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Author: Sharifah E. Albraiki Publisher: ISBN: Category : Electronic dissertations Languages : en Pages : 93
Book Description
Metastasis is the most clinically significant step in cancer progression. Migration and metastasis are not fully understood, but it is clear that the actin cytoskeleton plays an essential role. Palladin is specifically involved in metastasis of cancer cells, but also co-localizes with actin stress fibers in normal cells. The 90 kDa palladin is the only ubiquitously expressed isoform and contains three Ig domains and one proline-rich region. This proline-rich region has been shown to bind directly to the actin-regulating protein VASP. In a previous paper, our lab showed that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work we wanted to compare functions of the 90 kDa palladin to the isolated actin binding domain. Our hypothesis was that the 90 kDa palladin may be autoinhibited and may thereby block the binding site for monomeric actin. To understand the mechanism of action for how palladin can influence actin assembly, we used fluorescence spectroscopy to monitor pyrene actin polymerization. By using site-directed mutagenesis via PCR we were able to mutate the putative VASP binding site within the prolinerich region of the 90 kDa palladin. We then examined binding between VASP and WT or mutant palladin using a pulldown assay and far Western blot. Both palladin and VASP proteins are involved in the regulation of actin filaments and understanding the fundamental mechanism of these proteins may help us eliminate the progression of cancer invasion and metastasis. In addition, we sought to determine how palladin and VASP are involved in actin assembly required for cell motility. A facultative intracellular pathogen, Listeria monocytogenes, has been used to study the regulation of palladin in the regulation of actin dynamics. Our aim is to test the hypothesis that palladin promotes the nucleation, elongation and the stabilization of actin-based structure during cell motility. Understanding the role of palladin in actin cytoskeleton may help us prevent cancer cells from reaching the metastasis stage of cancer progression.
Author: Sharifah E. Albraiki Publisher: ISBN: Category : Electronic dissertations Languages : en Pages : 93
Book Description
Metastasis is the most clinically significant step in cancer progression. Migration and metastasis are not fully understood, but it is clear that the actin cytoskeleton plays an essential role. Palladin is specifically involved in metastasis of cancer cells, but also co-localizes with actin stress fibers in normal cells. The 90 kDa palladin is the only ubiquitously expressed isoform and contains three Ig domains and one proline-rich region. This proline-rich region has been shown to bind directly to the actin-regulating protein VASP. In a previous paper, our lab showed that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work we wanted to compare functions of the 90 kDa palladin to the isolated actin binding domain. Our hypothesis was that the 90 kDa palladin may be autoinhibited and may thereby block the binding site for monomeric actin. To understand the mechanism of action for how palladin can influence actin assembly, we used fluorescence spectroscopy to monitor pyrene actin polymerization. By using site-directed mutagenesis via PCR we were able to mutate the putative VASP binding site within the prolinerich region of the 90 kDa palladin. We then examined binding between VASP and WT or mutant palladin using a pulldown assay and far Western blot. Both palladin and VASP proteins are involved in the regulation of actin filaments and understanding the fundamental mechanism of these proteins may help us eliminate the progression of cancer invasion and metastasis. In addition, we sought to determine how palladin and VASP are involved in actin assembly required for cell motility. A facultative intracellular pathogen, Listeria monocytogenes, has been used to study the regulation of palladin in the regulation of actin dynamics. Our aim is to test the hypothesis that palladin promotes the nucleation, elongation and the stabilization of actin-based structure during cell motility. Understanding the role of palladin in actin cytoskeleton may help us prevent cancer cells from reaching the metastasis stage of cancer progression.
Author: Ravi Vattepu Publisher: ISBN: Category : Electronic dissertations Languages : en Pages : 153
Book Description
Palladin, an actin-binding and bundling protein, plays an important role in normal cell adhesion and motility via organizing the actin cytoskeleton. Palladin exists in multiple isoforms in humans and its canonical isoform contains five immunoglobulin (Ig) domains and Ig3 domain is the minimum requirement for actin-binding and bundling, while Ig4 does not bind directly to actin, the tandem Ig3-4 domain binds and bundles actin more efficiently than Ig3 alone. In our quest to understand palladin’s role in the actin cytoskeleton we have explored the following topics in this dissertation: actin–induced dimerization, phospholipid-binding and regulation of function, structural and functional outcomes of a recently identified point mutation of a critical tryptophan residue, and the role of the linker between the Ig3 and Ig4 domains. First, we demonstrated that actin induces dimerization in the actin-binding domain of palladin, which is confirmed by chemical crosslinking. Our results also provide biochemical proof that the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) functions as a moderator of palladin activity. A mutation in the Ig4 domain of palladin has been found in a pancreatic cancer cell line that displays increased cell motility. Our results reveal a severe disruption of Ig4 domain folding, stability, and actin bundling function. To gain insight into role of the linker between the Ig3 and 4 domain, we have generated a series of mutations to shorten the linker, swap domain linkers, and add phosphomimetic modifications that will allow us to study the effects on actinbinding, bundling, and polymerization. In this report we also highlight the development of a novel His-tag based fluorophore, a tool that will be useful in several future studies, and initial studies of a unique actin polymerization mechanism involving actin oligomerization. Our overall results provided conclusive evidence for Ig3-4 actin bundling mechanism and identified key residues involved in lipid-binding.
Author: C.G. dos Remedios Publisher: Springer Science & Business Media ISBN: 354046560X Category : Science Languages : en Pages : 272
Book Description
Actin is one of the most widespread proteins in eukaryotic cells. This book and its companion (Molecular Interactions of Actin. Actin-Myosin Interaction, Actin-Based Regulation) provide an authoritative and opinionated view of the structure and function of this essential protein. Each section includes an historical perspective and a detailed commentary on actin protein chemistry, molecular and cell biology of actin. While some chapters review the body of knowledge of the subject, others contain new experimental data. This book will appeal to research scientists seeking contemporary overviews of actin and its binding proteins. Contributors include senior scientists as well as the new breed of younger scientists.
Author: Cris dos Remedios Publisher: Springer Science & Business Media ISBN: 0387717498 Category : Science Languages : en Pages : 362
Book Description
There are scattered reports in the published literature citing relationships between actin, actin-binding proteins and disease. This volume brings this information together for the first time, with a focus on human disorders. The volume is relevant to a wide readership including cell biologists interested in understanding how structural and functional changes in proteins impact on the organism as a whole.
Author: Viviana Ioana Risca Publisher: ISBN: Category : Languages : en Pages : 187
Book Description
Mechanical cues affect a number of important biological processes in metazoan cells, such as migration, proliferation, and differentiation. Many of these processes are mediated by the cytoskeleton, an intracellular network of protein filaments that provides mechanical rigidity to the cell and drives cellular shape change. In particular, actin, a very highly conserved and abundant cytoskeletal protein, forms filaments that, when organized by a large and diverse group of actin-binding and regulatory proteins, self-assemble into dynamic and mechanically complex networks. The actin filament itself is polymorphic, with a structure and a set of mechanical properties that are modulated by the binding of regulatory proteins. Both the structure and the mechanical properties of actin filaments play an important role in determining the mechanical properties, architecture, and dynamics of the subcellular structure that result from self-assembly. We sought to investigate an important unanswered question: how do mechanical constraints help regulate the assembly of an actin network? This dissertation focuses on branched actin networks, which play a key force-generating role in the formation of membrane protrusions, in endocytosis, and in several types of intracellular motility. These networks are nucleated by the Arp2/3 complex and display adaptive behavior in response to compressive forces. They consist of Y-shaped branches formed by a pre-existing filament, the Arp2/3 complex bound to its side, and a new actin filament nucleated by the Arp2/3 complex. To investigate how the architecture of these networks is shaped by mechanical constraints, such as compressive forces arising from the resistance of cellular membranes to deformation, we devised a methodology for mechanically constraining single actin filaments while new branches are nucleated from their sides by the Arp2/3 complex. Branch nucleation on individual filaments was imaged with two-color fluorescence microscopy using a protocol that distinguishes constrained mother filaments from freshly nucleated daughter filaments. Combining this two-color assay with quantitative analysis of filament curvature, we show that filamentous actin serves in a mechanosensitive capacity itself, by biasing the location of actin branch nucleation in response to filament bending. We observed preferential branch formation by the Arp2/3 complex on the convex face of the curved filament. At radii of curvature of 1 micrometer, we observed approximately twice as many branches on the convex face as on the concave face. In the cellular context, where actin filaments tend to make a ~35 degree angle with the normal to the membrane, this observation suggests that compressive forces that bend actin filament tips away from the membrane would result in an enhancement of branching nucleated on the membrane-facing convex face of each filament. This effect constitutes a novel mechanism by which branched actin networks may be oriented toward membranes, as observed in vivo. Furthermore, in the context of a limited branching zone near the membrane, which is expected from the known biochemistry of the process, orientation of new branches toward the membrane also leads to an increase in network density in response to force, which has been documented in experiments with motility of bacteria in cytoplasmic extract. To explain the biased nucleation of branches on curved actin filaments, we propose a fluctuation gating model in which filament binding or branch nucleation by Arp2/3 occur only when a sufficiently large, transient, local curvature fluctuation causes a favorable conformational change in the filament. Using Monte Carlo simulations of a discretized worm-like chain model of the actin filament immobilized on a surface like the filaments in the constrained branching assay, we show that the fluctuation gating model can quantitatively account for our experimental data. Expanding the scope of the simulations beyond the in vitro experiment, we hypothesize that the curvature fluctuations of filaments in the cell may be modulated by the architecture of the actin network to which they belong. To test this hypothesis, we computationally explore how three types of mechanical constraints - buckling or bending of a filament end by a hard wall, bundling of filaments by a crosslinking protein, and uniaxial tension applied to a single filament - affect local curvature fluctuations. We find that bending of simulated filaments by a hard wall can significantly alter curvature fluctuations, the magnitude of which can be approximately calculated by the simple geometry of filament bending at the barrier. On the other hand, crosslinking of simulated actin filaments with crosslinking elements of physiologically relevant stiffness has surprisingly little effect on the small-scale local curvature fluctuations. Similarly, enclosure of a simulated filament bundle in a tube does not significantly affect curvature of filaments on the nanometer scale. Tension, however, in the range of 100 pN, does have a marked effect on curvature fluctuations in our simulations, suggesting that any interactions of actin-binding proteins with actin filaments that depend on bending fluctuations may be modulated by tension. This has been observed in several recent experiments, suggesting that the effects of tension on the biochemical interactions regulating actin network assembly and disassembly warrant further study. Overall, the results presented here demonstrate how filament curvature can alter the interaction of cytoskeletal filaments with regulatory proteins, suggesting that direct mechanotransduction by actin may serve as a general mechanism for organizing the cytoskeleton in response to force.
Author: Christine Chaponnier Publisher: Springer Science & Business Media ISBN: 0387336508 Category : Science Languages : en Pages : 154
Book Description
Tissue Repair, Contraction and the Myofibroblast summarizes the latest findings concerning the biology of the myofibroblast, a cell involved in the evolution and contraction of granulation tissue and of fibrotic changes. Coverage shows that the myofibroblast is responsible for the development of hypertrophic scars, pulmonary and renal fibrosis and bronchial asthma. Reviews the cell biology and pathology of the myofibroblast as well as mechanisms of fibrosis evolution in many organs and tissues.
Author: Thomas Kreis Publisher: Sambrook and Tooze Publication at Oxford University Press ISBN: 9780198599579 Category : Medical Languages : en Pages : 551
Book Description
Since the first edition, many new and important discoveries have been made in this expanding area of research. This updated edition details all that is currently known about the proteins involved in the microfilament, microtubule and intermediate filament systems in cells and their roles intension resistance and motor function. Biochemists will find this book particularly useful as will physiologists and biologists in fields in which these proteins are involved, such as neuromuscular research.
Author: Sharifah E. Albraiki
Author: Sharifah E. Albraiki
Author: Ravi Vattepu
Author: C.G. dos Remedios
Author: Cris dos Remedios
Author: Eric Alan Osborn
Author: 
Author: Viviana Ioana Risca
Author: 
Author: Christine Chaponnier
Author: Thomas Kreis