Jonathon Howard - US grants

The long-term objective of the proposed studies is to understand the physical chemistry of chemomechanical transduction, the conversion of chemical energy contained in high-energy phosphate bonds to mechanical energy used to power intracellular movement. This process is accomplished by several enzymes, termed motor proteins, that include myosin from muscle cells, dynein from cilia and flagella, and cytoplasmic dynein and kinesin from eukaryotic cells in general. Current models, such as the crossbridge model, postulate that the underlying transduction process is a cyclic reaction of a motor molecule with a cytoskeletal polymer, an actin filament in the case of myosin and a microtubule in the instances of dynein and kinesin. After binding to the filament, the motor protein is thought to undergo a conformational change, the power stroke, that produces an increment of movement. The protein then releases the filament before rebinding at another site along the filament and initiating another cycle. The specific aim of the proposed experiments is to test directly such models by making mechanical measurements of the transduction reaction at the single-molecular level. The movement of microtubules across surfaces coated at low density with purified kinesin will be visualized by dark- field microscopy. Special apparatus, previously used by the investigator to study force-sensitive ion channels in hair cells of the ear, will be used to exert forces and to measure displacements with subnanometer precision on a millisecond timescale. The distance that a single kinesin molecule moves a microtubule upon the hydrolysis of a molecule of ATP will be determined. After characterizing the movement of microtubules by single kinesin molecules, the nature of the interactions between several kinesin molecules moving one microtubules by single kinesin molecules, the nature of the interactions between several kinesin molecules moving one microtubule will be studied in order to predict the behavior of large assemblies of motor proteins such as those found in muscles and cilia. Because of the structural and biochemical similarities between kinesin and other motor proteins, the elucidation of the molecular events underlying transduction by kinesin should significantly increase the understanding of cellular motility in general. It is hoped that this understanding may lead to more rational treatments of muscle disorders such as heart disease, or to better methods of selectively interfering with pathological cellular movements such as the invasion and proliferation of tumor cells, and the transport of viruses between the cell membrane and the nucleus.

The long-term objective of the proposed studies is to understand how motor proteins work. These enzymes, which include myosin from muscle, dynein from cilia and flagella, and kinesin from eukaryotic cells in general, convert the chemical energy contained in the gamma phosphate bond of ATP into mechanical work used to power intracellular transport. Several molecular models for force generation, most notably the crossbridge-cycle model, have been formulated based on ATPase assays, mechanical recordings from muscle, and structural studies. The strategy of this proposal is to directly test these models by using recently-developed, highly-sensitive physical techniques to measure force and displacement at the single-molecule level. Single kinesin molecules will be placed under various known loads by challenging each one to pull on a microtubule attached to a minute calibrated flexible glass fiber. The motion of the motor will be measured by imaging the tip of the fiber onto a photodiode detector with subnanometer precision and submillisecond time resolution. The mechanical performance of individual motors will be tested under a wide range of loads, ATP concentrations, and orientations. The mechanical components of the motor, including the elastic element posited by the crossbridge cycle model, will be characterized physically; and the change in strain in this elastic element, the powerstroke, will be measured. A crucial prediction of the crossbridge cycle model will be tested by comparing the single-motor force with the product of the elastic element's stiffness and the powerstroke distance. Using site-directed mutagenesis we hope to identify which amino acids form the various mechanical components, and propose to determine the role of kinesin's two heads. Lastly, by combining biochemical techniques with the newly developed optical tweezer technology, we propose to measure the distance moved per ATP hydrolyzed: the simplest version of the model predicts that this distance should equal the 8-nm step size. Because of the structural and biochemical similarities between kinesin, myosin, and dynein, the elucidation of the molecular events underlying energy transduction by kinesin should significantly increase the understanding of cellular motility in general. It is hoped that this understanding may lead to more rational treatments of muscle disorders such as heart disease, or to better methods of selectively interfering with pathological cellular movements such as the invasion and proliferation of tumor cells, and the transport of viruses between the cell membrane and the nucleus.

The long-term objective of the proposed studies is to understand how motor proteins work. These enzymes, which include myosin from muscle, dynein from cilia and flagella, and kinesin from eukaryotic cells in general, convert the chemical energy derived from hydrolysis of the gamma phosphate bond of ATP into mechanical work used to power intracellular transport. The strategy of this proposal, which focuses on the microtubule-based motor kinesin, is to combine high-sensitivity single-molecule techniques with biochemical and protein engineering techniques in order to combine high-sensitivity single-molecule with biochemical and protein engineering techniques in order to identify the moving parts-the springs, levels, and axles- and to understand how their coordinated motion is coupled to the hydrolysis of ATP. Kinesin is a processive motor capable of making many steps along a microtubule without dissociating. We will test whether procesivity is due to mechanical coordination between kinesin's tow motor domains by measuring how force effects the dissociation of individual heads from the microtubule. Putative elastic elements will be localized, and a crucial prediction of the crossbridge cycle model will be tested by comparing the single-motor force with the product of the elastic element's stiffness and the powerstroke distance. We will directly determine whether changes in bound nucleotide alter the mobility of kinesin's two heads, by measuring the torsional stiffness of kinesin under different nucleotide conditions. Based on the approximately two-fold symmetry of dimeric kinesin when both its heads are in the same nucleotide conditions. Based on the approximate two-fold symmetry of dimeric kinesin when both its heads are in the same nucleotide state, we hypothesize that the power stroke is associated with a rotation of one head with respect to the other: we will use single- molecule fluorescence microscopy to visualize this rotation. To determine how tight is the coupling between chemical and mechanical steps, we will measure the effect of load on the ATP hydrolysis rate. A kinetic model will be developed to synthesize these mechanical results with biochemical of kinesin. Because of the structural and biochemical similarities between kinesin, myosin, and dynein, the elucidation of the molecular events underlying energy transduction by kinesin should significantly increase the understanding of cellular motility in general. It is hoped that this understanding may lead to more rational treatments of muscle disorders such as heart disease, or to better methods of selectively interfering with pathological cellular movements such as the invasion and proliferation of tumor cells, and the transport of viruses between the cell membrane and the nucleus.

Summary/Abstract A fundamental, but poorly understood, problem in cell biology is how the sizes of organelles are controlled. The lengths of mitotic spindles and axonemes, for example, vary by as little as a few per cent between cells of the same type. Furthermore, the correct size and morphology are essential for function-mitotic spindles for cell division and axonemes for motility. Cells regulate the sizes of these organelles by tightly controlling the lengths of their constituent microtubules. In the absence of a molecular ruler that templates microtubule length, it is thought that length control results from a delicate balance between polymerization and depolymerization of the microtubules. How this is achieved is not known. ! Based on our previous work in which we showed that the motor kinesin-8 Kip3 is a length-dependent microtubule depolymerase, we hypothesize that motor proteins, in conjunction with other microtubule- associated proteins (MAPs), can provide feedback between length and dynamics that tightly regulates the lengths of microtubules. ! The general aim of this grant is to use single-molecule techniques, together with mathematical modeling, to understand how two additional proteins-the yeast kinesin Kip2 and the yeast homolog of the vertebrate polymerase XMAP215, Stu2-together with Kip3, regulate the lengths of yeast microtubules. We have devised a novel purification scheme for native budding-yeast tubulin and this allows us to employ yeast as our model system, which has distinct advantages due to the small number of tubulin isoforms and the absence of potentially confounding post-translational modifications found in vertebrate, and in particular brain, tubulin. ! Our specific aims are to (1) characterize the acceleration of growth of yeast microtubules by Stu2, (ii) determine how Kip2 promotes microtubule assembly, and (iii) examine the precision with which Kip3, in combination with Kip2 and Stu2, controls microtubule lengths. These studies will provide important insight into the assembly and function of the mitotic spindle and establish principles of length regulation that will be applicable to other biomedically relevant organellar systems such axonemes, microvilli, stereocilia and filopodia.

DESCRIPTION (provided by applicant): The general problem that I will address is how neurons integrate their inputs and compute their outputs. Central to integration and computation is the morphology of neurons, and in particular that of their highly branched dendritic arbors, which receive synaptic input from other neurons or sensory input from the outside world. The connections between axonal and dendritic processes define the nervous system's structure, which is viewed as a prerequisite for understanding neural function; connectomics, the global study of neuronal connectivity, has emerged as a major goal of neuroscience. In this Pioneer proposal, I want to take an orthogonal approach to neuronal morphology. My hypothesis is that the cell biology of the neuron-the transport and turnover of materials-places very strong constraints on both building and maintaining dendrites. Furthermore, I propose that these constraints are so strong that they actually compromise the functioning of neurons: I hypothesize, for example, that the changes in diameters of dendritic processes across branch junctions are dictated by transport constraints and that they actually degrade signal propagation. If this is true, then morphology is a compromise between cell biology and neuronal function, and determining the nature of the tradeoff is likely to provide key insight into connectivity. The morphological rules that I will uncover will provide powerful a prioris for determining connectivit maps, and may help to solve a major problem in connectomics: how well does the connectivity map need to be in order to understand the function? To test this hypothesis, one needs a system in which morphology can be measured precisely (and in the most general sense, which includes protein localization), where it can be manipulated in a controlled way, and where morphology can be correlated with function. The Class IV dendritic arborization mechanoreceptor of Drosophila larvae meets these requirements, and will be the initial focus of study. The experimental goals are: (i) to use light and electron microscopy to discover the full set of branching rules?that is, how diameters, angles, branch lengths, and protein & organelle distributions change over branch points. And: (ii) to use calcium and voltage recordings, together with behavior, to characterize the function of the neuron. The measurements will be done in wild-type flies and in mutants, in which the morphology has been modified using precise genetic manipulations. The theoretical goal is to determine the extent to which the observed anatomical and functional characteristics optimize transport and developmental constraints on the one hand, and signal processing constraints on the other hand. The theory will be done in close coordination with experiments performed in the same laboratory. The nature of the tradeoff between these conflicting costs and benefits will provide tremendous insight into neuronal architecture. This research, via the combination of precise experimental measurement and theoretical modeling, will add inestimably to our understanding of the relationship between form and function in the nervous system. We hope that principles will be found that apply broadly across nervous systems and that the principles will have practical value in the determination of the structure of neural networks.

The well-being of humans and ecosystems relies on biodiversity on Earth. Diversity emerged because animals and plants use a fantastic variety of methods to survive in all forms of habitats. In the case of animals, survival depends crucially on their ability to move around. Most animals use fins in water, wings in air, and limbs on land. But these appendages have extraordinary morphological diversity and can be repurposed for novel functions such as using fins to walk and limbs to swim. This project will establish the Evolutionary Morphogenesis and Biodiversity (EMBody) Institute to drive discoveries on how appendages are formed and used in animals. Complex and poorly understood processes, ranging across levels of organization from molecules through cells to populations and in speed from milliseconds to hundreds of millions of years, drive the diversity of appendages. The EMBody Institute will use multi-disciplinary collaborations to produce novel techniques and tools to study the processes that shape animal appendages. Additionally, learning how animals move over diverse environments can lead to improvements in the design of robots used in disaster relief by land, sea, or air. Importantly, the Institute will foster a culture of inclusivity to broaden participation in research, education, and public engagement with science.

A central question in biology is how biodiversity on Earth emerged from the complex, multi-scale interactions of biological processes with the physical and chemical environment. The Evolutionary Morphogenesis and Biodiversity (EMBody) Institute will focus on animal locomotion and the remarkable diversity of propulsive appendages, essential for movement and survival in diverse habitats. This Design proposal aims to establish a collaborative community that integrates the multiple disciplines needed for propelling breakthroughs in understanding the evolution of morphogenesis in vertebrate appendages such as fins, limbs, and wings. Appendages develop through morphogenesis, a dynamical process that integrates genetic patterning with biochemical and mechanical regulation. Form enables function but does not dictate it. Rather, physical interactions with the environment, governed by mechanical principles and neural control, leads to function. Ultimately, natural selection operates on function, and the evolutionary transformation of ancestral gene regulatory networks yields novel forms and functions. From genes at the smallest level to selection on populations at the largest scale, this inextricable loop is the central theme of the EMBody Institute. The Institute will integrate experts from multiple disciplines and multiple levels of biological organization: (i) development that drives the emergence of diverse morphologies from shared gene networks through regulation, (ii) biomechanics that generates function by neural, musculoskeletal, and mechanical interactions, and (iii) evolution that transforms ancestral gene networks to yield novel morphology and function. The collaborative activities will generate and test novel hypotheses, innovative measurement methods, and unique datasets to benefit multiple scientific communities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY Microtubule Severing and Regrowth by Spastin Hereditary spastic paraplegia (HSP) is a neurodegenerative disease that causes progressive gait disorder. The most commonly mutated gene found in HSP patients encodes the microtubule- severing enzyme spastin, which can sever microtubule polymers into shorter fragments. While microtubule severing proteins?spastin, katanin and fidgetin?have been long thought to disassemble the cellular microtubule network, in vivo studies in various organisms have shown that they can actually increase the number of microtubules in cells. Our long-term goal is to establish a framework for how spastin regulates cellular microtubule networks, and how perturbation of spastin activity leads to neuronal degeneration. Recently, by reconstituting the activity of purified spastin, we discovered that the protein possesses a novel activity that promotes the regrowth of severed microtubules. This activity is independent of its canonical severing activity. We showed that the combination of severing and microtubule regrowth promotion can lead to an exponential increase in the number of microtubules and the amount of tubulin polymer. Based on this work, we hypothesize that spastin increases the amount of cellular microtubules by combining these two activities. How spastin perform these functions, however, is poorly understood. The overall objective of this project is to understand the molecular mechanisms of severing and regrowth using a combination of single-molecule fluorescence microscopy, force spectroscopy and mathematical modelling. Our two specific aims are: (i) dissect the molecular mechanics of spastin-dependent microtubule severing, and (ii) test competing models for how spastin promotes microtubule regrowth. Completion of these aims is expected to yield detailed kinetic and mechanical mechanisms for spastin?s activities and to generate reagents that will facilitate future structural and cellular studies, including on the pathophysiology of spastin.

PROJECT SUMMARY Dendrite structure: Data-Driven Models to Bridge from Molecules to Morphology The highly branched structures of dendritic arbors enable the extraordinary connectivity and information-processing power of the nervous system. Altered dendritic morphologies are often associated with neurological conditions and diseases. While we know many molecular components underlying dendritic growth and structure through genetic and cell biological studies, we still do not understand how molecular interactions generate dendritic arbors, which are thousands to millions of times larger than the constituent molecules. The overall goal of this application is to develop data-driven models that predict, quantitatively, dendritic growth in Drosophila Class IV da neurons. These cells are chosen because their dendrites can be imaged with outstanding spatial and temporal resolution, and the genetic tools in flies facilitate molecular manipulations. Our central hypothesis is that the growing and shrinking tips of dendrites constitute an intermediate level of organization between molecules and morphology. This allows us to divide the large gap between genotype and phenotype into two parts: the first is from molecules to dendrite tips, and the second is from dendrite tips to morphology. The second part will be bridged using models. To attain our overall objective, we will pursue the following three specific aims: (i) We will formulate kinetic rules underlying the dynamics of dendritic tips using high-resolution, live-cell imaging to measure the birth and death of tips through branching and retraction, and the transition rates between different velocity states. (ii) We will develop multi-scale mathematical models that take as input the data such as obtained in Aim 1 and predict morphologies, which will be compared to real dendritic arbors. (iii) We will genetically perturb cytoskeletal proteins and use the models to test whether the effects on tip dynamics account for the altered dendrite structures. The expected outcome is mechanistic understanding of how morphological phenotypes emerge from molecular processes occurring at the level of dendrite tips. These results will positively impact the field by bridging genotype to phenotype and by providing insight into the pathophysiology of genetic disorders that affect neuronal structures.

Biological cells are active materials meaning that they continuously turn over their constituent molecules as they dissipate energy obtained from metabolic processes. The physics underlying this non-equilibrium state—the material and energy fluxes together with the constraints that they impose on the organism—is only just beginning to be defined. Branched cells, such as those of the nervous and immune systems, pose especially difficult and interesting metabolic challenges. In neurons, branched dendrites collect synaptic or sensory information over a large area; yet the narrow, often bifurcated, dendritic processes must also supply materials and energy to all parts of the cell, especially to those locations undergoing growth or high activity. This project will pursue a physics-inspired approach to understand how this balance between the retrograde flow of information and the anterograde flow of nutrients impacts the morphology and function of dendrites. In this project, the group will formulate and test a supply-and-demand model that proposes that the supply of nutrients by transport processes such as molecular motor proteins match the metabolic demands of neuronal growth, activity and maintenance. The model predicts that the diameters of branched dendrites change across branch points according to specific laws that in turn depend on which cellular processes have the highest metabolic demands. These laws will be tested in living cells by combining state-of-the-art microscopy techniques with genetic and physical manipulations. It is anticipated that this project will provide insight into how the brain computes using so little energy (compared to man-made computers) and may elucidate principles that could be used in computing and engineering. The work may also improve our understanding of neurological diseases, which often arise due to the disruption of metabolic or transport processes. This work will entail training undergraduate, postgraduate and post-doctoral physics and biology students<br/><br/>What sets the profile of diameters in branched neurons? While electrical considerations must be crucial for setting diameters, it is also necessary that axons and dendrites be of sufficient girth to provide the flux of nutrients and energy to support the growth and activity of the cell, including cytoplasm, membrane, and synapses. What are the tradeoffs between information processing and material transport? Answering these questions is important for three reasons: It will provide design rules and models that increase our basic understanding of branched cells and tissues in general; it may facilitate the segmentation of neurons for making connectomic maps and classifying cell types; and it may provide insight into why aberrant dendritic morphologies are associated with disease. Reconciliation of the different interpretations of dendrite branching may give insight into how the brain computes so energy efficiently, a holy grail for engineers and computer scientists. Because of the deep mathematical connections between electrotonic spread and diffusion, and between action potentials and active intracellular transport, optimizing information processing and material flow may not be mutually exclusive. Indeed, shared signaling and transport constraints may have permitted the development of sophisticated brains that compute efficiently. This insight, if it holds up to the scrutiny of this project, may change the way we view evolution of the brain and may have applications in computing and engineering.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.