WorldCat Identities

Spudich, James A.

Overview
Works: 37 works in 60 publications in 1 language and 562 library holdings
Genres: Conference papers and proceedings  Periodicals 
Roles: Thesis advisor, Author, Editor, Other
Classifications: QH585, 574.87
Publication Timeline
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Most widely held works by James A Spudich
Dictyostelium discoideum : molecular approaches to cell biology by James A Spudich( Book )

12 editions published in 1987 in English and held by 228 WorldCat member libraries worldwide

Molecular genetic approaches to protein structure and function : applications to cell and developmental biology( Book )

2 editions published in 1989 in English and held by 109 WorldCat member libraries worldwide

The cytoskeleton( Book )

2 editions published in 1996 in English and held by 32 WorldCat member libraries worldwide

Annual review of cell biology and developmental biology( Book )

3 editions published in 1996 in English and held by 13 WorldCat member libraries worldwide

Annual review of cell developmental biology( Book )

1 edition published in 1998 in English and held by 10 WorldCat member libraries worldwide

Annual review of cell biology and developmental biology( Book )

1 edition published in 1995 in English and held by 9 WorldCat member libraries worldwide

Molecular genetic approaches to protein structure and function : applications to cell and developmental biology( Book )

4 editions published in 1989 in English and held by 6 WorldCat member libraries worldwide

Methods in cell biology / molecular approaches to cell biology / edited by James A. Spudich( Book )

4 editions published in 1987 in English and held by 5 WorldCat member libraries worldwide

METHODS IN CELL BIOLOGY, VOLUME 28: DICTYOSTELIUM DISCOIDEUM: MOLECULAR APPROACHES TO CELL BIOLOGY
Annual review of cell and developmental biology by James A Spudich( Book )

2 editions published between 1995 and 1998 in English and held by 4 WorldCat member libraries worldwide

Studying the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins by Megan Amanda Hartman( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

Molecular motors utilize energy from ATP hydrolysis to perform mechanical work, and examples include the myosins, a superfamily of proteins that utilizes actin filaments as a track for processive movement. There are many classes of myosins, and they share common elements necessary to regulate actin binding and force generation based on the identity of the nucleotide bound. Their diversity is most evident in their C-terminal tails; in the case of the unconventional myosins that do not form bipolar thick filaments, these regions mediate their associations with specific binding partners. Interactions between myosins and cognate adaptors allow for their recruitment onto cargoes, and it is these cargoes that define the functions of myosins in a cellular environment. Numerous studies have suggested developmental and cellular roles for myosins, but in general, few binding proteins have been discovered for most family members. In the case of mammalian myosin VI, yeast two-hybrid screening has revealed several adaptors that link this motor to vesicles, endosomes, and the Golgi, where it participates in a number of trafficking events. In contrast, very little is known about the proteins that bind to Drosophila myosin VI, despite the necessity of this protein for many essential processes during fly development. To better understand the specific pathways to which myosin VI contributes in flies, we set out to identify its cargoes. We used a combination of affinity chromatography and mass spectrometry in a proteomics-based screen to discover candidates, given the many advantages of this method over yeast two-hybrid and other approaches. Upon obtaining data indicating that over 1000 proteins could potentially associate with myosin VI, we were next charged with determining which interactions were specific and direct, and we chose to screen through select candidates with an in vitro assay. We identified a number of novel cargoes for myosin VI, including those associated with the Golgi, protein trafficking, microtubules, and others of unknown function. Next, we attempted to perform another screen to identify binding partners that might mediate the function of myosin VI in dorsal closure during embryogenesis. Because this motor is very concentrated at the leading edge of cells during this process, we developed an antibody to myosin VI for immunolocalization studies. We then examined embryos mutant for or depleted of myosin VI cargoes and assessed any effect on myosin VI localization. Although some showed slight differences in the staining pattern, we chose a different assay to test for a shared function between myosin VI and one of its novel cargoes. After commencing this project, we became aware of data indicating that myosin VI and Cornetto, one of the novel binding proteins we discovered, participate in Hedgehog secretion. Without further information about the assay used or the results obtained, we used cell culture to validate the original screen data and found that both proteins are indeed required for the timely secretion of exogenously expressed Hedgehog (Hh) from S2R+ cells. We then began examining embryos for defects associated with reduced Hedgehog secretion and found that a small percentage of flies expressing Cornetto RNAi or a dominant negative myosin VI truncation in Hh-producing cells indeed have the types of segmentation problems found in mild hedgehog mutants. Thus, we found a shared function for myosin VI and one of its binding proteins, which not only validates our screen data but also provides important information not previously available about their roles in development. In a second project, I turned my attention to another function for myosin VI to analyze its binding protein CG3529, which is orthologous to Tom1 family proteins involved in membrane trafficking. Based on information available about orthologs, I reasoned that CG3529 might mediate the involvement of myosin VI in asymmetric Notch signaling during pupal development, a role identified for this motor in another large-scale RNAi screen. Despite finding no evidence linking CG3529 to asymmetric trafficking of Notch in the dorsal epidermis as I had hypothesized, it was apparent that CG3529 depletion does affect mechanosensory bristle length, which had been previously noted for myosin VI as well. I then performed experiments to help determine by what pathway these proteins contributed to this process, but the data I collected did not indicate a shared function or binding interactions between CG3529 and the proteins to which its orthologs had been linked. Although it is still not clear what the function of this myosin VI adaptor is, I suspect that it is involved in protein trafficking at or near the endoplasmic reticulum, given that CG3529 appears to localize to this compartment. Beyond the proteins whose functions we were able to address, we obtained many other candidates that could potentially link myosin VI to a variety of cellular compartments and signaling pathways. The data presented here should be useful in future work to analyze the roles of myosin VI in specific systems upon utilization of information available about its novel binding proteins
Motors meet their cargo : establishing cellular functions of myosin VI through biochemical analysis of novel binding partners by Dina Finan( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

Molecular motors are proteins that use energy from ATP hydrolysis to walk along cytoskeletal tracks, often transporting cargo as they move. A substantial amount of work has been done to study their catalytic domains, obtaining in vitro biochemical, structural, and biophysical measurements of motors' stepping behavior. Compared to this wealth of data, much less is known about how they actually work in cells. Regions of the motor proteins called cargo-binding domains are responsible for mediating their interactions with other molecules, but we know relatively little about what specific cargoes they associate with, the biochemical basis of these interactions, how they are regulated, and thus the precise functions of the motors in vivo. We have focused on two unconventional myosin motors in Drosophila, V and VI, both of which play important roles in fly development, and whose cargo-binding domains have been relatively unexplored. We use a proteomics approach to identify multiple novel binding partners for both myosins. The identities and biochemical characterization of a small fraction of the candidate binding proteins have begun to shed light on pathways in which the motors are likely to be involved. We have focused on roles for myosin VI in protein secretion and intracellular trafficking, and it seems likely that myosin V will play distinct roles in trafficking of ribonucleoprotein complexes. The discovery of binding partners is an important step towards understanding what motors do in cells, and we expect that this method will be useful for other motors in the future. We also hope that these findings will continue to elucidate biological roles of myosin V and VI
The origin and evolution of alpha-catenin in epithelial cell polarity by Daniel James Dickinson( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex containing a classical cadherin, the Wnt-signaling protein [Beta]-catenin and the actin-binding protein [Alpha]-catenin. I have investigated the evolutionary origins of epithelial cell polarity and of the cadherin-catenin complex. I show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, I have identified and characterized an [Alpha]-catenin ortholog that binds a [Beta]-catenin-related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion and proper multicellular morphogenesis. Thus the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins
Annual review of cell biology( Book )

1 edition published in 1995 in English and held by 2 WorldCat member libraries worldwide

Understanding the molecular mechanisms of cardiomyopathy-causing mutations in sarcomeric proteins by Ruth Sommese( )

1 edition published in 2013 in English and held by 2 WorldCat member libraries worldwide

Heart disease is one of the leading causes of morbidity and mortality in the developed world. As such, significant amount of resources and energy are funneled into developing treatments and therapeutics. While genetically caused cardiovascular diseases manifest at the tissue level, the fundamental mechanism that triggers the secondary effects and tissue remodeling generally occurs at the protein level. It is therefore critical to understand the molecular changes on the basic biochemical and biophysical level and connect these to the cellular and developmental disease processes. One such cardiovascular disorder is hypertrophic cardiomyopathy or HCM, which has been estimated to affect approximately 1 in 500 individuals. It disproportionately affects young adults and is the leading cause of sudden cardiac death. Since the first case of genetically linked HCM was identified in the early 1990s, a significant amount of work has been done to understand the molecular mechanism of the disease. Mutations have been identified in the proteins comprising the contractile apparatus of the muscle, the sarcomere. One such protein is [beta]-cardiac myosin, which has been recognized as a significant culprit of genetically linked HCM (~30-50%). Many studies have been performed to understand how single point mutations can alter the enzymatic and mechanical properties of this motor protein, but there is no clear consensus about the molecular mechanism. This has been due mainly to the lack of available human [beta]-cardiac myosin and the use of non-human and non-[beta]-cardiac myosin. Non-human and non-[beta]-cardiac myosin from common animal models differ by>30 residues, and as clearly evident from the disease and from previous studies on myosin, a single amino acids mutation can significantly alter and disrupt myosin function. During my thesis work, I have performed the first biochemical and biophysical work in the field looking at cardiomyopathy mutations using human [beta]-cardiac myosin. I have shown that HCM-causing [beta]-cardiac myosin mutations result in a gain of function, increasing the power or work output of the myosin. I have also examined HCM-causing mutations in troponin T, a component of the thin filament regulatory unit, using human [beta]-cardiac myosin. In the muscle, the interaction of myosin and actin is regulated by calcium through the thin filament proteins, namely the troponin complex and tropomyosin. HCM-causing mutations in troponin T increase calcium sensitivity or the number of force-producing heads that can interact with actin, thereby also increasing force or power output in the muscle. My work not only sheds light on fundamental properties of thick and thin filament function in the human sarcomere and presents the first studies of HCM-causing mutants in a human background, it also establishes a new approach to the problem of cardiomyopathy that will be critical in truly understanding and targeting the disease
Single myosin molecule mechanics : a lecture( Visual )

1 edition published in 1995 in English and held by 2 WorldCat member libraries worldwide

Dynamics and mechanics of the actin cytoskeleton ex vivo by Mark Akira Tsuchida( )

1 edition published in 2012 in English and held by 2 WorldCat member libraries worldwide

In actin-based crawling motility, cells continuously build, reorganize, and disassemble an actin network in a process driven jointly by biochemical reactions and mechanical work. Herein, we use isolated actin cytoskeletons from fish epithelial keratocytes to study two aspects of this highly coordinated process. For the continuous actin network of a motile cell to drive translocation, net assembly of the actin network at the leading edge (which drives protrusion) must be balanced by net disassembly at the trailing edge. Although proteins such as ADF/cofilin and gelsolin are known to locally depolymerize actin filaments, it is not clear how such activity could be coordinated with assembly over the distance scale of the whole cell. Here we present experiments showing that activation of nonmuscle myosin II embedded in isolated cytoskeletons results in partial disassembly of the actin network. Taken together with prior work on the effect of myosin II inhibition in live cells, these results establish that myosin II contributes to actin network disassembly in the rear of motile cells, and suggest that gradual binding of myosin to a maturing actin network could serve as a mechanism to coordinate actin network assembly and disassembly over long distances. A thorough understanding of how forces produced by actin and myosin contribute to whole-cell movement will require detailed knowledge of the material properties of the cytoskeletal network at the relevant spatial and temporal scales. Measurements of mechanical properties have largely been limited to microscopic strains, whole-cell bulk measurements, or reconstituted gels that do not fully capture the cellular cytoskeletal organization. We have therefore sought to characterize the deformation of actin networks derived from motile cells under large applied strains. Strain could be applied to the lamellipodial actin network by displacing the cell body using a glass microneedle. The network behaves as a coherent material sheet that is mechanically well-coupled to the cell body and stretches to approximately 400% of its original dimensions. At smaller strains and time scales ranging from a fraction of a second to tens of seconds, the network behaves almost exclusively elastically, suggesting that the cytoskeletal network is capable of integrating and transmitting forces over large distances within the cell
Spatiotemporal dynamics from interlinked positive-and-negative feedback loops by Tony Yu-Chen Tsai( )

1 edition published in 2013 in English and held by 2 WorldCat member libraries worldwide

Positive and negative feedback loops are two of the most ubiquitous network motifs in biological systems. When a positive feedback loop and a negative feedback loop are coupled together, rich dynamics can emerge. Among these, oscillations in time and space, and pulses of protein activity are some of the commonly observed phenomena in a biological system. In this thesis, we started by computationally study the benefit of a positive feedback in an oscillator, and concluded that oscillators running on interlinked positive-and-negative feedback loops are more robust and tunable in period than the oscillators with only the negative feedback. We then focused on the Xenopus laevis embryonic cell cycle oscillator, a well characterized biochemical oscillator that runs on interlinked positive-and-negative feedback loops. We identified a transition of oscillator topology during Xenopus early development, from the long first cell cycle that depends critically on Wee1/Cdc25 mediated positive feedback, to the short subsequent cell cycles in which the positive feedback is dispensable. Using the human neutrophil-like HL60 cells, we studied the spatiotemporal dynamics of cytoskeleton proteins. We identified a perfect coupling between the protruding front and the retracting rear of a migrating HL60 cells. Myosin exhibits dynamic flashes at the rear of the cell, and the time scale of the flashes depends on local positive and negative feedback loops. When the duration of the myosin flashes is perturbed, the cell exhibits difficulties in turning and has inefficient chemotaxis. We also performed a statistical study comparing the shape and migration dynamics of HL60 cells, and provided evidence of a close correlation between the two phenotypes. Our results showed that the spatiotemporal protein dynamics allowed by proper regulation of interlinked positive-and-negative feedback loops is important in many examples in biology
From single molecules to single cells : mechanistic studies of myosin VI and cardiac myosin by Peiying Chuan( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

Myosins are a superfamily of molecular motors that couple the chemical energy derived form adenosine triphosphate (ATP) hydrolysis to precise mechanical movements along the filamentous cytoskeletal protein actin. There exist more than 20 distinct classes of myosins within this superfamily, and their specific biophysical and biochemical properties likely allow them to function accordingly in vivo. My research has focused on understanding the how the structural and kinetic characteristics of a particular myosin, myosin VI, relate to its proposed trafficking and tethering functions. In the first section of this dissertation, I perform single molecule studies on the contribution of an unexpected structural domain in the myosin VI tail to the efficiency of processive stepping, especially against external forces. I additionally use optical trapping to examine the load-dependent kinetics of the motor in the second section, and propose that external load in vivo likely regulates myosin VI's dual functions as cellular transporter and cytoskeletal anchor. The specific cellular cargoes as well as oligomerization states of different myosins are defined by their tail domain, which is the most divergent structural region among myosin classes. Myosin VI, shown to be monomeric when purified in its native form, likely dimerizes upon cargo binding. As most in vitro work on myosin VI so far have been performed on a truncated, artificially forced myosin VI dimer, I discuss in the third section steps taken toward achieving a more native, biochemically reconstituted full-length myosin VI molecule bound to its bona fide cargo. Having gained a better understanding of the biophysics, kinetics and biochemistry of myosin at the single molecule level, I moved on to studying myosin function at the cellular level. In the forth section of this dissertation, I examine the effects of a familial hypertrophic cardiomyopathy (FHC) mutation, R403Q in cardiac myosin, on the contractile function of single mouse cardiomyocytes. I find that the functional effects of the R403Q mutation in the diseased FHC heart are manifest at the level of cardiomyocytes as well, suggesting that these effects are cell autonomous. From these studies, we can better appreciate the structural and kinetic properties of myosin VI that make it uniquely suited for its functions. This knowledge is also beneficial in aiding the understanding of other molecular motors that may employ similar mechanisms. We are now also aware of the cell-autonomy of particular dysfunctions exhibited by diseased R403Q FHC hearts, and are better poised to understand the complex disorder as a whole
Myosin regulatory proteins mediate hypertrophic cardiomyopathy and mitochondrial homeostasis by Sadie Rae Bartholomew Ingle( )

1 edition published in 2013 in English and held by 2 WorldCat member libraries worldwide

The eukaryotic actin and microtubule cytoskeleton is extremely dynamic and complex. Actin and microtubule filaments, together with their accessory proteins, perform structural roles and provide tracks for molecular motors to move along. Converting chemical energy released upon nucleotide hydrolysis into mechanical work, molecular motors provide the driving force behind most non-Brownian movement within a eukaryotic cell. Myosins are a type of molecular motor that undergo conformational changes upon the hydrolysis of ATP and product release that enables them to translate along actin filaments. Myosin motors are responsible for the sliding of actin filaments during muscle contraction, as well as transport and anchoring of organelles, vesicles, and other intracellular components (Hartman et al., 2011). Identified for its role in muscle contraction, muscle myosin II (MII) hydrolyzes ATP to generate force and drive the sliding of actin filaments in the sarcomere (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954). MII functions in both skeletal and cardiac muscle, and is responsible for the muscle contraction, isometric force generation, and the contraction force associated with each heartbeat. In myocytes, MII is highly organized with about a dozen other proteins into sarcomeric units that form a near crystalline structure and comprise about 50% of myocyte volume. Availability of MII binding sites along the actin filament is regulated by MII accessory proteins in a Ca2+-dependent manner. Thus, MII-generated force and muscle contraction is dependent upon intracellular Ca2+ concentration and cooperation of accessory proteins. Human mutations in MII or its accessory proteins can yield a variety of disease states, depending on the mutated protein and the tissue in which it is expressed. In this work, we characterize four different mutations in the MII accessory protein troponin I that cause hypertrophic cardiomyopathy (HCM). A condition affecting approximately 1 in 500 individuals in the United States, the underlying mechanisms of HCM remain elusive (Maron et al., 1995a). Using a six-component system, we reconstituted the sarcomere in vitro and examined biochemical properties of the system with wild type and mutant troponin I protein. We found that three of the four mutations significantly increase the affinity of regulated actin ("regulated thin filament") for Ca2+. The fourth mutation we examined did not affect the affinity of the system for Ca2+, but rather affected the mechanochemical cycle of the MII motor. Because no therapeutics currently exist to treat the underlying causes of HCM, these studies highlight the complexities underlying HCM and the importance of understanding the complete biochemical and biophysical nature of the mutations that cause it. We next explore the role of myosin Va (MVa) and its accessory proteins in neuronal function. Mice expressing mutant MVa have marked neurological defects and die prematurely (Mercer et al., 1991). While new biological roles of MVa are emerging, it is likely that there are many that are yet to be discovered. To identify novel MVa functions, we chose to determine the proteins to which it binds in the cell. Affinity chromatography was used to ascertain candidate MVa binding partners from brain extract. Each candidate was tested for direct binding, and the protein spire1 was found to be a direct binder to MVa. Spire1 is an actin nucleator that was originally identified in Drosophila, and few mammalian functional studies had been performed at the start of this work. We serendipitously amplified a novel isoform of spire1 from mouse brain cDNA that targeted to mitochondria. Additional work with MVa-null mice showed that this mitochondrial localization was independent of MVa, and any potential role spire1 may play in mediating mitochondrial dynamics or homeostasis is also MVa-independent. Detailed analysis of mammalian spire1 isoforms showed that at least 3 exons are differentially-spliced to form spire1 of various compositions. Interested in the potential functional differences among isoforms, we created spire1 constructs lacking each of the 3 alternate exons, and found alternate exon C to be necessary and sufficient for mitochondrial localization of spire1. After confirming the expression of exon C-containing spire1 in multiple mouse tissues, we probed functional roles for spire1 by expressing different truncations in mammalian cells. We identified a striking mitochondrial phenotype that strongly suggests spire1 mediates mitochondrial fission and/or fusion in an actin-dependent manner. Finally, we studied the biochemical link between spire1 and mitochondria. We found alternate exon C to bind directly and specifically to cardiolipin, a mitochondrial-specific lipid in eukaryotes. We performed affinity chromatography to look for potential proteins that interact with exon C and tether it to mitochondria, but the purified spire1 protein with which we were working was not tractable for these experiments. This work highlights the similarities and differences between two different myosins, their accessory proteins, and their diverse biological functions
Methods in Cell Biology, 28 by James A Spudich( )

1 edition published in 1987 in English and held by 0 WorldCat member libraries worldwide

METHODS IN CELL BIOLOGY, VOLUME 28: DICTYOSTELIUM DISCOIDEUM: MOLECULAR APPROACHES TO CELL BIOLOGY
 
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The cytoskeleton
Alternative Names
James A. Spudich US-amerikanischer Biochemiker

James Spudich Amerikaans biochemicus

James Spudich biochimico statunitense

詹姆斯·斯普迪赫

Languages
English (42)

Covers
The cytoskeletonAnnual review of cell biology and developmental biologyAnnual review of cell developmental biologyAnnual review of cell and developmental biologyMethods in Cell Biology, 28