James A. Spudich - US grants

The molecules involved in cell movement and changes of cell shape assume a variety of supramolecular forms during the cell cycle. For example, actin exists as monomers, filaments, and filament-bundles. These assemblies are transient and their formation must be under strict cellular controls. The filament-bundles appear to be part of a membrane-associated cytoskeleton which may be related not only to cell movements and cell shape, but also to cell adhesion and contact inhibition. Our approach is to purify the components involved in these cellular events and to study them biochemically and structurally, with emphasis on factors controlling their interaction and their assembly into higher order supramolecular forms. We have extensively purified and characterized actin and myosin from amoeba of Dictyostelium discoideum. Purification and characterization of various factors that affect actin assembly is underway. Furthermore, we have recently discovered that phosphorylation of Dictyostelium myosin heavy chain inhibits the assembly of myosin into bipolar thick filaments. The cellular controls acting on this phosphorylation of myosin are currently being examined.

1. Antisera raised in mice against purified nuclear and cytoplasmic poly(A)-binding proteins have been used to isolate genes for these proteins from yeast. DNA sequence determination and mutational analysis of the genes are in progess. 2. Preliminary studies have demonstrated the capacity of synthetic peptides to serve as signal sequences for the transport of proteins by nuclear membranes. We propose to exploit this finding to isolate the nuclear signal receptor, determine its location in the nuclear envelope, isolate the gene for the receptor, and reconstitute the transport process in vitro.

One of the early events after fertilization of sea urchin eggs is the outgrowth of more than 105 microvilli, which contain actin filaments in the form of a bundle (Burgess and Schroeder. 1977. J. Cell Biol. 74, 1032). The detailed substructure of these bundles has been determined by image processing (Spudich and Amos. 1979. J. Mol. Biol. 129, 319), and reconstitution of the bundles from actin and one other protein has been achieved by Bryan and Kane (1981. J. Mol. Biol. 125, 207). Begg and Rebhun (1979. J. Cell Biol. 83, 241) reported that cortices from unfertilized eggs do not contain actin filaments, but that filaments appear upon raising the pH to 7.5. This transformation can be readily studied since eggs can be obtained in large quantities and about 20% of the actin of the unfertilized egg is associated with the cortical layer (Spudich and Spudich. 1979. J. Cell Biol. 82, 212). We are analyzing the actin of unfertilized and fertilized eggs biochemically and structurally. Accessory components that modify the actin by binding to it or enzymatically altering it are being purified and characterized. In addition, we will work to establish an assay for the elongation of microvilli in permeabilized cells. Our objective will be to extract components essential to this elongation and identify them byreconstitution experiments. Other experiments will be designed to determine the direction of growth of the actin filaments in the sea urchin egg microvilli.

Muscle contraction and other forms of cell motility are believed to be driven by myosin molecules pulling themselves along actin filaments. Although the understanding of ATP-dependent movement of myosin along actin filaments is paramount to the understanding of motility, the molecular basis of this movement remains obscure. Recently, Sheetz and Spudich (1983. Nature 303:31-35) directly visualized the movement of myosin in vitro and devised a quantitative assay to measure this movement. The development of this assay provides the basis for this proposal. The overall goal is to define in molecular terms the parameters of the myosin molecular necessary for its ability to move. First we will attempt to develop a totally defined assay for myosin movement using an organized polar array of purified actin filaments. We will then characterize the biochemical and biophysical parameters of the myosin movement and search for activities in crude extracts of both Dictyostelium amoebae and skeletal muscle that modify the rate of the myosin movement. Such regulatory activities will be purified and characterized. Of primary importance will be studies to determine the regions of the myosin necessary for or involved in movement. For this part of the overall program we will examine the effects of various monoclonal antibodies against Dictyostelium myosin, the movement capabilities of myosin fragments prepared by proteolysis, and the movement capabilities of myosins altered by site-specific mutagenesis using a molecular genetics approach.

1. Antisera raised in mice against purified nuclear and cytoplasmic poly(A)-binding proteins have been used to isolate genes for these proteins from yeast. DNA sequence determination and mutational analysis of the genes are in progess. 2. Preliminary studies have demonstrated the capacity of synthetic peptides to serve as signal sequences for the transport of proteins by nuclear membranes. We propose to exploit this finding to isolate the nuclear signal receptor, determine its location in the nuclear envelope, isolate the gene for the receptor, and reconstitute the transport process in vitro.

Muscle contraction and many other forms of cell movement and changes in cell shape are believed to be driven by myosin molecules pulling themselves along actin filaments. Although the understanding of the ATP-dependent movement of myosin along actin is paramount to the understanding of cell motility, the molecular basis of this movement remains obscure. We have developed several in vitro assays for myosin movement on actin, two of which use purified actin and myosin. These assays allow us to quantitate the rate of movement of various forms of myosin. We are now in a position to determine which regions of the myosin molecule are required for motive force production and what aspects of those regions are important for movement. One overall goal of this proposal is to define in molecular terms the parameters of the moysin molecule necessary for its ability to move. We have succeeded in generating movement with short HMM a proteolytic fragment of mammalian muscle myosin missing the carboxy-terminal 110 nm of the 150 nm rod. Further work with proteolytic fragments of myosin will be carried out to determine, for example, whether two heads are necessary for short HMM to move. We will also use molecular genetic approaches to examine the movement capabilities of myosins altered by site-directed mutagenesis. Other experiments will be designed to determine the minimum number of myosin molecules needed for movement of a single actin filament. We also propose to examine whether organelle movements in vivo involve myosin- driven translocations analogous to the myosin-coated bead movements on actin filaments in vitro.

The ability to target specific mutations into the yeast genome has been an extremely powerful tool for the study of many cellular processes. The application of this methodology has been limited exclusively to yeast. Our initial description of gene targeting in Dictyostelium has made possible the use of this approach in an organism that displays many cellular processes that are not found in yeast. Dictyostelium is an excellent candidate for biochemical and molecular genetic studies of many cell biological processes found in higher eukaryotes. The two major goals of this proposal are to explore in detail the phenomenon of plasmid integration by homologous recombination in Dictyostelium and to use this approach to study the function of conventional myosin and single-headed myosin in vivo. 1. The requirements for efficient targeted integration will be determined: we will study the efficiency of targeted integration by linear and circular plasmids, the relationship of integration efficiency and homologous insert length, the integration efficiency of different portions of the mhcA gene, the use of carrier DNA to improve transformation efficiency, and the development of other selectable markers. 2. Gene-targeting will be used for the study of myosin function in vivo: myosin heavy chain (mhcA) null mutants will be constructed either by gene replacement or by plasmid integration, mutant mhcA genes will be introduced into the null mutant strains and the effect of specific site-directed mutations and also random mutations will be studied, the myosin I (single-headed myosin) heavy chain gene will be isolated and used to construct a null mutant, all the myosin mutants will be characterized by studying their cytoskeletal structure by electron microscopy and their behavior during growth, chemotaxis, and development by time- lapse video microscopy and by Nomarski DIC video microscopy.

Interaction of the molecular motor myosin with actin drives muscle contraction and various changes in cell shape and movements that are fundamental to the biology of nonmuscle eukaryotic cells. Essential to the function of myosin in nonmuscle cells is the regulation of its assembly into filaments and higher order structures such as the contractile ring, the assembly of which is spatially and temporally controlled. Little is known about the molecular basis of such spatial and temporal control but phosphorylation of the myosin molecule appears to play a key role. Light chain phosphorylation may regulate myosin filament assembly in some systems, but may primarily control the motor function of myosin. Heavy chain phosphorylation may be primarily involved in regulation of myosin filament assembly. The specific aim of this proposal is to study the roles of myosin phosphorylation in Dictyostelium, an organism that allows the convergence of biochemical, molecular genetic, and physiological approaches. It therefore offers a unique opportunity to investigate the molecular basis of how a cell moves, divides, and changes shape in response to cellular and developmental signals. The experimental plan for the next five year period can be divided into three parts: 1. Biochemical, structural, and molecular genetic studies on the kinases and phosphatases that are responsible for the control of Dictyostelium myosin phosphorylations. 2. Biochemical and structural studies on the effects of these phosphorylations on the functions of purified myosin. 3. Characterization of the effects of phosphorylation on the in vivo behavior and function of the myosin. The latter is made possible by recent applications of sophisticated molecular genetic techniques to Dictyostelium.

In muscle and nonmuscle cells the molecular motor myosin plays crucial roles in contraction, various forms of cell movement, and changes in cell shape. In spite of decades of investigations on myosin, the molecular basis of how the chemical energy of ATP hydrolysis is converted into mechanical movement is not understood. The development of quantitative in vitro assays for myosin movement along actin filaments has provided a new powerful tool to examine the molecular basis of this movement. Of these assays, the 'myosin-coated surface assay' has proved the most useful. These developments were the foundation of the origin of this grant. The specific aims of this competing renewal are to develop more refined in vitro assays for myosin movement and to use these assays and the actin- activated myosin ATPase assay to define the mechanical and biochemical parameters of native and mutated forms of myosin. We propose to develop the in vitro motility assay to measure individual steps in the motion produced by one or a few myosin molecules. We also propose to develop an assay for measuring force produced in vitro using purified actin and myosin. We have already used molecular genetic approaches to express large amounts of functional forms of native Dictyostelium myosin and its head fragments in Dictyostelium. Mutated forms of myosin and its fragments will be obtained using the same approaches. These various forms of myosin and its head fragments will be purified to homogeneity and will then be analyzed in vitro using actin-activated ATPase assays and in vitro motility assays to determine parameters of both velocity and force. Such correlations of ATPase activity, force, and velocity should lead to a better understanding of the molecular basis of myosin movement.

This proposal originates from our discovery in 1987 that the eukaryote Dictyostelium can be used for high-efficiency gene targeting. At that time disruption of the myosin heavy chain gene provided the first elucidation of the phenotype of a cell lacking this form of myosin. Since then Dictyostelium molecular genetic tools have been developed that have allowed the exploration in Dictyostelium of the in vivo roles of a variety of proteins in ways that have been readily accessible only in yeast. Whereas yeast lacks many phenotypes common to higher eukaryotic cells, including a multicellular developmental pathway, Dictyostelium manifests cellular and developmental behavioral aspects which are very similar to those of mammalian cells. The long term goal of this proposal is to exploit Dictyostelium as a model system to study the molecular basis of fundamental cell and developmental processes. The primary aims for the next five years are: (1) to define structure/function relationships of myosin II in regard to its in vivo roles in cytokinesis and other myosin II-dependent events, (2) to identify and isolate genes that code for proteins that are intimately involved in myosin II-dependent events, and (3) to study the in metro and in vivo properties of a protein tyrosine kinase that is essential for the final stages of the Dictyostelium developmental cycle. In the context of these three research foci, we will continue to expand further the repertoire of molecular genetic methods available for Dictyostelium. Aim 1 will involve both site-directed and random mutagenesis of Dictyostelium myosin II, including the isolation of cold-sensitive missense mutations obtained by a novel azide selection scheme. Aim 2 will involve three approaches for identifying and cloning new genes involved in myosin-related functions: use of the azide selection scheme to generate mutants that behave phenotypically as a myosin-minus cell but are mutations in other loci; application of the REMI (restriction enzyme mediated integration) technique for knocking out and tagging genes having myosin-associated function; and development of an antisense-driven technique for identifying and cloning genes involved in cytokinesis and other myosin II-dependent functions. Aim 3 will involve application of the repertoire of molecular genetic methods available for Dictyostelium to study an important member of the protein tyrosine kinase family.

One of the most fundamental processes in cell biology is the division of a mother cell into two daughter cells, resulting in the faithful partitioning of the cytoplasm and genetic material into two equal parts. While this process of cytokinesis clearly involves the contractile proteins actin and myosin, which accumulate in the furrow as a contractile ring, virtually nothing is understood at the molecular level about any of the key issues surrounding this process. How much of the actin and myosin concentrates in the furrow region of the dividing cell? How do these proteins know to concentrate there at exactly the right place and at the right time in the cell cycle? What is the detailed molecular architecture of the contractile ring? What turns on the contractile event and what terminates it? What limits the velocity of the furrowing process? How does the contractile ring get out of the way in the final stages of cytokinesis so as not to inhibit the necessary membrane fusion event to yield two independent daughter cells? What role, if any, does traction on a surface play in the cell division process? These are just some of the many questions that need to be answered in molecular terms. Key to a long term undertaking to solve this problem is the judicious choice of a model system. Dictyostelium discoideum is a model of choice because it's behavioral characteristics during mitosis and cytokinesis are extremely similar to mammalian cells. It is, however, a much simpler eukaryotic cell with only 1 percent of the genome size of a mammalian cell. Furthermore, it has single copy genes, it is haploid, molecular genetic approaches have been well worked out, and homologous recombination is very efficient. In addition, a wealth of information about a key player in cytokinesis, the myosin molecule, is already available. This proposal represents a long-term commitment to study cytokinesis in molecular detail. To lay crucial groundwork towards this long-term goal, detailed characterization of the dynamic relocalization of myosin as cells enter mitosis and proceed through cytokinesis will be accomplished. The structure/function relationships of myosin in regard to its in vivo role in cytokinesis will be examined. The goal of identifying other key genes that are involved and isolating all of the essential proteins will be pursued. Finally, synchronization of cytokinesis in populations of Dictyostelium cells for biochemical studies will be attempted.

PI's Name, Institution: Thomas Kenny, Stanford University
Proposal Number: 9980838
Proposal Title: Micromechanical Structures for Experiment in Biology
Project Abstract
The goal of this Engineering Microsystems: "XYZ" on a Chip project is to investigate development of micromechanical structures for use in conducting biological measurements. Many of the most interesting current problems in microbiology are related to the geometry and function of molecular and cellular structures. There is great controversy in the interpretation of measurements of protein folding and cellular adhesion, and also great opportunity for research based on new measurement approaches. Biological folding and adhesion problems may be viewed from a mechanical engineering perspective as a simple matter of forces and energies - all of which might be measured by direct mechanical means. Experimental matters are complicated by the small size of the forces at work in molecular biology - ranging from nano-Newtons to less than femto-Newtons. However, these are forces that can be measured with MEMS devices. We propose to look specifically at experiments to measure folding forces in ribosomal RNA, ligand receptor binding forces, and cellular adhesion forces. In each case, new micromechanical instrumentation will be designed and used to carry out these measurements. These experiments will provide new understanding of the mechanics, dynamics, and other characteristics of these fundamental biological interactions. Throughout this work, research will take place at the interface between engineering and biology, bringing excitement and expertise from both domains. Engineering students will learn about fundamental biological phenomena. Biology students will learn about the design and fabrication of MEMS structures for experiments. Together, they will design and carry out novel fundamental experiments and learn important engineering methods. Students emerging from this program will possess unique interdisciplinary skills suitable for leadership roles in the emerging area of "BioEngineering". Our primary goal for this program is to establish a world-leading program in the application of micromechanical structures for Biological measurements, and the world-leading source of young scientists trained in the issues and disciplines surrounding these experiments.

[unreadable] DESCRIPTION (provided by applicant): Myosin molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in myosins. Examples from this family of molecular motors include myosin II, a non-processive motor that drives cytokinesis and muscle contraction, and myosins V and VI, processive motors that drive vesicular movements in neurons and other cell types. Comparative studies of these three family members have revealed key features of how myosins transduce the chemical energy of ATP hydrolysis into the mechanical energy of movement. The molecular basis of myosin function has been analyzed in detail, following the advent of in vitro motility and laser trap assays, the latter of which allows direct measurement of force and displacement produced by a single myosin molecule pulling on a single actin filament. Data from these assays, among others, provide firm evidence supporting the lever arm hypothesis for the origin of directional movement. Nevertheless, many pivotal issues remain. For instance, we are beginning to understand the importance of tension sensing for modulating the activities and functions of myosin motors. In the coming grant period, we will fully characterize the processive stride of myosin VI, the motor that has most challenged conventional views of myosin structure and function. Using functional analyses of monomeric constructs, we will determine the structural basis of the power stroke and diffusive components of the myosin VI processive stride. We will also determine the molecular basis of tension sensing by myosin V and myosin VI and its significance in processivity. This effort will require our single molecule assays to be further developed using the most current available components. Importantly, we will also develop new technologies to directly visualize the power stroke and nucleotide dynamics of myosins in general to rigorously test current models of myosin movement. PUBLIC HEALTH RELEVANCE All forms of human movement, from the beating of your heart to the processes of individual cell division or cell migration, involve tiny molecular motors that burn the cell's fuel, ATP, in order to produce mechanical force. A wide variety of diseases, including congestive heart failure, cancer, deafness, and a number of neurological syndromes, are the result of defects in these motor proteins. Understanding how they work is therefore fundamental to understanding both their normal and pathologic behaviors. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Myosin-II bipolar thick filament formation is highly regulated in cells and is required for cytokinesis as a key component of the contractile ring. The long-term objective of this application is to understand in molecular terms the regulation of myosin-II bipolar thick filament assembly, and to elucidate the spatial and temporal control of contractile ring formation, maintenance, and dissolution during cell division. D. discoideum has a number of advantages for the study of cytokinesis, and will be used as the model system. The research plan is designed to answer the following questions. How does myosin-II heavy chain phosphorylation control the assembly of myosin-II bipolar thick filaments at the molecular level? How are the three known myosin-II heavy chain kinases organized during the cell cycle, and how does that organization relate to the dynamics of the myosin-IIcontaining contractile ring? What are the other essential proteins involved in the establishment, maintenance and dissolution of the myosin-II-containing contractile ring, and what are their cellular organizations and dynamics during cell division? Molecular genetic approaches will be used to create directed mutations in the myosin-II tail domain, to test specific hypotheses of the mechanism of regulation of thick filament assembly. These mutant myosin-IIs will be transformed into Dictyostelium myosin-II null cells to test for rescue of cytokinesis in suspension. Fragments of the myosin-II tail will be analyzed in vitro by biochemical and biophysical methods to examine phosphorylation-dependent changes in conformational states, and to characterize the kinetics and thermodynamics of the assembly process. Several approaches will be used to identify proteins that are crucial players in myosin-II-dependent cytokinesis. cDNA complementation will define suppressors of myosin-II -impaired mutant strains of Dictyostelium. Proteins that bind directly to the myosin-II tail domain will be pursued by affinity column chromatography. Finally, Dictyostelium genes homologous to cytokinesis-related genes in other organisms will be searched for. All identified new proteins will be characterized in vitro and in vivo. The spatial and temporal organization of proteins of the contractile ring and of proteins involved in its formation will be followed in live cells by fluorescent tagging ans visualization in vivo using computer-linked, low-light-level imaging. Total internal reflection fluorescence microscopy allows visualization of single myosin-II bipolar thick filaments in the cell cortex just beneath the cell membrane. This method will allow dual visualization of myosin-II and other fluorescent-labeled cytokinesis-related proteins.

DESCRIPTION (provided by applicant): The myosin family of molecular motors plays crucial roles in contraction, diverse forms of cell movement, and changes in cell shape. Myosin II is a non-processive motor that drives cytokinesis and muscle contraction, and myosin V and VI are processive motors that drive vesicular movements in neurons and other cells. The molecular basis of myosin function has reached a detailed level of analysis, due to the advent of in vitro motility assays, total internal reflection fluorescence microscopy for measuring movement of single molecules, and the establishment of laser trap assays that allow the direct measurement of force and displacement produced by a single myosin molecule pulling on a single actin filament. The specific aims of this proposal are to continue to develop these assays, and to use them and other approaches to examine the mechanical and biochemical parameters of native and mutated forms of both myosin V and myosin VI. We will also initiate studies on other members of the myosin family. Specific goals involving myosin V include preparation and characterization of a two-headed construct of myosin V joined by a totally flexible hinge, to test the role of the proximal "hip" region in processive movement. We will visualize individual fluorescent nucleotides as they come on and off myosin V during the stepping process to test models of chemomechanical coupling. These studies will be complemented with determination of the effects of both backward and forward external loads on the behavior of myosin V stepping. Similar studies will be carried out with myosin VI and other myosin family members, including preparation of altered forms of the motors to examine the roles of various domains. A primary goal for this grant period is to determine the molecular basis of movement of myosin VI, which is a non-classical myosin that moves by an unknown mechanism, but may involve the melting out of some part of the protein in a nucleotide dependent manner. We will use multiple approaches to reveal regions that become exposed during its chemomechanical cycle.

ATP phosphohydrolase; ATPase; ATPase, Actin-Activated; Actin-Binding Protein; Actins; Actomyosin; Adenosine Triphosphatase; Adenosine Triphosphatase, Myosin; Adenosinetriphosphatase; Arm; Binding; Binding (Molecular Function); Biological; Body Tissues; Cell Locomotion; Cell Migration; Cell Movement; Cellular Migration; Class; Collaborations; Complement; Complement Proteins; Complex; Crystallization; Cysteine; Data; Docking; Elements; Enzymes; F-Actin; Family; Family dynamics; Filamentous Actin; Fluorescence Spectroscopy; Goals; Grant; HMWMI; Half-Cystine; Head; High Molecular Weight Myosin I; L-Cysteine; Light; MLPH protein, human; MYH12 protein, human; MYO5A protein; MYO5A protein, human; MYO6 gene product; Measurement; Mechanics; Modification; Molecular; Molecular Interaction; Molecular Motors; Motility; Motility, Cellular; Motor; Myosin ATPase; Myosin Adenosinetriphosphatase; Myosin IV; Myosin Type IV; Myosins; Numbers; Photoradiation; Position; Positioning Attribute; Radiation, X-Rays; Radiation, X-Rays, Gamma-Rays; Receptor Protein; Resolution; Risk; Roentgen Rays; SLAC2-A protein, human; Specificity; Spectroscopy, Fluorescence; Structure; Tail; Tissues; Upper arm; Work; X-Radiation; X-Rays; Xrays; cell motility; dilute protein; family structure/dynamics; human MYO5A protein; melanophilin; melanophilin protein, human; member; monomer; mutant; myosin ATP phosphohydrolase (actin translocating); myosin VA (heavy polypeptide 12, myoxin) protein, human; myosin VI; myosin Va; myoxin; polymerization; receptor; receptor binding

PROJECT SUMMARY/ABSTRACT The overarching goal of this project is to use myosin as a model system in which to address the fundamental biological question of how alterations in tissue organization and function can arise from often subtle changes in function at the molecular level. Force generation by myosin is required not only for the physiological functions of skeletal muscle and the heart, but also for the proper development and maintenance of these tissues during embryogenesis and beyond. Our team aims to develop a detailed mechanistic understanding of how force generation by myosin acts to regulate muscle tissue development and homeostasis. We examine this general question through the lens of asking how seemingly small changes in the activity of individual myosin molecules can drive dramatic changes in tissue-level organization and function, for example in the context of inherited disease. In Aim 1, we will determine how structural changes in myosin affect the chemo-mechanical properties of the myosin-actin interaction for individual and small assemblies of motor proteins. This aim will leverage innovative techniques developed by our team to quantify biomechanical changes induced by myosin mutations at the single molecule level and the corresponding consequences for sarcomere-level structure and function. In Aims 2 and 3, we will determine how changes in myosin kinetics and force production influence the growth, maturation, and function of cells and tissues, using cardiomyocytes and skeletal myocytes as model systems. These aims will leverage CRISPR-editing to introduce myosin mutations in isogenic hiPSC-derived cardiac and skeletal myocytes. We will then be able to compare biomechanical alterations at the individual molecule level with those in sub-cellular organelles (myofibrils), cells and micro-tissues. We expect to answer basic mechanistic questions as to how alterations in protein structure and function affect cell and tissue function, changing force and plasticity, and provide a window into understanding how cells adapt to alterations in changing mechanical forces. We will then be positioned to utilize our hiPSC platforms for high-throughput screens to develop novel therapies targeted to phenotypic subgroups of myosin mutations. Another major goal of our Research Program is to support Early Stage Investigators (ESI). We will support pilot studies from ESI investigators that explore innovative research questions relevant to our Research Program. Critical to the NIGMS mission, our team?s multi-disciplinary integrated approach, spanning the scale from individual molecules to sub-cellular structures to whole cells to engineered micro-tissues, will serve as a prototype for teams undertaking future studies using hiPSCs to explore other biological protein assemblies, using human disease-producing mutations as perturbations to define their molecular and functional mechanisms across organ systems.