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Author: Jason Harris Publisher: ISBN: Category : Languages : en Pages :
Book Description
"Inaccurate segregation of chromosomes during cell division is thought to lead to tumour formation. Microtubules play a key role in chromosome separation and segregation. The study of yeast spindles offers a convenient and versatile system to study the processes that impact cell division. One protein found to have an integral role in proper spindle assembly is yeast [gamma]-Tubulin (Tub4). Tub4 is a 53kDa protein that contains a flexible C-terminal tail. Removal of this tail has shown to be lethal in vivo. Mutation of a DYSL motif in the tail has also been shown to influence recruitment of proteins to the opposite end of a microtubule. Phosphomimetic mutations of the tyrosine in the DYSL motif have been shown to increase the number of microtubules that do not attach to kinetochores. NMR is ideally suited to study the effects of phosphorylation on the dynamics of the last 39 residues of Tub4. In doing so, we will gain information on how changes in the dynamics of Tub4 correlate to changes in protein function, which in turn influence the cell as a whole.We have used NMR spectroscopy to characterize the differences between the wild type(WT) Tub4 tail and a mutant containing the Y445D phosphomimetic substitution. Gradient diffusion experiments were employed to measure the hydrodynamic radii of the proteins, revealing that both WT and Y445D constructs diffuse more slowly than expected. The dynamics of the two constructs were analyzed using 15N and 2H spin relaxation measurements. Spectral density mapping and analysis of backbone dynamics show no change in the fast timescale dynamics by the Y445D mutation. Interestingly, CPMG analysis revealed that the Y445D mutation changed the milli-microsecond timescale dynamics of the protein. This change is observed throughout the entire protein and was determined to be a global exchange between a major and minor state. This suggests that the mutation causes the protein to exchange between collapsed and extended conformations. In this model, the extended conformation increases the capture radius in which it can interact with potential binding partners." --
Author: Jason Harris Publisher: ISBN: Category : Languages : en Pages :
Book Description
"Inaccurate segregation of chromosomes during cell division is thought to lead to tumour formation. Microtubules play a key role in chromosome separation and segregation. The study of yeast spindles offers a convenient and versatile system to study the processes that impact cell division. One protein found to have an integral role in proper spindle assembly is yeast [gamma]-Tubulin (Tub4). Tub4 is a 53kDa protein that contains a flexible C-terminal tail. Removal of this tail has shown to be lethal in vivo. Mutation of a DYSL motif in the tail has also been shown to influence recruitment of proteins to the opposite end of a microtubule. Phosphomimetic mutations of the tyrosine in the DYSL motif have been shown to increase the number of microtubules that do not attach to kinetochores. NMR is ideally suited to study the effects of phosphorylation on the dynamics of the last 39 residues of Tub4. In doing so, we will gain information on how changes in the dynamics of Tub4 correlate to changes in protein function, which in turn influence the cell as a whole.We have used NMR spectroscopy to characterize the differences between the wild type(WT) Tub4 tail and a mutant containing the Y445D phosphomimetic substitution. Gradient diffusion experiments were employed to measure the hydrodynamic radii of the proteins, revealing that both WT and Y445D constructs diffuse more slowly than expected. The dynamics of the two constructs were analyzed using 15N and 2H spin relaxation measurements. Spectral density mapping and analysis of backbone dynamics show no change in the fast timescale dynamics by the Y445D mutation. Interestingly, CPMG analysis revealed that the Y445D mutation changed the milli-microsecond timescale dynamics of the protein. This change is observed throughout the entire protein and was determined to be a global exchange between a major and minor state. This suggests that the mutation causes the protein to exchange between collapsed and extended conformations. In this model, the extended conformation increases the capture radius in which it can interact with potential binding partners." --
Author: Brandon Payliss Publisher: ISBN: Category : Languages : en Pages :
Book Description
"Microtubules are highly dynamic and essential cytoskeletal structures. They play key roles in cell division and are essential components of the mitotic spindle, which facilitates chromosome separation and segregation into daughter cells. One of the key components in driving the assembly of the mitotic spindle in Saccharomyces cerevisiae is a protein known as [gamma]-tubulin, which has a folded globular domain, followed by an intrinsically disordered carboxyl terminus (CT) rich in glutamate and aspartate residues. Deletion of the last 35 residues of the [gamma]-tubulin CT (or [gamma]-CT) is lethal,1 and mutations in the DSYL motif of the [gamma]-CT can cause spindle defects.2 In this study, we used nuclear magnetic resonance (NMR) spectroscopy and video particle tracking (VPT) microrheology to study the biophysical behaviour of the wildtype [gamma]-CT peptide (WT), but also three mutants where the DSYL motif had been substituted to a DSDL, DDYL, or AAAA, which we have termed the YD, SD, and 4A mutants. One-dimensional NMR diffusion experiments and 1H-15N Heteronuclear Single Quantum Correlation (HSQC) spectroscopy were used to evaluate the impact of increasing acidity on both the diffusion coefficient, as well as the shielding environment of the peptide backbone. Our results show that the WT populates more condensed conformational states with increasing acidity, and that the extent of peak movement for backbone amides in 1H-15N HSQC spectra correlates with the position of ionizable side-chains as pH is reduced. Using VPT microrheology to track 0.2 [mu]m fluorescent beads, we have also studied the viscoelasticity of WT, YD, SD, and 4A peptide solutions. Interestingly, both WT and YD peptide solutions demonstrated subdiffusive properties at pH 7.2 with peptide concentrations 0.1 mM, indicative of liquid-liquid phase separation and/or formation of a system-spanning network. Our results show that subdiffusivity can be ranked WT YD > 4A > SD. Moreover, these results suggest that the [gamma]-CT could play a key role in vivo by promoting liquid-liquid phase separation at the centrosome, and that mutations in the CT could affect these properties." --
Author: Anne Straube Publisher: Humana Press ISBN: 9781493961856 Category : Science Languages : en Pages : 319
Book Description
Microtubules are at the heart of cellular self-organization, and their dynamic nature allows them to explore the intracellular space and mediate the transport of cargoes from the nucleus to the outer edges of the cell and back. In Microtubule Dynamics: Methods and Protocols, experts in the field provide an up-to-date collection of methods and approaches that are used to investigate microtubule dynamics in vitro and in cells. Beginning with the question of how to analyze microtubule dynamics, the volume continues with detailed descriptions of how to isolate tubulin from different sources and with different posttranslational modifications, methods used to study microtubule dynamics and microtubule interactions in vitro, techniques to investigate the ultrastructure of microtubules and associated proteins, assays to study microtubule nucleation, turnover, and force production in cells, as well as approaches to isolate novel microtubule-associated proteins and their interacting proteins. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Definitive and practical, Microtubule Dynamics: Methods and Protocols provides the key protocols needed by novices and experts on how to perform a broad range of well-established and newly-emerging techniques in this vital field.
Author: Publisher: Academic Press ISBN: 0123813506 Category : Science Languages : en Pages : 549
Book Description
Microtubules: in vivo includes chapters by experts around the world on many aspects of microtubule imaging in living and fixed cells; assays to study microtubule function in a wide array of model organisms and cultured cells; high resolution approaches to study of the cytoskeleton. The authors share their years of experience, outlining potential pitfalls and critical factors to consider in experimental design, experimental implementation and data interpretation. - Includes chapters by experts around the world on many aspects of microtubule imaging in living and fixed cells; assays to study microtubule function in a wide array of model organisms and cultured cells; high resolution approaches to study of the cytoskeleton - The authors share their years of experience, outlining potential pitfalls and critical factors to consider in experimental design, experimental implementation and data interpretation
Author: Andriy Sibirny Publisher: Springer ISBN: 303021110X Category : Science Languages : en Pages : 568
Book Description
This volume scopes several aspects of non-conventional yeast research prepared by the leading specialists in the field. An introduction on taxonomy and systematics enhances the reader’s knowledge on yeasts beyond established ones such as Saccharomyces cerevisiae. Biotechnological approaches that involve fungal utilization of unusual substrates, production of biofuels and useful chemicals as citric acid, glutathione or erythritol are discussed. Further, strategies for metabolic engineering based on knowledge on regulation of gene expression as well as sensing and signaling pathways are presented. The book targets researchers and advanced students working in Microbiology, Microbial Biotechnology and Biochemistry.
Author: John J. Correia Publisher: Academic Press ISBN: 0124078885 Category : Science Languages : en Pages : 472
Book Description
There continues to be intense interest in the microtubule cytoskeleton; the assembly, structure and regulation of microtubules; and the numerous motors and accessory proteins that control cell cycle, dynamics, organization and transport. The field continues to grow and explore new aspects of these issues driven immensely by developments in optical imaging and tracking techniques. This Second Edition brings together current research and protocols in the field of microtubules in vitro and will serve as a valuable tool for cell biologists, biophysicists and pharmacologists who study the microtubule cytoskeleton, as well as for researchers in the biomedical and biotechnology communities with interest in developing drugs that target microtubules, MAPS and motors. - Chapters reflect experimental procedures and new developments in the field of microtubule in vitro research - Combines classical approaches and modern technologies - Presents easy-to-use protocols and thorough background information, compiled by leaders in the field
Author: Sergey Edward Lyshevski Publisher: CRC Press ISBN: 1420008145 Category : Technology & Engineering Languages : en Pages : 912
Book Description
There are fundamental and technological limits of conventional microfabrication and microelectronics. Scaling down conventional devices and attempts to develop novel topologies and architectures will soon be ineffective or unachievable at the device and system levels to ensure desired performance. Forward-looking experts continue to search for new paradigms to carry the field beyond the age of microelectronics, and molecular electronics is one of the most promising candidates. The Nano and Molecular Electronics Handbook surveys the current state of this exciting, emerging field and looks toward future developments and opportunities. Molecular and Nano Electronics Explained Explore the fundamentals of device physics, synthesis, and design of molecular processing platforms and molecular integrated circuits within three-dimensional topologies, organizations, and architectures as well as bottom-up fabrication utilizing quantum effects and unique phenomena. Technology in Progress Stay current with the latest results and practical solutions realized for nanoscale and molecular electronics as well as biomolecular electronics and memories. Learn design concepts, device-level modeling, simulation methods, and fabrication technologies used for today's applications and beyond. Reports from the Front Lines of Research Expert innovators discuss the results of cutting-edge research and provide informed and insightful commentary on where this new paradigm will lead. The Nano and Molecular Electronics Handbook ranks among the most complete and authoritative guides to the past, present, and future of this revolutionary area of theory and technology.
Author: Jason Harris
Author: Jason Harris
Author: Jeffrey Moore
Author: Brandon Payliss
Author: Anne Straube
Author: 
Author: Urvashi Patel
Author: Andriy Sibirny
Author: John J. Correia
Author: 
Author: Sergey Edward Lyshevski