Samara L. Reck-Peterson - US grants

DESCRIPTION (provided by applicant): The goal of this project is to characterize the mechanochemical properties of the molecular motor dynein using a structure- function approach in the model system, S. cerevisiae. This work is relevant to human disease because humans lacking functional axonemal dynein develop Kartagner syndrome, a disease resulting in chronic respiratory problems, infertility and mirror- image organ positioning. Cytoplasmic dynein is thought to be present in all eukaryotic cells and has multiple, essential roles in cell growth and division. Research in the dynein field has been hampered by the lack of a system in which mutant proteins can be made and analyzed in vitro. The first aim of this project is to develop in vitro assays to study the motile properties of affinity purified yeast cytoplasmic dynein. Such biophysical assays have been pioneered by the Vale lab and others. These assays will be used to test hypotheses described in the following two aims. Unlike other cytoskeletal molecular motors, dynein contains multiple nucleotide binding sites that may bind and hydrolyze ATP to produce the force that drives movement. The second aim of this project is to determine the relative contribution of each ATP binding site in dynein function by mutating conserved residues predicted to be involved in ATP hydrolysis. Finally, the role of dynein-associated proteins in dynein-mediated motility will be analyzed. One such associated protein is the Liscencephaly 1 protein, which, when defective causes a severe brain developmental disease in humans. As dyneins are evolutionarily distinct from the molecular motors kinesin and myosin, these studies represent a frontier in the motor field and may reveal novel mechanisms for motor protein function.

Summary The microtubule (MT) cytoskeleton and the molecular motors that move along it-dynein and kinesin-are responsible for powering the movement of chromosomes during mitosis and of organelles, signaling molecules and RNAs in the cytoplasm. The spatial and temporal regulation involved in transporting these cargos at the cellular level remains one of the big unsolved questions in the field of cell biology. I propose to use the filamentous fungus, Aspergillus nidulans, as a model system to dissect the molecular mechanisms of MT-based transport, with a combination of approaches ranging from genome-wide screens to single- molecule biophysics. Aspergillus polarized hyphae, whose rapid growth requires MT-based transport, and its high frequency of homologous recombination make it an ideal model organism for studying transport. Importantly, the number and types of cargo transporting motors present in Aspergillus are more similar to mammalian systems than to yeast-like fungi. We will identify all the organelles transported by the Aspergillus motors; this will constitute the first inventory of cargos carried by MT-based motors in a single cell. After identifying these cargos, we will create a complete gene disruption library that will be used to perform high-throughput microscopy-based screens to identify novel molecules required for dynein- or kinesin-based motility. In parallel with screening, we will purify the native Aspergillus motors and determine their properties in vitro using single molecule motility assays. Hits from our screens that pass secondary rounds of screening will be tested in these assays for roles in regulating motor function or cargo binding. Ultimately, we aim to reconstitute motor-cargo transport in vitro and to develop methods to observe the dynamics of transport in vivo with nanometer precision. We expect to identify novel conserved paradigms regarding the mechanism of MT-based cargo transport.

DESCRIPTION (provided by applicant): The long term research goal of this project is to understand how cytoskeletal motors power the transport of diverse macromolecules within eukaryotic cells, enabling them to effectively organize their contents, move, divide, and respond to signals. This proposal focuses on cytoplasmic dynein, the largest, most complex, and least understood of the cytoskeletal motors. The specific objectives are to determine how single dynein dimers move processively, how ensembles of motors efficiently move cargo, and the role of processive movement in cells. A significant obstacle to understanding these important features of motility is a lack of tools to precisely control motor-motor and motor-cargo interactions in vitro. Using DNA nanotechnology, we have developed methods to achieve this. First, we generate stable, functional dynein heterodimers through DNA base pairing. Second, using three-dimensional (3D) DNA nanotechnology, we build synthetic cargo to which DNA-linked dynein or kinesin motors can be attached with defined numbers and spacing. To determine how dynein takes consecutive steps along microtubules, single-molecule techniques, including high-precision, multi-color fluorescence microscopy and single-molecule Forster resonance energy transfer (smFRET), will be applied to track the behavior of individual moving dynein molecules. The results of these experiments will be used to construct a model for how dynein moves processively on microtubules. To determine how coordination among dynein motors or between dynein and kinesin motors affects cargo motility, varying numbers of dynein or dynein mixed with kinesin will be attached to a 3D, synthetic DNA cargo. By analyzing the behavior of both the cargo and individual, cargo-attached motors in single-molecule motility assays, the biophysical properties of multi-motor-based transport will be determined. Long distance transport is thought to require processive motility. However, we recently discovered that dynein is sub-maximally processive. Using in vivo and in vitro approaches, we will test the hypothesis that sub-maximal processivity is especially critical for cytoplasmic dynein. Because cytoplasmic dynein is encoded by only a single gene in all eukaryotes but carries out a wide range of tasks, sub-maximal processivity may allow it to be tuned to perform a variety of cellular functions. This research will provide fundamental, mechanistic insights into how the ubiquitous and essential dynein motor works. In addition, the DNA nanotechnology tools generated here will serve as general engineering principles for studying the oligomerization state of other proteins or for studying arrays of any molecular motor in a more physiologically relevant manner.

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand how the microtubule (MT)-based motor cytoplasmic dynein (dynein) is regulated. Dynein is the largest and most complex of all cytoskeletal motors; this >1 MDa dimeric complex contains numerous mechanical elements whose movements must be coordinated over strikingly long molecular distances to achieve processive motility along MTs. In addition to its enormous size, dynein is also the most versatile of the molecular motors; in sharp contrast to the 45 kinesins and 39 myosins present in humans, a single dynein gene product is responsible for transporting macromolecules within neurons, constructing the mitotic spindle, polarizing cells, and anchoring mRNAs during development. To give dynein the functional plasticity necessary for carrying out its many roles, several ubiquitous co-factors interact with dynein, including Lis1 and the dynactin complex. This project will apply our combined expertise in biochemistry, single-molecule biophysics and cryo-electron microscopy to address the structural and mechanistic bases of dynein's interaction with MTs and its regulation by Lis1 and dynactin. We recently showed that binding of dynein to MTs is accompanied by conformational changes in its MT- binding domain and that Lis1 acts as a clutch to uncouple MT binding and release from ATP hydrolysis, promoting a strongly MT-attached state. Dynactin, a 1.2 MDa complex, enhances dynein's processivity and is required for nearly all dynein functions in cells, but its mechanism o action is poorly understood. This grant will address major mechanistic questions about dynein and its regulation. What are the structural and mechanistic bases of MT binding (Aim 1), and of regulation by Lis1 (Aim 2) and dynactin (Aim 3)?

SUMMARY The spatial and temporal organization of intracellular components by the microtubule cytoskeleton is required for cell division, many aspects of development, and neuronal function. Defects in the microtubule cytoskeleton cause neurological diseases in humans. The long-term goal of this research program is to determine how the multiple components of this transport system?the motors and their tracks, cargos, cargo adaptors, and regulators?work together. Microtubules are dynamic polar structures, with ?plus? ends usually located near the cell periphery and ?minus? ends embedded in internal microtubule organizing centers. Dyneins move towards the minus ends of microtubules, whereas most kinesins move in the opposite direction. In humans, a single dynein (cytoplasmic dynein-1) and ~15 kinesins are responsible for the interphase transport of organelles, proteins, and mRNAs. Viruses hijack these same motors. How does a small subset of motors transport such a large and diverse set of cargos? Understanding this is one of the major frontiers in the transport field. The goal of this proposal is to determine how cargo specificity is achieved for cytoplasmic dynein-1, the major minus- end-directed microtubule-based motor in eukaryotic cells. Traditional biochemical approaches have yielded surprisingly little information about these mechanisms. To solve this problem, we are using both genetic and proteomic discovery approaches to identify motor-cargo interactions and to determine how they are regulated. Genetic approach: Using a forward genetic screen in the filamentous fungus Aspergillus nidulans we co- discovered that peroxisomes ?hitchhike? on early endosomes to achieve motility. Previously, the paradigm was that each cargo directly recruited the transport machinery. We will investigate the mechanism of hitchhiking and determine if it is widely used for organelle motility across eukaryotes. Proteomic approach: Mammalian dynein requires the dynactin complex and a coiled coil containing ?activator? to achieve processive motility. Using proximity-dependent biotinylation we identified the components of the human dynein proteome in human interphase embryonic kidney cells, including novel dynein activators. We will use these proteomic approaches to identify additional novel dynein activators in different cell types and at different stages of the cell cycle. We will use these novel dynein activators as ?stepping stones? to identify their proteomes and determine which cargos they transport.

PROJECT SUMMARY The contents of eukaryotic cells are highly dynamic, yet organized spatially and temporally. This is achieved primarily by the microtubule cytoskeleton and associated transport machinery, whose fundamental nature is highlighted by the many neurological diseases caused by mutations in them. The overarching goal of my research program is to understand how this system works at the molecular, cellular, and organismal scales. My team is highly interdisciplinary and we use in vitro biochemical reconstitution, protein engineering, single-molecule imaging, proteomics, live-cell imaging, and fungal genetics to achieve our goals. Through collaborative projects we use cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to incorporate a structure-guided approach to understanding intracellular transport, and we develop testable quantitative physical models of transport. We have made major contributions to determining how the dynein motor works and is regulated, to developing tools and screening strategies to study bi-directional movement of cargos on microtubules, and to understanding the regulation of intracellular transport in cells. Fundamental questions that we will address here include: (1) How does the dynein motor work? Our earlier work revealed how Lis1, a protein mutated in the neurodevelopmental disease lissencephaly, interacts with dynein and regulates its mechanochemical cycle. Here, we will focus on determining the mechanistic underpinnings for how Lis1 promotes the formation of activated dynein/dynactin complexes. We will also explore a new direction?the role of RNA editing?as a previously undescribed mechanism to regulate dynein and kinesin motors. Microtubule-based motors move dozens if not hundreds of cargos. (2) How is cargo-specificity achieved? Our past work used two complementary discovery-based approaches?genetics and proteomics?to identify molecules responsible for specifying dynein?s many functions. One mechanism revealed by our past work is organelle hitchhiking, where cargos link to motors indirectly, by attaching themselves to other cargos that are directly bound to the motors. A second strategy for achieving cargo specificity is the expansion of dynein activating adaptor genes in vertebrates. However, the molecular connections between most activating adaptors and dynein?s cargo are unknown. Here, we will determine the mechanisms underlying hitchhiking and the linkages between the Hook and Ninein families of activating adaptors and their cargos. As an additional approach to understand how dynein and kinesin link to their cargos, we will visualize these connections in cells in three dimensions using cryo-electron tomography of endosomes in Aspergillus nidulans and melanosomes in Xenopus laevis melanophores, two systems where we can use exquisite genetics or chemical tools to control microtubule- based motility.