Timothy J. Mitchison - US grants

[unreadable] DESCRIPTION (provided by applicant): Summary. When cells divide they build a transient structure called the mitotic spindle to separate their chromosomes into two equal groups. Spindle assembly is a fundamental and fascinating process. We will study the molecules required to build mitotic spindles, and how they generate structures and forces within spindles. Blocking spindle assembly is an effective way of killing cancer cells, and understanding basic mechanisms of spindle assembly will reveal targets for more effective anti-cancer drugs. In the last grant period, we discovered that an unusual molecule called poly(ADP-ribose) is an essential building block of spindles, and we hypothesize that it may be part of a mysterious "spindle matrix" that helps physically organize spindles. We will look for proteins that bind to this molecule, and determine how it contributes to structures and forces within the spindle. The most studied components of spindles are microtubules, long filaments made of the protein tubulin, which grow and shrink rapidly and generate force on chromosomes. We will investigate the biochemical reactions that create new microtubules in the spindle. We will also use new microscopy methods to visualize how microtubules move within the spindle, and mathematical models to understand how the creation, movement and loss of microtubules generates the characteristic size and shape of spindles. Relevance Our studies will address major unresolved questions about how spindles self-organize to generate a stereotyped structure that nevertheless adjusts itself depending on circumstances, and how they perform their essential biological functions. These discoveries will have immediate relevance to the development of novel cancer drugs. [unreadable] [unreadable] [unreadable]

DESCRIPTION:(from abstract) The ability of eukaryotic cells to move over their substrate is important for such processes as embryonic development, wound healing and cancer metastasis. The mechanism is not known; however, it is clear that the action cytoskeleton plays a central role. One important aspect of cell movement is protrusion of the leading edge, which is dependent on actin polymerization. The overall aim of this application is to understand at a molecular level how polymerization of actin is regulated, and how it generates force for movement. Much of the work will be on the mechanism of the movement of a pathogenic bacterium, Listeria, which uses host cell actin polymerization to move through the cytoplasm and spread from cell to cell. The investigators propose to use biochemical fractionation to determine how Listeria induces polymerization of an actin tail. They also propose to determine the structure of the Listeria actin tail and how filaments in the tail depolymerize. They then intend to use what they learn in Listeria and apply it to understanding the movement of whole cells. One focus will be the mechanism by which the filopodia of neuronal growth cones extend and retract.

The aim of our program is to develop a novel approach to discovering molecular mechanism in biology, chemical genetics, and to change shape; and vesicle trafficking, the study of how cells selectively import and export proteins in different compartments. For both of these biological areas, chemical tools are particularly important because the events we wish to study happen in seconds or minutes, and most other methods of study are slow. The chemical genetics approach aims to emulate classical genetics as a discovery tool, by using chemicals that alter the functions of specific proteins as a way to understand the functions of these proteins. In particular, we want to use chemicals to identify new proteins, not previously known to be important in the function of the cytoskeleton or in vesicle trafficking. We therefore plan to accumulate large collections of chemicals (chemical libraries) and use high-throughput screening to identify chemicals that interfere with these processes, and only then identify the proteins that the chemicals bind to. In parallel, we will screen for inhibitors of active chemicals to share with the whole scientific community. In Projects 1 and 2, we will discover chemicals that inhibit cell migration and several key proteins involved in formation and function of the cytoskeleton. In Project 3, we will identify novel inhibitors of in-bound and out-bound vesicle traffic. In Project 4, we will develop new methods for identifying the targets of those chemicals, and new ways to assess the usefulness of a chemical library for biological screening. In Project 5, we will construct a chemical library patterned after alkaloids, which we expect to be a rich source of biologically active chemicals. Three Cores will support this work: Core A will provide administrative support, Core B will make the screening and database functions of the Program possible, and Core C will support all projects with synthetic chemistry expertise.

DESCRIPTION (provided by applicant): Cytokinesis is the process by which a cell divides into two daughters. When mitosis is complete, a cleavage furrow assembles in the middle of the cell and this furrow constricts to divide the cell into two equal parts. Cytokinesis is fascinating from a basic cell biology perspective, since it requires concerted activity of the cytoskeleton and membrane systems of the cell. It is also a process that could potentially be targeted in anti-cancer therapy. We are interested in two questions: how does the cell position the cleavage furrow exactly in its middle and how does the furrow assemble? We will address these by a combination of biochemistry, microscopy and addition of drugs. We will continue our biochemical analysis of two interesting cleavage furrow proteins, anillin, which we discovered, and septins, a family of GTPase proteins that we showed could assemble into filaments. We want to know what these proteins do in the cell and how they influence each other. We will study how new membrane is targeted to the cleavage furrow using extracts from frog eggs and the role of anillin in this process. Building a furrow at the center of the cell depends on microtubules that provide the positional information. We will probe how the microtubules associated with cytokinesis are organized in the cell and what mechanisms they use to target anillin and myosin II to the center of the cell to build a furrow there.

In the last funding period we developed a platform to allow post-docs and graduate students to construct libraries of potentially biologically active small molecules in a format that allows efficient screening. In this funding period, we will capitalize on the investment already made by developing libraries based on two approaches in which cycloaddition is followed by specific bond breakage: (1) a macrocyclic scaffold derived from a steroid ring system by sequential cycloaddition and retrocycloaddition; and (2) a library of biaryl macrocycles derived from a cycloaddition/fragmentation strategy. We also plan to perform follow-up chemistry on a number of dihydropyrancarboxamides identified as inhibitors of mitosis during the previous funding period. A second set of technologies developed in the previous funding period allows our library molecules to be arrayed on glass slides and probed with labelled proteins. We plan to exploit these fast, cheap binding assays as a general route to target identification for compounds identified as biologically active in a phenotypic assay.

DESCRIPTION (provided by applicant): Our goals are to develop "chemical genetics", an approach to solving mechanism in cell biology based on use of small molecule tools, and to discover small molecule tools that perturb cell division by novel mechanisms. The small molecules we discover will impact on cancer by revealing target protein/small molecule pairs as the starting point for anti-mitotic drug design. We will develop diversity-oriented synthesis (DOS) pathways that allow synthesis of large libraries of structurally diverse small molecules with complex stereochemistry. Library design will be guided by principles from cheminformatics to maximize diversity. We will screen these libraries for small molecules that perturb cell division, using automated fluorescence microscopy of treated cells, followed by computational analysis of images, to find hits that cause specific phenotypic effects. We will also screen for small molecules that inhibit the function of key proteins known to be involved in cell division using enzymatic assays and assays of protein binding to small molecules immobilized as microarrays. Library synthesis and screening will use a technology platform we developed in the previous funding period. We will optimize the affinity of interesting hits by synthesizing and screening "tuning" libraries that sample chemical space around the original hit. We will start by optimizing a hit we found from a DOS library that targets Eg5, a motor protein required for cell division. We will develop a new method for finding the protein targets of small molecules that cause interesting phenotypic effects, based on in vitro translation of cDNA libraries, and selection of the target protein by its binding to the small molecule immobilized on glass. Having found small molecule tools that perturb cell division by novel mechanisms, we will test their ability to kill cancer cells in vitro.

DESCRIPTION (provided by applicant): We request funds to purchase a computer controlled electron microscope that is equipped with a large CCD camera. This state of the art instrument will be used for basic biomedical projects by a large user group divided into four loose focus areas. The projects range in scale from cellular to molecular, and cover most modern methods of specimen preparation. A digital electron microscope is required because it will allow rapid image acquisition for a large number of projects, some of which require large numbers of images for quantitative analysis. The automated controls and digital image acquisition will also be optimal for introducing new users to electron microscopy and for minimizing maintenance requirements that might arise from operator inexperience.

In the last funding period we developed a platform to allow post-docs and graduate students to construct libraries of potentially biologically active small molecules in a format that allows efficient screening. In this funding period, we will capitalize on the investment already made by developing libraries based on two approaches in which cycloaddition is followed by specific bond breakage: (1) a macrocyclic scaffold derived from a steroid ring system by sequential cycloaddition and retrocycloaddition; and (2) a library of biaryl macrocycles derived from a cycloaddition/fragmentation strategy. We also plan to perform follow-up chemistry on a number of dihydropyrancarboxamides identified as inhibitors of mitosis during the previous funding period. A second set of technologies developed in the previous funding period allows our library molecules to be arrayed on glass slides and probed with labelled proteins. We plan to exploit these fast, cheap binding assays as a general route to target identification for compounds identified as biologically active in a phenotypic assay.

[unreadable] DESCRIPTION (provided by applicant): When animal cells divide they first round into a ball, that cleaves in the middle to form two daughter cells, then the two daughters spread out again. Understanding how these shape changes are controlled will provide basic knowledge relevant to human development and tissue formation, and might provide clues to new methods for blocking cell division in cancer cells. Cell shape is governed by the cytoskeleton, a system of fibers in the cytoplasm made of the subunit proteins actin and tubulin. Cell probably change shape during division controlling addition and loss of subunits from these fibers. We will use biochemistry and microscopy o understand the addition and loss reactions. The food-poisoning bacterium, enters human cells and generates a "comet tail" of actin fibers that propels the bacterium. By studying actin fiber formation and loss n these tails we will learn how subunit addition and loss is normally controlled during cell division, n aim 1 we will analyze how actin subunits are lost from polymers, both in single filaments and in Listeria tails. For example, are subunits lost from the ends of actin filaments, or does the whole filament break apart as it disassembles? We have purified three proteins that catalyze subunit loss, and we will determine how they work. In aim 2 we will ask how nucleation of new actin filaments in the cytoplasm is controlled during cell division, using extracts from frog eggs. We hypothesize that nucleation is globally stimulated in mitosis, and inhibited during cleavage. We will test this idea, and determine its biochemical basis. In aim 3 we will ask how an asymmetric assembly of microtubules call the midbody is formed as cells cleave, and how the polymers in this architecture are stabilized. Understanding midbody assembles will reveal principles for generating asymmetric assemblies in the cytoplasm, and will tell us how the cleavage furrow is positioned. [unreadable] [unreadable] [unreadable]

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DESCRIPTION (provided by applicant): This application proposes a systematic effort to collect and analyze multi-factorial "Pharmaco-Response Signatures" (PRSs) for 15 therapeutic small molecules across a bank of 80 cancer cells lines for which genomic data is becoming available. The signatures will be used to elucidate response mechanisms, identify specific determinants of drug sensitivity or resistance at the cellular level, and create new response classifiers. PRSs will be based on high dimensionality measurements of phenotypes in single cells collected using high content microscopy supplemented by biochemical and plate-based assays, all performed at multiple times following exposure to drug at multiple doses (for a total of ca. 5 x105 unique measurements). The data will be analyzed using a variety of mathematical modeling methods that incorporate more or less prior knowledge and will, in all cases, be combined with gene sequence and transcriptional data on pre-treatment state. Regions of the drug/cell line/dose response landscape that are particularly rich will be subjected to in-depth biochemical analysis aimed at creation of detailed mechanistic models of response pathways. By providing a more effective means to prioritize lead compounds, PRSs should help to overcome substantial obstacles to the development of therapeutic small molecules. Looking forward, such signatures should also be useful in monitoring patients during the course of therapy. For example, applying PRSs to measurements made on circulating tumor cells would fundamentally advance the personalization of cancer therapy. The assembly and analysis of sophisticated new signatures of drug response will involve close collaboration between seven investigators with expertise in medicinal chemistry, systems biology, genomics and high content screening and could not be undertaken in the absence of RC2 funding. Pharmaco-response signatures are expected to find a wide audience in industry and academe and to meet a critical knowledge gap;their creation will require new informatics approaches, reduction to practice of diverse measurement technologies and application of innovative mathematical modeling- the essence of a "grand opportunity." PUBLIC HEALTH RELEVANCE: Project Narrative The proposed development of pharmaco-response signatures is directly relevant to NIH goals of developing better anti-cancer drugs and identifying those patients most likely to benefit from specific therapies;it also meets the GO (RFA-OD-09-004) requirement that a unique information resource be developed through collaborative attack on a fundamental problem in translational drug development. The work can be initiated immediately and substantially completed within two years.

Our overall goal is to understand the mechanisms by which dividing cancer and normal cells are killed, or escape death, when treated with diverse anti-mitotic drugs. We seek understanding both at the level of cell population behaviors, and the molecular mechanisms that give rise to those behaviors. We are especially interested in understanding how different classes of anti-mitotic drug differ in their ability to kill cancer cells, and using that information to design better future drugs. We will address these goals in 4 specific aims: Aim 1. How do drugs with different anti-mitotic mechanisms differ in their ability to kill cancer cells at the level of cell population behavior? We will use microscopy with fluorescent reporters for key molecular events to measure single-cell responses to four anti-mitotic drugs across a panel of cell lines chosen to vary in apoptosis sensitivity. We will also test the effects of blocking different aspects ofthe drug response. Aim 2. Elucidate the molecular mechanism of cell death during mitotic arrest. To determine how the intrinsic apoptosis pathway is activated by prolonged mitotic arrest we will test candidate regulators of apoptosis, and pursue an unbiased biochemical approach. We will also Identify an alternative cell death pathway cells use when they cannot escape mitotic arrest, and apoptosis is blocked. Aim 3. Develop small molecule inhibitors of mitotic exit that work independent of the SAC. Our preliminary data suggest that novel anti-mitotic drugs with this mechanism would kill cancer cells more effectively than current drugs. We will test this concept, and identify druggable targets in the mitotic exit pathway, by small molecule screening in Core B using a cell-based assay, followed by identification of protein targets of hits. Aim 4. Compare drug responses in mouse tumors to those seen in cell culture. Drug response mechanisms elucidated in cell culture in aims 1-3 may not translate to real tumors. We will test whether cells that die during mitotic arrest in mouse tumors cause bystander killing of non-dividing tumor cells, which could explain why some tumors with low mitotic index can be treated with anti-mitotic drugs. Working with Core C we will probe responses to anti-mitotic drugs in mouse tumors at the single-cell level by intravital imaging.

DESCRIPTION (provided by applicant): The long-term goal of this Program Project is to understand, in precise quantitative terms, how individual cancer cells and tumors respond to drug treatment, from target engagement to induction of apoptosis to eventual tumor regression. This will improve patient care by allowing improved prediction of drug responses and rational design of combination therapies, and by identifying targets for better future drugs. We will address this goal in the context of two drug classes that trigger apoptosis in cancer cells, anti-mitotic drugs, and targeted apoptosis inducers, including TRAIL and ABT737. Experiments will be performed in cell culture and mouse tumors. We aim for an understanding of the cellular response to these drugs that is (i) mechanistic in explaining cellular phenotypes in terms of interactions among specific proteins and other bio-molecules (ii) quantitative in applying mass-action kinetics and other mathematical formalisms to predicting the behavior of ensembles of interacting proteins from knowledge of their individual biochemistry (iii) probabilistic in accounting for the variability from one cell to the next in responses to drugs with the attendant likelihood that only a fraction of tumor cells will arrest or die in response to treatment with a chemotherapeutic drug (iv) post-genomic in analyzing diverse cell lines (and ultimately patient samples) with knowledge of their genetic differences and with the possibility of applying powerful knock-out/in and RNAi strategies to alter genotype (v) integrative in assuming that determinants of drug response are multi-factorial and that multiple interacting pathways rather than single genes or proteins must be studied. We will address these goals in four Program Specific Aims: In aim 1 we will determine the molecular mechanisms that regulate MOMP in response to anti-mitotic drugs and ABT737. In aim 2 we will investigate the causes of variation in cell responses to anti-mitotics and targeted inducers of apoptosis. In aim 3 we will ask to what extent drug responses are the same in cell culture and mouse tumors, using intravital imaging and other methods. In aim 4 we will pursue several approaches towards translating mechanistic understanding from aims 1-3 into improved patient care.

The Program consists of three projects and 5 cores, distributed between 4 sites in Boston, all affiliated with Harvard Medical School. The Administrative Core will support all these sites with help in communications, meetings organization, web design and recruitment. It will support financial administration and purchasing for Projects 1 and 2 and Core B, and will interface with local grants management teams for financial administration of Project 3 and Cores C-E.

DESCRIPTION (provided by applicant): This proposal will create a center for Large Scale Production of Perturbagen-lnduced Cellular Signatures at Harvard Medical School and collaborating institutions, with a focus on perturbations provoked by small molecule drugs and cellular signatures measured using diverse biochemical and single-cell assays. The result will be a large, self-consistent and diverse set of network-centric Pharmacological Response Signatures that provide unique insight into disease processes, drug mechanism/selectivity and ultimately patient-specific responses to therapy. The initial focus of the Center will be small molecule kinase inhibitors, versatile perturbagens with high translational potential. We will use known inhibitors and also expand dramatically the publicly documented collection of inhibitors through new medicinal chemistry and use of kinome-wide selectivity assays. The responses of a large collection of human tumor cells and some primary cells to kinase inhibitors, will be assayed using multiplex biochemical assays (for 20-100 proteins) involving bead-based sandwich immunoassays and reverse-phase lysate microarrays, and single-cell assays (using imaging and flow cytometry) for cell cycle state, commitment to senescence or apoptosis, mesenchymal vs. epithelial phenotype and markers of primitive (stem-cell) status. Data will be collected, integrated and distributed using a series of novel, interoperable software tools that manipulate semantically-typed data arrays based on a new XML/HDF5 format. A multi-faceted informatics program will link these phenotypic and biochemical measures of cellular response to a rich and growing set of genomic data being collected by others. These goals will be met through pursuit of six linked specific aims. Aim 1 will focus on existing - largely clinical grade - kinase inhibitors and a set of 45 cell lines that are known to display diverse drug responses and for which extensive genomic data are available. Aim 2 will enlarge the set of perturbagens by developing a large library of kinase inhibitors using new and existing chemistry and profiling biochemical specificity across the kinome. Aim 3 will combine existing and novel compounds in a dose-response analysis across a set of >1000 tumor cell lines to identify representative cell lines and outliers which, in Aim 4, will subjected to detailed analysis at a single-cell level. Aims 5-6 will develop and deploy the information processing systems needed to collect, systematize and distribute diverse data types. This will involve a novel set of interoperable software tools that incorporate emerging no-SQL and semantic web concepts. Methods for adaptive experimental design will be developed to focus data collection on those areas of the dose response landscape where signatures are most informative. The final product will be a large publicly available data set radically different from, but highly complementary to, the expression profiles and genome data that are the primary focus of current high-throughput biological studies on perturbagen-induced cellular signatures.

? DESCRIPTION (provided by applicant): This proposal requests expanded support for a pioneering predoctoral training program in systems biology. The Harvard Ph.D. Program in Systems Biology attracts unusually adventurous and analytically confident graduate students, with a demonstrated determination to cross disciplines. Our central goal is to help these students identify biological questions to which an interdisciplinary approach can provide uniquely satisfying answers, and to prepare them to identify and address such questions independently in their future careers. The Program draws on the intellectual and practical resources of the entire Harvard scientific community to help students develop a broad, rigorous and creative approach to solving challenging problems in biology and medicine. A two-part Preliminary Qualifying Exam is central to our approach. Part 1 requires the student to propose a quantitative, computational, or theoretical approach to solving a biological problem. This tests the student's ability to conceive, articulate, and exercise quantitative and theoretical ideas and methods as applied to questions in biology. This section of the exam demands creativity and thoughtful analysis of what theory and computation can offer, as well as general knowledge about biology. Part 2 evaluates the student's plan for dissertation research and understanding of experimental logic and methods. Our students enter the Program with a wide variety of backgrounds. Students may take any of a range of science courses offered by Harvard or MIT (through cross-registration). The first year student faculty advisors work with entering students individually to help them determine which courses will best complement their existing training, and to help them to identify potential rotation labs. Five courses on different aspects of systems biology are offered by Program faculty. In addition all students are required to take a course on communication that culminates in writing a fellowship proposal, and a course on biomedical research ethics. Our program aims to educate students in the current state of the art in systems biology, and to encourage them to reach higher, expanding the use of systems biology approaches in biology and medicine. Our students have published many high-quality papers on systems ranging from bacterial pathogens to humans. An average student graduating from the program will have published 3.5 papers, of which 2 are first-author papers. We believe that the students we attract and the mentoring that we give them are both outstanding, and that we have the ability to recruit additional exciting students into the Program who could benefit greatly from the opportunities we can offer them. We are therefore requesting an increase from 6 training slots to 8 for this funding cycle. Students will be funded in their first and second year of graduae studies.

Project Summary We propose the existence of a ?Microtubule Integrity Response? (MIR), akin to the DNA damage and unfolded protein responses. The MIR network, in our hypothesis, comprises a system of sensor molecules that detect perturbation of microtubules and/or soluble tubulin by environmental insults and normal physiological inputs. In response, they control downstream signaling and gene expression. We will combine biochemical and genetic approaches to identify MIR sensors, determine how they control signaling and gene expression, and explore the consequences for microtubule physiology and pharmacology. We will identify a hypothetical microtubule- bound kinase whose phosphorylation of MAPs and tip-tracking proteins depends on intact microtubules, and test its role in homeostatic regulation of microtubules. We will determine how multiple Jnk-dependent phosphosites in the nucleus are increased following microtubule stabilization, and the consequences for cell physiology. We identified a robust MIR mRNA signature in which mRNAs for multiple ??? and ?-tubulins are counter-regulated by MT stabilizing vs destabilizing drugs across multiple cell types. We will use this signature in bioinformatics searches to discover novel microtubule physiology, e.g metabolic regulation of microtubules. We will develop imaging biosensors to measures soluble Tb and its spatiotemporal regulation in single cells, and homeostatic response to pertrubation. Finally, we will test the hypothesis that microtubules serve as sensors for mechanical cues such as cell shape, substrate stiffness and cytoplasmic crowding, and control cell responses to these cues via MIR signaling. Elucidating the molecular- and cell biology of the MIR will open new directions in fundamental cytoskeleton research, help us understand the therapeutic and toxic actions of microtubule targeting drugs using in cancer and inflammation, and reveal novel human physiology.

Project Summary/Abstract: We will investigate the cell and chemical biology of microtubules in order to answer fundamental questions of cell organization and improve treatment of human diseases. Microtubules are dynamic, linear polymers of the protein tubulin. They physically organize the cytoplasm of most human cells, and serve as transport tracks for moving cellular components. They are particularly important during cell division, when they build mitotic spindles which separate chromosomes, and in neurons, where they provide transport tracks for supplying distant synapses with new building blocks. We will probe cell division mechanism using frog eggs as a model system. Our work may help treat infertility and understand mistakes in cell division that give rise to birth defects. We k now most of the proteins required for cell division, but we do not know how they work together to build spindles or position cleavage furrows. We will use a new tool, quantitative mass spectrometry, to simultaneously measure hundreds of proteins in mitotic spindles in frog egg extracts, and how they compete for binding sites on microtubules. We will also combine microscopy, biochemistry and mathematical modeling to learn how the spindle communicates with the cell surface to position cleavage furrows. In neurons, we will investigate how tubulin is transported down axons, and test a new hypothesis in which stathmin proteins serve as transport adapters. This work will address a fundamental question in neuronal cell biology, and may help treat motor neuron disease (ALS). Drugs that target microtubules and are used as medicines can provide important insights into microtubule biology in adult human tissues. These include paclitaxel, which is used to treat breast and lung cancer, and colchicine, which is used to treat gout and other inflammatory diseases. We understand the molecular actions of these drugs on microtubules in detail, but not how they act in the human body to treat disease. We have developed new hypothesis for the therapeutic action of both drug classes, and will test these in cell culture and rodent models. This work could lead to new uses of old drugs, for example we suspect low doses of colchicine might be useful to prevent heart attacks and slow the progression of lung cancer. It could also lead to replacement drugs that are more active and less toxic. PHS 398/2590 (Rev. 11/07) Continuation Format Page

Project Summary We propose the existence of a ?Microtubule Integrity Response? (MIR), akin to the DNA damage and unfolded protein responses. The MIR network, in our hypothesis, comprises a system of sensor molecules that detect perturbation of microtubules and/or soluble tubulin by environmental insults and normal physiological inputs. In response, they control downstream signaling and gene expression. We will combine biochemical and genetic approaches to identify MIR sensors, determine how they control signaling and gene expression, and explore the consequences for microtubule physiology and pharmacology. We will identify a hypothetical microtubule- bound kinase whose phosphorylation of MAPs and tip-tracking proteins depends on intact microtubules, and test its role in homeostatic regulation of microtubules. We will determine how multiple Jnk-dependent phosphosites in the nucleus are increased following microtubule stabilization, and the consequences for cell physiology. We identified a robust MIR mRNA signature in which mRNAs for multiple ??? and ?-tubulins are counter-regulated by MT stabilizing vs destabilizing drugs across multiple cell types. We will use this signature in bioinformatics searches to discover novel microtubule physiology, e.g metabolic regulation of microtubules. We will develop imaging biosensors to measures soluble Tb and its spatiotemporal regulation in single cells, and homeostatic response to pertrubation. Finally, we will test the hypothesis that microtubules serve as sensors for mechanical cues such as cell shape, substrate stiffness and cytoplasmic crowding, and control cell responses to these cues via MIR signaling. Elucidating the molecular- and cell biology of the MIR will open new directions in fundamental cytoskeleton research, help us understand the therapeutic and toxic actions of microtubule targeting drugs using in cancer and inflammation, and reveal novel human physiology.

Project Abstract: We seek eight training slots to support students during their first and/or second year of training in pharmacological sciences. The Therapeutics Graduate Program (TGP) recruits students from multiple Harvard PhD programs to build a diverse and inclusive community focused on therapeutic science, with a strong focus on professional skills development and career mentoring. The TGP provides rigorous training in the science of drug discovery, evaluation, and clinical use through a required core curriculum, paracurricular activities focused on professional skills development, and a signature required internship, where students spend 2?4 months in an industrial or regulatory science setting before the end of their 4th year. Recruitment: The TGP will recruit students and faculty from multiple HMS and Harvard programs, including Biological and Biomedical Sciences (BBS), Chemical Biology, and Systems Biology graduate programs at Harvard Medical School (HMS), Harvard University, and the HMS-affiliated teaching hospitals. Faculty will be evaluated for scientific relevance and mentoring skills, and openness to student-centric training experiences. Diversity/inclusion: We are committed to building a diverse, inclusive, and supportive training community. Intellectual diversity will benefit by recruitment from multiple PhD programs. We will boost social and ethnic diversity by emphasizing how the TGP prepares students for careers with direct societal impact and provides clear and direct career preparation through internships. Professional skills and career development: We involve industry professionals in teaching 1st year classes and co-organizing paracurricular activities focused on skills development. Our required internship experience provides trainees with hands-on exposure to industrial or regulatory science work environments, and provides strong networking opportunities. Internships also help mentors understand the benefit of our commitment to student-centric training. Scientific rigor and responsible conduct: These components are emphasized throughout the curriculum, and reinforced by mentorships where students are exposed to team-based science in a professional setting. Training benefit: There is a strong demand for PhD-level scientists who have been rigorously trained in the core concepts of drug discovery. Demographic trends are increasing the need for drug treatments, and there is optimism for treating the most feared diseases based on recent progress and emergence of new drug modalities such as antibodies and oligonucleotides. Many research schools and hospitals are building translational research centers, often in collaboration with local industry, and these need to recruit faculty and PhD-level staff. The pharma/biotech industry has exploded in Massachusetts in the last decade, and many PhD-level positions remain unfilled. TGP trainees will lead in this growing field.

Abstract Molecular trafficking between the nucleus and the cytoplasm is essential for cellular health and is tightly regulated in all cell types including those in the brain. Recent publications demonstrated nuclear transport defects in neurons in Alzheimer?s disease (AD) and related dementias (ADRDs). AD/ADRDs are caused, in part, by misfolded tau protein, suggesting misfolded tau may impair nuclear transport by aberrantly interacting with nuclear pore proteins. We propose that late-onset neurodegenerative disease, such as AD, reflects two vulnerabilities: (i) At the cellular level, intrinsic loss of nuclear import efficiency during the neural differentiation program sensitizes the neurons to damages associated with misfolded tau. (ii) At the molecular level, nuclear pore complexes (NPCs) are selectively vulnerable to disruption by misfolded proteins because their activity depends on exposed hydrophobic phenylalanine-glycine (FG) repeats that are easily disrupted by misfolded AD/ADRD-tau and turn over very slowly. To test these hypothesis, we developed novel optogenetic nuclear transport assays, based on photo-activatable NLS/NES elements. We now propose to combine Mitchison group?s expertise in advanced microscopy and image analysis with Song group?s expertise in neuron cell biology and pathology models to measure rates of nuclear import and export in living neurons and test the effects of neural differentiation, misfolded tau, and drug candidates that may alleviate the effects of misfolded tau on nuclear transport. We will (i) characterize the change in nuclear transport rates during neural differentiation, (ii) compare the sensitivity of nuclear transport to AD/ADRD-related misfolded tau challenges (e.g. G272V-tau and P301S-tau) in neurons and neural progenitors, and (iii) investigate the underlying molecular mechanisms using super-resolution microscopy and immunoassays. The transport assays will also enable future translational programs aimed at rescuing nuclear transport in aging neurons. As a test case, we will characterize drugs that increase O-linked b-N-acetylglucosamine (O-GlcNAc) modification of intracellular proteins. This modification is thought to inhibit aggregation of misfolded proteins such as tau. However, FG repeat in NPC are among the most O-GlcNAc modified proteins. We propose that the function of this druggable modification is to protect the intrinsic vulnerability of NPCs to damage by misfolded proteins. Success on this R21 pilot will set the stage for moving our optical reporter strategy into mouse models of brain aging and degeneration, and for identifying drug targets and testing candidate therapeutic molecules in high- content assay formats.

Abstract This proposal requests additional support to implement a multi-faceted meditation program that cultivates wellness, resilience and leadership among Harvard graduate students in the biomedical sciences. The SKY Campus Happiness program teaches students tools to cultivate wellbeing and resilience, create positive-minded community, and nurture empathetic leadership. The SKY Campus Happiness program is a psychosocial, three component curriculum that incorporates evidence-based breathing techniques, meditation, belongingness and leadership. The SKY Program?s cornerstone practice is an evidence-based, rhythmic breathing meditation called Sudarshan Kriya Yoga (SKY). We will collaborate with the Chemical Biology Ph.D. program and Molecules, Cells, and Organisms Ph.D. program on pilot programming, which will be available to their students as well as others in the Harvard Integrated Life Sciences Consortium (HILS). The full curriculum will consist of 3 components: a weekend workshop where students learn the SKY technique, a Silent Retreat, and a Leadership Training. Students are not required to complete all three components, and will have the opportunity for deeper self-reflection and development through the second and third components. This supplement will be used to support the implementation of SKY Campus Happiness programming on Harvard?s campus and ongoing activities to support the student community. The mental health of college students has steadily declined over the last decade, with increased prevalence of depression and rates of suicide. University counseling centers are struggling to serve a growing demand. Students need a broader set of tools and community to support their well-being. The SKY Campus Happiness Program will broaden the tools available to support mental well-being and resilience at Harvard to better prepare students for competitive careers in a variety of settings.