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 specifying the position of the cleavage furrow. Specialized arrays of microtubules are also used for ciliary and flagellar beating.
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. We also are using biochemical approaches to dissect the mechanism of T cell 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 (University of California, San Francisco) and Ron Milligan (The Scripps Research Institute), we determined the structure of the kinesin motor domain, analyzed how it binds to microtubules, and identified nucleotide-dependent conformational changes that power its movement along a microtubule. We also have shown that intramolecular tension, conveyed through mechanical elements called the neck linkers, allows the two motor domains in the kinesin dimer to communicate with one another. We are currently analyzing how the ATPase cycles of the two motor domains are coordinated.
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 a system in budding yeast 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 highly processive, have resolved individual steps taken by the dynein motor domains, and used optical trapping to analyze how dynein responds to force. We obtained the first crystal structures of dynein's microtubule-binding domain (in collaboration with Ian Gibbons, at the University of California, Berkeley) as well as the entire ~300 kDa motor domain. These studies provided insights into dynein's domain organization and potential mechanisms for ATP-dependent conformational changes. We are currently developing a more refined structural model for dynein-dependent movement and investigating how its motility is regulated by dynactiin and other adapter proteins.
Cytoskeleton Organization and Cell Division
Microtubules create the mitotic spindle, the central structure that facilitates cell division. From a genome-wide RNAi screen, we identified new proteins that are involved in mitosis and microtubule functions in cells. Through this screen, we discovered a large, eight-subunit protein complex, that we named augmin. Augmin docks the γ-tubulin ring complex (γ-TuRC, which nucleates new microtubules) to preexisting microtubules in the mitotic spindle. We have shown that augmin (along with γ-TuRC) gives rise to a branching pattern of microtubule nucleation, which plays an important role in amplifying the number of microtubules in the spindle. Another protein identified through the screen is patronin, that we found specifically binds to microtubule minus ends and protects them against disassembly. This function appears to play an important role in stabilizing the microtubule cytoskeleton in interphase and mitosis. We are interested in the structural features of augmin and patronin that allow these proteins to execute their activities. We are also studying structural diversity of tubulins, since mammals have many different tubulin genes and different post-translational modifications. We are preparing purified recombinant tubulins to probe how genetic variations or post-translational modifications affect tubulin and microtubule functions.
Recently, we also have developed a new technology for imaging single motor proteins (or any protein of interest) in living cells. This method (called "SunTag") involves recruiting up to 24 copies of a GFP-tagged single-chain antibody to a repeating epitope tag on a polypeptide. We (in collaboration with Jonathan Weissman) also used this method to recruit transcriptional activators to Cas9 to robustly activate the transcription of single genes in the genome.
Mechanism of T Cell Signaling
By combining tools of biochemistry and microscopy, we are dissecting the mechanism of the T cell signaling cascade. We initially entered this field by studying the organization of signaling molecule on the plasma membrane of T cells using single-molecule and confocal microscopy. We found that signaling molecules are organized into plasma membrane microdomains, which are held together by a dense network of protein-protein interactions, and not by phase separation of lipids into lipid raft domains. More recently, we are developing approaches to reconstitute the signaling pathway, so that we have control over each component of the pathway. By introducing the T cell receptor (TCR) and several proximal signaling molecules into a non-immune cell, we have been able to reconstitute peptide-major histocompatibility complex (pMHC) dependent phosphorylation of the TCR. This work allowed us to dissect the mechanism of TCR phosphorylation and our results support a model in which membrane bending by a TCR-pMHC interaction leads to a phase separation of plasma membrane proteins, which triggers TCR phosphorylation. Also, we are purifying the TCR signaling proteins and attaching them to membrane vesicles, or supported lipid bilayers, to study kinase-phosphatase reactions and to reconstitute downstream events in the signaling pathway (the latter work being conducted as part of an HHMI Collaborative Innovation Award).
Grants from the National Institutes of Health provide support for the work on mechanisms of dynein.
As of March 14, 2016