the face recognition task. Fusion of face and fingerprint by SVM (Support Vector Machine) fusion strategy which introduced a new normalization method improved the accuracy of identity authentication system. Furthermore, key techniques such as fast and robust face detection algorithm and dynamic fingerprint detection and recognition method based on gray-Level histogram statistic are accepted to guarantee the fast and normal running. Practical results on real database proved that this authentication system can achieve better results compared with face-only or fingerprint-only system. Consequently, this system indeed increases the performance and robustness of identity authentication systems and has more practicability. [ABSTRACT FROM AUTHOR]