Ronald S. Rock - US grants

[unreadable] DESCRIPTION (provided by applicant): In the crowded environment of a typical eukaryotic cell, any object larger than 50 nm is effectively immobile and cannot rely on diffusion to arrive at its destination. As a result, myosin motors evolved to generate force for the transport of cargoes, cell motility, and cell division, processes that are critical for life itself. These motors convert chemical free energy by changing shape in a controlled manner while bound to actin filaments. A fundamental feature of all motor proteins is that their movements are carefully coordinated, so that the motor travels through a specific sequence of coupled biochemical and mechanical states. The nature of this coordination remains obscure, but it is clearly essential for proper motor function. The recent recognition that each myosin class has evolved specific structural and kinetic features allows for the comparison of different coordination mechanisms across the myosin super family. These individual adaptations may now be characterized by a host of advanced motility assays. The work proposed here will determine the allosteric coordination mechanisms using new single-molecule manipulations to alter the intramolecular strain felt by the motor. The role of the motor track in transmitting strain will be established, by mechanically altering the actin filament geometry. In addition, communication mechanisms will be identified for myosin V and myosin VI through load dependent stepping measurements under conditions where the two heads of the motor are uncoupled. These mechanisms will be contrasted with those for myosin X, a motor with unusual in vivo motility that suggests a novel form of strain sensing. The long term goal of this research is to reveal how motors, and systems of motors, have been optimized for their cellular trafficking tasks. Myosin motility is required for various normal and pathological cell functions, including development of polarized cellular structures, formation of cellular adhesions and tissue development, wound healing, intracellular trafficking, and cell division. This research will address critical questions about the underlying biology behind motor-related disease, including, among others, several forms of inherited deafness related to the proper development and maintenance of stereo cilia in sensory hair cells. [unreadable] [unreadable] [unreadable]

Post-translational modification of proteins by tyrosine phosphorylation is a critical feature in the evolution of multicellular metazoans. Recognition of phosphotyrosine-containing sequences is accomplished primarily by SH2 domains. These modular protein domains emerged in the simplest animals and have co-evolved with tyrosine kinases into complex and essential signaling systems. This project seeks to establish an integrated program of research and education into protein-protein interactions in signal transduction using approaches at the interface of the physical and biological sciences. Bioinformatic approaches, cell biology, peptide chemistry and genetics will be integrated to examine SH2 domains from a systems level, an evolutionary perspective and in terms of their role in promoting self-assembling molecular machines for signal transduction.

Intellectual goals for the project are:

1. Following on Dr. Nash's bioinformatic analysis of the human complement of SH2 domains, his research group will analyze the genomes of 14 species that cover the full range of SH2 domain evolution from the first appearance of functional phosphotyrosine signaling in social amoeba, through to humans. The development of novel SH2 protein architectures along with duplication and divergence will be traced to evolutionary events and changes in organismal complexity in terms of body plans and the development of new systems such as the adaptive immune system. His SH2 database and website (http://sh2.uchicago.edu/) will be extended to contain updated information regarding SH2 domain structures, targeted gene disruptions, alignments, tree structures, and evolutionary.

2. To define the specificity of a wide range of SH2 domains for physiological peptide ligands, an array of 192 peptides has been developed that comprehensively defines early events in Fibroblast growth factor, Insulin and Insulin-like growth factor signaling. By probing such arrays with some 50 SH2 domains that represent the various classes of SH2 domains, one can both identify potential interactions as well as use the ensemble data to define the specificity of SH2 domains for physiological ligands. The role of non-permissive residues and context-specific interactions in governing selectivity for physiological ligands will be investigated.

Intellectual merit
Successful completion of this project will provide understanding of the evolution of phosphotyrosine signaling in metazoans, how physiological interaction specificity has evolved to detail complex signaling networks and how the SH2 interaction-space has evolved to generate diversity in cellular signaling. The proposed work will extend our understanding of both individual SH2 domain-containing proteins as well as how phosphotyrosine based signaling functions have evolved to promote assembly of the molecular machines of signal transduction.

Broader Impact
This work will have broad implications in terms of the understanding of phosphotyrosine signal transduction as well as understanding of specificity and diversity among protein interaction domains. As interaction domains are increasingly implicated in generating combinatorial complexity required in the evolution of higher organisms, it is increasingly necessary to fully develop paradigms of this sort. Educationally, this project will expose a number of young scientists to experimental and symbolic approaches at the interface of biology, chemistry and computational/bioinformatic sciences. It will assist in training a future generation of scientists equipped to tackle complex, multi-factorial problems utilizing systems approaches

DESCRIPTION (provided by applicant): Cells perform diverse processes, such as cell division, growth, motility, formation of adhesions, and tissue morphogenesis, under a wide range of mechanical environments. Central to these processes are mechanical forces, which may come from outside the cell or may be generated internally and which are integrated with signaling pathways to guide the cellular process. The cell's macromolecular cytoskeletal machinery, including the actin-based myosin II motors and actin crosslinking proteins, assemble, function and then disassemble in response to these forces and signaling pathways. This dynamic force-responsive assembly provides self-tuning of the machinery, leading to natural positive and negative feedback and further allows mechanical inputs to be converted (transduced) into signaling outputs. Our collective research spans from single molecule to whole cell level functions with an emphasis on how contractile systems operate to drive cytokinesis and motility and to provide mechanosensory functions for the cell. In this application, we propose studies of two major model protein systems that capture key aspects of force-sensitive macromolecular assembly. Substantial published and unpublished data, including quantitative cell imaging combined with mechanical and genetic perturbations and coupled with computational modeling motivate the questions in this proposal. In particular, we aim to determine the molecular basis for force-dependent assembly of the myosin II bipolar thick filament (BTF). In Aim 1, we will determine the compliance within the BTF and then determine how this compliance restricts the activity of the myosin heavy chain kinase, which tracks the assembled BTF and phosphorylates it to promote BTF disassembly. Quantitative imaging will test how these mechanisms allow for force-dependent BTF assembly in vivo. In Aim 2, we will examine different isoforms of the actin crosslinker alpha-actinin which, based on their in vitro measured kinetic properties, are predicted to display different degrees of mechanosensitive sub-cellular accumulation. We will compare the mechanosensitive accumulation of each alpha-actinin isoform (human ACTN1 and ACTN4 as well as amoeboid ACTN). Because computational modeling supports a catch-slip behavior and/or structural cooperativity as the physical basis of mechanosensitive accumulation, we will determine the force-dependent binding lifetimes for each isoform using single molecule methods. In sum, this research effort will decipher key principles of force-dependent cytoskeletal assembly, which guide cellular processes such as cell division, cell motility, stem cell divisions, and tissue morphogenesis and homeostasis.

Abstract A critical function for all living organisms is the ability to move when needed. These movements--intracellular trafficking, cell division, muscle contraction, and cell motility-- are driven by molecular machines that exert an amazing amount of force considering that they are only a few nanometers across. Given the variety of motor proteins in the cell, a key question is how motors cooperate and compete while moving cargoes and applying forces. An emerging paradigm is the notion of specialized motors, or motors that are fine-tuned to perform a specific function. Despite the importance of these motor proteins, relatively little is known about their individual adaptations and how these relate to the motility patterns found in the cell. This work focuses on myosin-6 and myosin-10 and their unique forms of cellular regulation. Myosin-6 plays essential roles in organelle morphology, cell morphology, cytokinesis, autophagy, and endocytosis, while myosin-10 delivers essential cargoes such as integrins, cadherins and netrin receptors to filopodia at the leading edge of the cell. Both are overexpressed in tumors, owing to their roles in membrane trafficking, cell migration, and metastasis. The work will develop new approaches to control myosin-6 and isolate its activity in cells. One of the main approaches will be to sequester myosin-6 with light, making cells that have an optically switchable Snell's waltzer (myosin-6 null) phenotype. Experiments are designed to identify when myosin-6 acts as a passenger, an anchor, or a transporter in the cells, and if direct handoff from one cargo adaptor to another is required for function. The proposed work will also investigate how cargo binding and myosin quaternary structure tune myosin-10's motility. An integrated approach is developed, combining structural studies with functional reconstitution and single molecule motility assays. This proposal will test the hypotheses that cargo and extracellular ligand binding are both required for myosin-10 activation, and that cargo can steer myosin-10 from one type of actin network to another. Completion of this study will yield a comprehensive view of how cytoskeletal motor proteins are activated and regulated for distinct tasks in the cell. Motor protein regulation is a process of fundamental biological importance, but is poorly understood. This work will direct future efforts to understand activation in multiple contexts.

Abstract A critical function for all living organisms is the ability to move when needed. These movements--intracellular trafficking, cell division, muscle contraction, and cell motility-- are driven by molecular machines that exert an amazing amount of force considering that they are only a few nanometers across. Given the variety of motor proteins in the cell, a key question is how motors cooperate and compete while moving cargoes and applying forces. An emerging paradigm is the notion of specialized motors, or motors that are fine-tuned to perform a specific function. Despite the importance of these motor proteins, relatively little is known about their individual adaptations and how these relate to the motility patterns found in the cell. This work focuses on myosin-6 and myosin-10 and their unique forms of cellular regulation. Myosin-6 plays essential roles in organelle morphology, cell morphology, cytokinesis, autophagy, and endocytosis, while myosin-10 delivers essential cargoes such as integrins, cadherins and netrin receptors to filopodia at the leading edge of the cell. Both are overexpressed in tumors, owing to their roles in membrane trafficking, cell migration, and metastasis. The work will develop new approaches to control myosin-6 and isolate its activity in cells. One of the main approaches will be to sequester myosin-6 with light, making cells that have an optically switchable Snell's waltzer (myosin-6 null) phenotype. Experiments are designed to identify when myosin-6 acts as a passenger, an anchor, or a transporter in the cells, and if direct handoff from one cargo adaptor to another is required for function. The proposed work will also investigate how cargo binding and myosin quaternary structure tune myosin-10's motility. An integrated approach is developed, combining structural studies with functional reconstitution and single molecule motility assays. This proposal will test the hypotheses that cargo and extracellular ligand binding are both required for myosin-10 activation, and that cargo can steer myosin-10 from one type of actin network to another. Completion of this study will yield a comprehensive view of how cytoskeletal motor proteins are activated and regulated for distinct tasks in the cell. Motor protein regulation is a process of fundamental biological importance, but is poorly understood. This work will direct future efforts to understand activation in multiple contexts.