Laura Lowery, PhD - US grants

DESCRIPTION (provided by applicant): This proposal is designed to study the mechanisms underlying initial brain ventricle inflation, using a genetic approach in zebrafish. The brain ventricles are a conserved system of cavities containing cerebrospinal fluid that are required for normal brain function. Abnormalities in brain ventricle structure can lead to hydrocephaly and are correlated with many mental health disorders including autism and schizophrenia. The molecular mechanisms underlying brain ventricle development are poorly understood. The zebrafish is an excellent model for this study, as the brain ventricles are visible throughout development, and many genetic, molecular, and embryological tools are available. Research in the Sive laboratory has demonstrated that brain ventricle formation is a multi-step process, involving at least two steps: formation and maintenance of epithelial integrity by junction-associated proteins, and initial inflation of the ventricles with fluid, which requires the snk (atp1a1a.1) gene encoding a Na K ATPase. This proposal will test the hypothesis that the Snk protein functions in initial brain ventricle inflation by acting locally. Tissue transplants and mosaic analysis will be used to ask whether limited wild-type Atp1a1a.1 function can rescue snk mutants. Additionally, this proposal will test the hypothesis that multiple genes synergize with Snk to direct initial brain ventricle inflation. Five mutants isolated from chemical screens and two from an insertion mutagenesis screen show a putative brain inflation phenotype. These will be tested for synergistic effects with the Snk protein. This analysis will exploit the zebrafish to determine the mechanisms underlying brain ventricle inflation and that are required to build normal brain structure, which may be perturbed in mental health disorders.

DESCRIPTION (provided by applicant): Accurate axon pathfinding is an essential yet highly complicated process during nervous system development. The mechanisms by which axons form complex functional neuronal networks are still a major puzzle and are relevant to understanding how abnormalities in neuronal development arise and also to nerve regeneration therapeutics. My long-term goal is to define the logic by which guidance information is integrated at the level of cytoskeletal dynamics control during axon pathfinding. As a starting point to address this issue, I will study the role of Msps and TACC, two microtubule-associated proteins which have been recently identified as suppressors of the Abl tyrosine kinase effector protein Orbit which regulates microtubule dynamics in the growth cone and mediates midline axon repulsion in the Drosophila nervous system. I will use genetic, biochemical, proteomic, and cell biological assays to investigate Msps, TACC and Orbit function in the growth cone to define the network of interactions which coordinate positive and negative microtubule dynamics during axon guidance. Specifically, I will: 1) define potential genetic pathways of interaction between Msps, TACC, and the Abl Kinase pathway (e.g. Slit, Robo, Orbit), using axonal pathfinding phenotypes in the Drosophila embryonic nervous system as an assay, testing the hypothesis that Msps and TACC function opposite of Orbit and distinguishing between possible genetic models;2) use biochemical and proteomic analysis to determine if there are direct physical interactions between Msps/TACC, Orbit, and Abl, as well as to expand and define the Msps and TACC interaction networks involved in regulating microtubule dynamics in a Drosophila cell culture line and in neurons;and 3) define the cellular mechanisms of action of Msps and TACC, using highresolution live imaging in Xenopus growth cones. In particular, I will determine if Msps and TACC play a functionally antagonistic role to Orbit, by promoting MT extension towards the growth cone leading edge, or whether they have a different effect on MT dynamics in Xenopus growth cones. Abnormalities in axon guidance have been associated with multiple hereditary neurological disorders and thus this work may shed light on how these defects arise and possibly how to prevent them. Furthermore, mechanisms involved in axon guidance are thought to influence the ability of axons to regenerate after neural injury and so we may be able to use this information to design treatments to allow regeneration in the future. Finally, the proteins studied here are also misregulated in certain cancers. Thus, the research proposed here is of broad biomedical significance.

The long-term goal of Dr. Laura Anne Lowery is to obtain a tenure-track faculty position at a research university and develop a comprehensive, multi-faceted research program that investigates the logic by which guidance information is integrated at the level of cytoskeletal dynamics during axon pathfinding. To this end, she has constructed an extensive career development and research training plan which will facilitate her success and complement her previous training experiences. She received her BS and MS in biology from UCSD, where she worked with Dr. William Schafer on the neural circuitry controlling C. elegans behavior. This work resulted in two papers (including first-author in Journal of Neurobiology). She received her PhD in Biology at MIT under the mentorship of Dr. Hazel Sive. Supported by a pre-doctoral NRSA, she made significant progress defining the genes essential for early brain morphogenesis, including the identification of several genes required for normal neurogenesis and axon pathway formation. This work resulted in five first- author publications in journals such as Development. In July 2008, Dr. Lowery joined the Van Vactor lab in the Department of Cell Biology at Harvard Medical School, where she began a project to identify new interactors of an intriguing cytoskeletal regulator that functions downstream of axon guidance cues, called CLASP. This work, supported by a post-doctoral NRSA, has thus far resulted in 2 first-author publications (in Genetics and Nature Reviews). Dr. Lowery's immediate goal is to gain new expertise in quantitative cytoskeletal imaging and analysis using Xenopus growth cones, in order to investigate the roles of specific microtubule regulators during axon guidance. While in the mentored K99 phase, Dr. Lowery will continue to benefit from the mentorship of Dr. Van Vactor, a leader in the field of genetic analysis of axonal growth and guidance. Additionally, Dr. Lowery will receive new training and support from co-mentor Dr. Gaudenz Danuser, one of the world's leaders in quantitative cytoskeletal analysis. Both Drs. Van Vactor and Danuser have excellent mentoring records and are committed to fostering Dr. Lowery's training and independence. This environment is an ideal setting for her transition to independence, as Harvard Medical School is one of the strongest biomedical research facilities in the country and is perfectly suited to facilitate the goals in this proposal. Her development will be enhanced by additional microscopy and computation courses, as well as support from an advisory committee of expert investigators of axon guidance and the cytoskeleton. The new skills, techniques, and experimental data she acquires during the K99 phase (Aims 1, 2) are essential to the research planned for the independent R00 phase (Aim 3). The research objective in this application is to determine how a specific group of microtubule 'plus-end tracking proteins' (+TIPs) localize, interact, and function, within the growth cone downstream of guidance cue signaling. Initial work has identified +TIP XMAP215 and its co-factor Maskin as potent antagonists of the +TIP and Abl signaling substrate, CLASP. Furthermore, XMAP215 and Maskin are required for accurate axon guidance decisions in vivo, and XMAP215 antagonizes Abl's in vivo axon guidance function. These preliminary findings, combined with knowledge from non-neuronal studies of +TIP function, have led to the working model that, within the growth cone, XMAP215 and Maskin interact with microtubules (MTs) in a functionally-distinct manner compared to CLASP, and that Abl signaling leads to differences in the ability of these +TIPs to interact with each other and with microtubules, thereby driving changes in cytoskeletal dynamics and growth cone directionality downstream of guidance cues. This will be tested using a combination of quantitative imaging, genetic manipulations, and biochemical approaches, to pursue three specific aims. Aim 1) How do +TIPs behave and co-localize with each other and with microtubules inside the growth cone? +TIP localization and MT dynamic instability parameters will be quantified using computational analysis, following acquisition of high-resolution live imaging data of +TIPs and MTs within cultured Xenopus growth cones. Aim 2) How does +TIP function influence MT dynamics and growth cone motility? This aim will use loss-of-function and gain-of-function genetic strategies in Xenopus combined with the imaging platform established in Aim 1 to identify the functional roles of XMAP215 and Maskin, compared to CLASP, within the growth cone. Aim 3) How is +TIP function within the growth cone regulated by upstream guidance signaling? In part 3A, biochemical experiments using Xenopus embryonic lysates will be performed to assess the regulation of +TIP binding events in vitro and to determine the structural domains that modulate those interactions. In part 3B, high-resolution live imaging will allow visualization of +TIP/MT interactions as the growth cone encounters guidance cues in culture, as well as after direction manipulation of Abl signaling. This approach is innovative because it will, for the first time, combine state-of-the-art imaging and analysis tools to pioneer the elucidation of quantitative global MT and +TIP behavior within cultured growth cones during decision-making events. The proposed research is significant because it is an important step in a continuum of research that will illuminate how the growth cone cytoskeleton is coordinated during axon guidance, the knowledge of which may eventually be applied to understanding the basis of neurodevelopmental and mental health disorders.

? DESCRIPTION (provided by applicant): This proposal focuses on the fundamental question of how neuronal growth cones are accurately and precisely guided to their targets. It has long been established that growth cone navigation depends on regulated changes in both F-actin and microtubule (MT) dynamics in response to external guidance cues. However, the mechanisms by which these cues bring about specific changes in growth cone MT dynamics are an unresolved issue in the field. This proposal takes aim at that void, by investigating the functions of two interacting microtubule 'plus-end tracking proteins' (+TIPs), TACC3 and XMAP215, and their regulatory mechanisms. These two +TIPs uniquely bind to the extreme end of the MT, in front of all known others, and their binding to MTs is regulated by phosphorylation. Based on our preliminary data, we hypothesize that major guidance cue signaling pathways converge on TACC3 and XMAP215 to control MT plus-end dynamics and steer the growth cone. We will test this in Xenopus laevis using an array of complementary cell-based and biochemical approaches. The specific aims are: Aim 1 - Test the hypothesis that TACC3 and XMAP215, in response to guidance cues, facilitate axon guidance through spatial regulation of MT polymerization in growth cones. We will use in vivo gene transfer and time-lapse imaging of live axons in the brain of Xenopus laevis embryos, as well as high-resolution quantitative imaging of MT and +TIP dynamics in cultured neurons grown on stripes of guidance cues, to test the hypothesis that TACC3 and XMAP215, in response to guidance cues, facilitate axon guidance through spatial regulation of MT polymerization in growth cones. Aim 2 - Determine how kinase signaling controls TACC3 and XMAP215 function during growth cone navigation. We will use small molecule modulators, phosphomutant forms of TACC3 and XMAP215, and quantitative imaging analysis of +TIP dynamics in cultured neurons, along with biochemical approaches, to discern the effects of kinase regulation on +TIP activity in growth cones, to test the hypothesis that discrete kinase signaling pathways modulate TACC3 and XMAP215 regulation of MTs. Aim 3 - Define how TACC3 and XMAP215 directly control MT dynamics in vitro and in vivo. We will use multi-wavelength TIRF microscopy with in vitro MT reconstitution assays, structured illumination microscopy (SIM), and quantitative analysis of MT dynamics in growth cones, to test the hypothesis that TACC3 promotes guidance-mediated growth cone steering by locally enhancing the ability of XMAP215 to drive MT polymerization, while reducing the ability of XMAP215 to bind the MT lattice and couple MTs with F-actin retrograde flow. The results of these Aims will reveal direct mechanistic links between guidance cue signaling and regulation of the only well-characterized MT polymerase (XMAP215) and its key targeting factor (TACC3), all within the context of growth cone steering. As such, this proposed work has the potential to bring long-needed mechanistic understanding to the question of how extracellular cues govern MT dynamics to steer growth cones during neurodevelopment.

An award is made to Boston College to support the acquisition of a Super-Resolution Structured Illumination Microscope that will be shared by researchers in the Biology, Chemistry, Physics and Psychology departments. Each of the co-PIs on this grant trains multiple undergraduates every year, and these undergraduates will benefit dramatically from experience using a super-resolution microscope. Furthermore, this microscope will be used as a general training and recruitment platform to draw more undergraduates from diverse backgrounds into life-science. Specifically, the microscope will be directly incorporated into the undergraduate curriculum via the Advanced Cellular Imaging Course which enrolls 24 students annually. Additionally, the microscope will be incorporated into many outreach efforts run by faculty in the department including Research Day for Under-represented Students and the Women in Science and Technology Program. Finally, a collaboration has been initiated with Wellesley College, a highly competitive college for women, which will have access to this microscope for research projects, and will include in the class Modern Biological Imaging, which trains 12 women every year. Thus, enrichment of undergraduate training in general, and for women and under-represented groups specifically, is a primary function of this instrument.

Super-resolution microscopy is crucial to cell biological research today. This fact is highlighted by entire sessions at international meetings such as the American Society for Cell Biology Annual Meeting being devoted to the technique, and by the volume of manuscripts that would not be possible without the spatial resolution made possible by super-resolution microscopy. Boston College has a critical mass of cell biologists, and a well-equipped, University-funded, imaging facility. The addition of Super-Resolution Microscopy capabilities will expand the questions that can be asked, and the depth to which questions can be addressed. Furthermore, it will be a tool for the recruitment of new faculty, postdoctoral fellows, and graduate students. In conclusion, super-resolution capabilities are necessary, not only to drive cell based research today, but to properly train future scientists. The SR-SIM system at Boston College will be maximized for both goals and will additionally be used as a tool to develop collaborations between faculty and students at a number of area institutions.

This REU Site award to Boston College, located in Boston, MA, will support the training of 10 students for 10 weeks during the summers of 2017 - 2019. This project is supported by the Divisions of Biological Infrastructure (DBI) and Chemistry (CHE). Projects will generally pair students from different disciplines to collaborate on solving fundamental science problems with an impact on society. These include understanding the growth of neurons, generating clean energy from water, identifying proteins responsible for infectious disease, developing nano-structures for the brain, and making interfaces for quantum computation. These projects include mentors from the physics, chemistry, biology, mathematics, and psychology departments. The program will include training in user facilities, graduate school preparation, and oral/written communication. Potential participants should submit an application electronically including: a resume, college transcript, two letters of recommendation, and indication of research interests, career goals, prior experience, and preferred project. Participant selection will be conducted by the PI and Co-PI in consultation with faculty mentors. Emphasis will be placed on students interested in integrated science with an impact on society.

It is anticipated the program will train a total of 30, primarily underrepresented minority and first generation college students from schools with limited research opportunities. The REU will provide training by technical staff in user facilities, scientific communication, and will include student seminars with feedback and preparation for taking the GRE. Students will thus receive professional and scientific skills training, including opportunities to present their research at professional conferences. Combined with networkng activities and suite living, these experiences will give students a sense of belonging in STEM, along with individual and scientific growth.

A common web-based assessment tool used by all REU programs funded by the Division of Biological Infrastructure (Directorate for Biological Sciences) will be used to determine the effectiveness of the training program. Participants will be tracked after the program in order to determine student career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://reu.bc.edu, or by contacting the PI (Dr. Burch at ks.burch@bc.edu) or the co-PI (Dr. Lowery at laura.lowery@bc.edu).

? DESCRIPTION (provided by applicant): This proposal focuses on the fundamental question of how cranial neural crest cells migrate to form facial structures during embryonic development. In particular, we will elucidate the role that TACC3 (transforming acidic coiled-coil containing protein 3) plays during craniofacial development. The TACC3 gene maps to a region of the genome that is deleted (or sometimes duplicated) in Wolf-Hirschhorn Syndrome, a complex disorder with a distinctive craniofacial phenotype. While other genes have been implicated in this disorder, it has recently become apparent that these genes are not acting alone, and others must contribute to the syndrome phenotypes. TACC3 has previously been overlooked, likely due to a lack of mechanistic insight into its possible contribution. However, our recent studies of TACC3 have uncovered novel cell biological functions that may directly contribute to the Wolf-Hirschhorn Syndrome phenotype. Specifically, our work suggests that TACC3 is an important microtubule regulator that has a direct effect on cell motility, including migration of cranial neual crest cells. The central hypothesis that we will test in this proposal is that TACC3 dysfunction contributes to Wolf-Hirschhorn syndrome by playing a critical role in cranial neural crest cell migration. We will this with the following aims: Aim 1: Quantify the craniofacial defects following TACC3 manipulation in Xenopus Our preliminary data suggest that craniofacial defects are present when TACC3 levels are manipulated in Xenopus embryos, a useful model for dissecting details of cell biological mechanisms during development. We will test the hypothesis that TACC3 regulates specific craniofacial characteristics that are abnormal in Wolf-Hirschhorn syndrome, by using TACC3 genetic manipulation strategies and cell transplantation techniques, along with morphometric quantification of craniofacial features and analysis of in vivo cranial neural crest cell migration patterns. embryos. Aim 2: Define the effects of TACC3 manipulation on cranial neural crest cell motility. We find that TACC3 manipulation results in cranial neural crest cell motility defects. We will test the hypothesis that TACC3 regulates specific parameters of cranial neural crest cell motility, by measuring multiple parameters of cell motility and focal adhesion turnover after TACC3 manipulation, in cultured cranial neural crest cells of Xenopus and neural crest cells derived from human ES cells. Aim 3: Determine the structural domains required for TACC3 localization in cranial neural crest cells. Our recent work demonstrates that TACC3 localizes to two distinct sub-cellular domains in neural crest cells - microtubule plus- ends and focal adhesions. We will test the hypothesis that different structural domains mediate TACC3 accumulation at microtubule plus-ends versus focal adhesions. This will be accomplished by mapping the domains required for localization using a series of TACC3 deletion constructs. The experiments in this proposal represent an essential first step in establishing whether TACC3 may be a critical player in Wolf- Hirschhorn syndrome specifically and in cranial neural crest cell migration more generally.

Project Abstract It has long been established that growth cone navigation depends on regulated changes in both F-actin and microtubule (MT) dynamics in response to external guidance cues. However, the mechanisms by which these cues bring about specific changes in growth cone MT dynamics are a fundamental unresolved issue in the field. The parent research grant takes aim at that void, by investigating the functions of two interacting microtubule `plus-end tracking proteins' (+TIPs), TACC3 and XMAP215, and their regulatory mechanisms. These two +TIPs uniquely bind to the extreme end of the MT and their binding to MTs is thought to be regulated by phosphorylation. Our data support a model in which TACC3 and XMAP215 mediate changes in MT dynamics downstream of guidance cue signaling. We are currently funded to test the central mechanistic hypothesis that major guidance cue signaling pathways converge on TACC3 and XMAP215 to control MT plus-end dynamics and steer the growth cone. This supplement will fund the research training and career mentorship of a highly driven and enthusiastic first- generation female Hispanic undergraduate student, who has a strong interest and potential in eventually attending a graduate program in Biology. The undergraduate student will be trained not only in many diverse bench techniques, but she will be intensively mentored in how to be a successful biomedical scientist and succeed in a graduate PhD program in biology. With this supplement, she will work with a supportive team to make new insights into the mechanistic regulation of TACC3 during neural development, using a series of complementary approaches over the next three years. First, she will determine whether four critical phosphorylated amino acids are important for TACC3 biochemical interaction with XMAP215. Then, she will determine whether these amino acids affect the ability of TACC3 to bind to and regulate microtubules in neuronal growth cones. Finally, she will investigate whether these amino acids are important for TACC3 to promote normal swimming behaviors in Xenopus tadpoles as a readout of brain development. The undergraduate student will have extensive contact with her advisor as well as others on the parent grant (including a senior scientist, current PhD student, and senior undergraduate students). In the course of her undergraduate studies, she will have many opportunities to discuss and present her research in multiple forums, developing her skills not only as a bench biomedical researcher but as a scientific communicator. She will receive intensive mentoring from her advisor, and will participate in numerous additional training opportunities to strongly prepare her for the rigors of a graduate program.