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Microtubule-Based Motility
Summary: Ron Vale studies the molecular motors that power the movement of membranes, mRNAs, and chromosomes inside of cells as well as the role of the cytoskeleton in cell division and cell shape.
The ability of cells to move their internal contents has fascinated biologists for centuries. Our research focuses on motility generated by mechanochemical enzymes, termed motor proteins, that travel along microtubules, 25-nm-diameter polymers found in all eukaryotic cells. During interphase, the orchestrated efforts of microtubules and motors are required for organizing large intracellular membrane compartments and for transporting small membrane carrier vesicles in the endocytotic and secretory pathways. During mitosis, microtubules are the primary constituents of the mitotic spindle and are needed for proper segregation of chromosomes and for specifying the position of the cleavage furrow. Specialized arrays of microtubules are also used for ciliary and flagellar beating.
Although many microtubule motors have been identified in recent years, their precise biological roles and the mechanism by which they generate movement remain to be elucidated. Through investigations of the microtubule motors kinesin and dynein, we hope to develop a detailed understanding of the energy transduction pathway used by ATP-hydrolyzing protein motors. We are also interested in how molecular motors transport membrane organelles and how motors and numerous other cytoskeletal proteins govern complex cell behavior such as mitosis, migration, cell morphology, and signaling.
Elucidating the Force-Generating Mechanism
The biological machines that generate cell migration and intracellular movements consist of a protein motor that hydrolyzes ATP and a filamentous polymer (either actin filaments or microtubules) that provides a track for the unidirectional motion of the motor. We are studying biological movement generated by the microtubule motor kinesin. Kinesin's motor domain represents the smallest known molecular motor (~350 amino acids). Together with Robert Fletterick's laboratory (University of California, San Francisco), we have determined the atomic resolution structure of the kinesin motor domain and discovered unexpectedly that it is similar in structure to myosin, an actin-based motor. We also use custom-built microscopes that can measure the force, steps, processivity, and velocity produced by single kinesin molecules.
Using a combination of spectroscopy, cryoelectron microscopy (with Ron Milligan's group, Scripps Research Institute), pre-steady-state kinetics, and mutagenesis techniques, we identified a critical mechanical element in kinesin (called the neck linker) and showed that it undergoes nucleotide- and microtubule-dependent conformational changes. This allowed us to construct the first structural model that explains the known features of kinesin movement. We are testing this model by introducing fluorescent sensors into the kinesin protein so that we can observe its conformational changes while it is moving along the microtubule.
More recently, we have turned our attention to understanding the mechanism of dynein, the least understood of the cytoskeletal motor proteins. Dynein is a large molecule: the minimal motor domain is approximately 10-fold greater in size than kinesin's motor domain. Studying dynein has required strategies to express and mutagenize its large motor domain and to develop single-molecule assays to study its activity. We have developed budding yeast as a system for expressing and purifying the dynein motor. Using in vitro motility assays, we have observed single-molecule motility of cytoplasmic dynein and find that it is more processive than kinesin and generates a comparable amount of force. We also find that processivity (long-distance movement along a microtubule without detachment) requires two motor domains working in a coordinated manner. By attaching bright fluorescent markers (quantum dots), we also have clearly resolved individual steps taken by dynein motor domains. Our results indicate that the two motor domains shuffle past one another in an alternating manner as the dynein advances along the microtubule track. We have been able to coax dynein to take such steps by pulling on it with an optical trap without providing chemical energy (ATP). This result has led to new model for dynein stepping. We are currently studying the structural changes that drive dynein motility.
Cytoskeleton and Membrane Organization
The cytoskeleton creates much of the asymmetry observed in nature. Controlling the dynamic assembly and disassembly of the cytoskeleton, therefore, is crucial for cell function. For the microtubule cytoskeleton, an important class of proteins binds to the growing plus ends of microtubules, facilitating microtubule assembly. The "plus-end binding proteins" also recruit various signaling molecules to the growing microtubule ends and carry them to the cell cortex. We are using a combination of structural, biochemical, and cell biological approaches to study the operation of these plus-end binding proteins. We have solved the atomic structures of the microtubule-binding domains of several of these proteins (EB1, Clip170, XMAP215). Although their structures differ, a common theme of these proteins is that they are dimeric or multimeric, which allows them to bind to two or more tubulin dimers. The ability to bind multiple tubulin subunits is critical for their tracking along microtubule plus ends and enhancing microtubule assembly. We also have solved the atomic structure of the cargo-binding region of one of these proteins (EB1) and identified several new proteins that interact with this region. (The National Institutes of Health provided partial support for these projects.)
We also have been using single-molecule and confocal microscopy to explore organization of the plasma membrane. Using T cells as a model system, we have found that the plasma membrane is dotted with microdomains enriched in molecules that facilitate signal transduction pathways. These microdomains are held together by a dense network of protein-protein interactions, and not by phase separation of lipids into lipid raft domains. By fluorescence imaging, we can watch single proteins bind to and dissociate from these signaling microdomains. Our recent results also show that T cells can form multiple, distinct microdomains during signaling, each with a distinct protein composition. Our next goals are to understand how these microdomains form and how this spatial organization contributes to T cell signaling.
Identifying New Proteins Involved in Cytoskeletal Function
Recently, we have combined the gene-silencing strategy of RNAi (RNA interference) with high-resolution microscopy to identify roles of cytoskeletal proteins in determining cell shape and governing cell division. Using Drosophila S2 cells, we are performing whole-genome (14,000 genes) RNAi screens and using high-throughput microscopy to identify genes involved in cytoskeletal function. To better understand the roles of genes identified by such screens, we are tagging the proteins with green fluorescent protein (GFP) to determine their intracellular localization and dynamics. We are also performing live-cell imaging of the cytoskeleton after gene knockdown. We have just completed a whole-genome screen for mitotic spindle shape, which led to discoveries of several novel genes involved in mitosis, including genes that participate in microtubule formation, centriole assembly, chromosome alignment, spindle shape, and the spindle assembly checkpoint. In addition to continuing our studies on these genes, we are launching new screens on other cytoskeletal functions.
In collaboration with Alejandro Sánchez Alvarado (HHMI, University of Utah), we have also launched a new project, using the light microscope, to study the cellular changes that underlie regeneration in planarians.
Last updated: January 29, 2008
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