University Of California Berkeley

NIH Research Projects · FY 2024 · 2020-09

Summary: Spectacular recent advances in single cell genomics have provided high-resolution information about cell identity, however relating molecular information to the spatial and temporal context remains a major challenge. This is particularly relevant for the immune system, since immune responses occur in highly organized tissue environments, and involve tightly-controlled changes in cell state over time. Here we propose to develop new approaches to increase the spatial and temporal resolution of single cell analyses, and to use these methods to generate and then disseminate a 4-dimensional (time and 3D spatial coordinates) map of T cell development and the associated microenvironment in the thymus. In Aim 1, we will use simultaneous measurement of mRNA and surface proteins on single cells, together with computation and experimental approaches to develop a temporal map of cell state transitions during T cell development in the thymus. In Aim 2, we will use coherent Raman and multiphoton microscopy of living thymic tissue slices, together with laser microdissection, to isolate functionally relevant regions of tissues. We will then perform single cell analyses of individual cells within these defined regions and use computational analyses to define cell types and resolve cellular cross-talk. In Aim 3, we will increase the value of this resource to the scientific community, we will make the data readily accessible to researchers via a user-friendly interface. !

NIH Research Projects · FY 2024 · 2020-09

Predicting and controlling polygenic health traits using probabilistic models and evolution-inspired gene editing PROJECT SUMMARY: New mutations are a source of adaptive evolutionary novelty but can also cause genetic diseases and cancer. While we can now correct detrimental mutations using CRISPR/Cas9 technologies, DNA modifications can have unintended consequences through seemingly unpredictable epistatic and environmental interactions, as could well be the case for the presumed HIV-resistance mutations in CCR5 recently CRISPRed into humans. In higher eukaryotes, fitness or health traits such as adaptability or disease susceptibility appear to be controlled by numerous mutations acting in concert – they are so-called polygenic or complex traits. Such mutations might even manifest detrimental in some environments while beneficial in others, therefore also called antagonistic pleiotropic. The main goal of the proposed work is to use the versatile model plant Arabidopsis thaliana to enhance the predictability and control of the polygenic and antagonistic fitness effects of mutations. Results from this project will provide universal principles to deepen our understanding of complex human genetic disease and inform the safe correction or avoidance of harmful mutations in the future. Specifically, I will pursue the following aims: 1) predicting polygenic fitness effects across environments, 2) improving fitness by controlling deleterious and beneficial mutations using multiplexed genome editing and mutator alleles. Arabidopsis thaliana is an ideal model to tease apart the fitness effects of mutations in complex environments due to its high malleability to engineered mutations, and its extensive community and resources. The 1001 Arabidopsis Genome Project and a genome-wide Knock-Out (KO) collection allow for quantifying fitness of thousands of publicly available natural and artificial mutations across environments. Building a global network of Arabidopsis researchers, we have started an experiment with the same natural strains in 45 locations, which I will use to quantify environment-associated mutation effects. Integrating this with information of relevant KO lines, I will build on my previous predictive models to understand the effects of mutations on fitness across environments, and the features that make them deleterious. Such a deep understanding of mutation effects will ultimately allow us to alter fitness in predictable ways. I will test this in two ways: First, using multiplexed CRISPR base-edits, I will substitute detrimental for beneficial mutations. Second, to study how accumulating mutations impact fitness and to learn how to correct this, I will engineer plants with known mutator and anti-mutator alleles. These alleles, associated with the DNA repair machinery and cancer susceptibility, can increase or decrease the mutation rate in A. thaliana, helping us explore mutation accumulations up to lethal levels in many mammals. Overall, my research will provide fundamental insights into the genetic control of complex fitness traits, ultimately paving the way to improving personalized genomic disease risk predictions and safely probing the limits of poly-gene therapies.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY In our daily lives, we choose between different courses of action with the hope of achieving desired positive outcomes and avoiding feared negative outcomes; this is complicated by various forms of uncertainty that impact the probability that any given action will result in a particular outcome. Within NIMH’s RDoC framework, studies of the mechanisms involved in reward-based action valuation and choice have informed constructs listed under the Positive Valence Systems (PVS) domain. Associated paradigms examine how differences in outcome probability (first order uncertainty) and action-outcome contingency uncertainty (second order uncertainty) impact choice between alternate options. Within the Negative Valence Systems (NVS) domain, the construct of potential threat (anxiety) does not include consideration of the impact of potential threat, its probability and action- outcome contingency uncertainty, upon action valuation or choice; in addition paradigms listed under the NVS domain have no choice (instrumental) element and use physiological indices as dependent measures. These differences between constructs and tasks across the PVS and NVS domains hinder attempts to elucidate whether psychopathology-related deficits in probabilistic decision-making and the factors influencing action valuation and choice are common across both domains or unique to one or the other. Here, we will address this by creating equivalent PVs (reward) and NVS (shock) versions of two probabilistic decision-making tasks. We will use a hierarchical Bayesian computational framework to model behavioral and brain (functional magnetic resonance imaging) data from PVS and NVS versions of each task. This data will be acquired from healthy adult humans with a range of anxiety and depressive symptomatology. In addition to group-level analyses, we will use bifactor analysis to examine the latent factors underlying variance in anxiety and depressive symptomatology across participants and will relate scores on these factors to parameter estimates obtained by modeling of behavioral and brain data. Using this approach, we will examine commonalities and differences in the mechanisms supporting probabilistic decision-making when potential outcomes are aversive versus rewarding and alterations to these mechanisms as a function of anxiety and depressive related symptomatology. We hope that this will advance our understanding of the aspects of decision-making disrupted in anxiety and depression and the potential consequences for daily life. An additional goal of this research is to provide tasks and models that can be used in future clinical studies of probabilistic decision-making across both PVS and NVS domains.

NIH Research Projects · FY 2025 · 2020-08

PROJECT SUMMARY/ABSTRACT Chimeric protein-protein conjugates provide a wide variety of successful platforms for immunotherapy, tar- geted drug delivery, cell biology studies, and vaccine development. However, many desirable constructs cannot be produced using genetic methods alone, and the targeted coupling of two or more proteins using chemical methods is still very challenging. In this program, a new approach will be explored for the rapid coupling of pro- teins using native amino acids. Tyrosinase enzymes will be used to oxidize solvent-exposed tyrosine residues on protein and peptide substrates to generate ortho-quinones that react rapidly with strategically placed cyste- ine residues in other proteins. Importantly, tyrosine residues that extend from the N- or C-terminal positions on proteins are oxidized readily, but internal tyrosine residues are unaffected during the reactions. The cysteine residues can be placed anywhere on the surface of the second protein target, allowing many different linkage locations to be accessed. Previous NIH-funded studies have shown that this approach can generate complex, multifunctional constructs from individual proteins in under 1 h at room temperature despite the high degree of steric interactions that are inherent in these reactions. The reaction strategy has been used to generate antibody drug conjugates, bispecific cell engagers with new geometric relationships, and multidomain protein chimeras. Due to these features, this method stands alone in its simplicity and flexibility for making complex protein con- structs, and thus it is greatly expanding the range of bioconjugates that can be accessed for biotechnology ap- plications. The first Specific Aim of the proposed research will capitalize on newly-available charge-sensitive tyrosi- nase mutants to make double- and triple-domain protein constructs. A central element of this work will be a systematically exploration the new protein-protein linkages that this chemistry can uniquely create, with the goal of developing heuristic models for linking proteins with desired geometries and flexibilities. This will be explored using a combination of experimental and computational approaches in the context of bispecific and trispecific cell engagers. The second Specific Aim will explore the ability of this chemistry to generate intramolecular crosslinks within a protein of interest, providing easy access to circular and knotted proteins that are envisioned to have greater stability and proteolysis resistance. We will do this in the context of the enzyme luciferase, with the goal of gen- erating imageable proteins that can be specifically activated in the presence of cancer-specific proteases. We will also explore this chemistry as a way to stabilize CRM197, which is a common platform for vaccine preparation. The third Specific Aim will focus on identifying new tyrosinase enzymes with desirable properties, and the engineering of these enzymes to achieve the activation of different tyrosine-containing sequences. This work will combine rational design with fitness landscaping methods to identify tyrosinase mutants with new specificities, higher stabilities, and other useful properties.

NIH Research Projects · FY 2024 · 2020-07

DESCRIPTION (provided by applicant): The goal of the Northern California NC-ERC, a consortium of programs of the University of California, is to train professionals as practitioner and research leaders in occupational safety and health by offering graduate degrees, residency training, clinical experiences, and research mentorship to trainees. The aim of the NC-ERC Is to provide a broad, multidisciplinary education experience involving student and faculty collaborations in the classroom and on research and service projects. Activities are grounded in multi-campus, interactive teaching programs that translate knowledge Into Information that can be used to improve worker safety and health. In addition, through the Confining Education and Outreach components of the Labor Occupational Health Program, the NC-ERC provides continuing education courses and outreach activities to other health professionals. The Center aims to provide an educational bridge from the University to external constituencies to ensure that practicing professionals, workers, their representatives, supervisors, and other educators benefit from the University's occupational health and safety expertise. The NC-ERC strives to integrate an occupational safety and health perspective in all of its activities, including such activities as the Short Term Educational Experiences for Research (STEER) program, summer internships funded by the National Institute for Environmental Health Sciences, designed to encourage students to consider further study in one of the NC-ERC programs. The Northern California NC-ERC trains professionals in the following areas: Industrial Hygiene (UC Berkeley) - MPH, MS, PhD degrees; Epidemiology (UC Berkeley) - MPH, MS, PhD degrees; Occupational and Environmental Health Nursing (UCSF) - MS, PhD degrees; Occupational and Environmental Medicine (UCSF) - Residency Training. MPH degrees; Ergonomics (joint program at UCB/UCSF) - MS/MPH, PhD degrees; Targeted Research Training (joint program at UCB/UCSF) and proposed Agricultural Safety & Health Program - PhD degree. DESCRIPTION (provided by applicant): The aim of the NC-ERC training program is to produce graduates with strong problem solving skills and the ability to synthesize diverse information in order to effectively address both typical and unusual problems that arise in the technically, institutionally, and culturally complex workplaces that characterize the current economy. Issues facing low-wage and immigrant workers are particularly important to the NC-ERC, as are health and safety issues in emerging sectors such as green jobs.

NIH Research Projects · FY 2026 · 2020-06

Illness and death caused by infectious diarrheal disease agents, like Vibrio cholerae, are major threats to public health and significant barriers to socioeconomic development worldwide. Natural disasters and continuing conflict in some depressed regions threaten to exacerbate the already rising incidence of cholera globally. Comparative genomics of V. cholerae isolated over the last century reveal recurring episodes of novel genetic variants with new repertoires of mobile genetic elements (MGEs) spreading globally from where they initially emerged in the Bay of Bengal. For unknown reasons, a hallmark of this pattern is that previously prevalent strains completely disappear when new variants emerge. The arms race between viruses and their host organisms is a key driving force in the evolution of all cellular life. Successful strains of epidemic V. cholerae must defend against the ubiquitous threat of predatory phages in aquatic reservoirs and the intestinal tract during human disease. In collaboration with the icddr,b, we have established a longitudinal collection of clinical phages and V. cholerae isolates to serve as a tractable, clinically relevant platform to study mechanisms driving reciprocal adaptations in ongoing phage-bacterial conflict. Using this platform, we discovered fluctuating MGEs in epidemic V. cholerae that provide robust protection against co-circulating phages. One such family of anti-phage MGEs, called PLEs, provides exquisitely specific protection against ICP1, the predominant phage in cholera patient stool samples. We discovered novel mechanisms that ICP1 evolved to counter PLEs and other V. cholerae defenses. Although we have made critical advances in understanding mechanisms underpinning reciprocal adaptations that contribute to some of the observed diversification and dynamics of PLEs and ICP1 in nature, many observations from our extensive collection of contemporary clinical isolates remain unexplained. To reveal molecular mechanisms contributing to the selection of emergent phage and epidemic V. cholerae genotypes, we will pursue the following specific aims: 1) We will examine how a novel PLE variant blocks phage despite phage-mediated degradation of PLE. 2) We will interrogate barriers limiting the acquisition and maintenance of co-resident PLEs in V. cholerae, and 3) We will define the array of defenses and counter-defenses in a clinical collection of V. cholerae and phage. The proposed studies will advance the understanding of factors influencing population shifts in epidemic V. cholerae and reveal novel mechanisms underpinning phage-bacterial coevolution. This knowledge will further enhance our understanding of phage-mediated perturbations to microbial populations in healthy and diseased states and advance our capacity to manipulate these communities for therapeutic or prophylactic benefit.

NIH Research Projects · FY 2025 · 2020-05

Project Summary/Abstract The Computational Social Science Training Program (CSSTP) at UC Berkeley provides training in advanced analytics to predoctoral students in the social and behavioral sciences who investigate health topics covered by the Eunice Kennedy Shriver National Institute for Child and Human Development. CSSTP combines Berkeley’s long-standing strength in quantitative social and behavioral science with its nationally-recognized campus programs in data science education, practice, and research. It serves five entering trainees per year over five years. The training faculty includes 30 social scientists who have exemplary records of developing and applying novel statistical methods to health-related social/behavioral science problems, as well as 19 data scientists who are leading figures in the foundations of mathematics, statistics/biostatistics, and computer science. Trainees, who are drawn from a diverse pool of students in six social science doctoral programs, are provided with a rigorous and tailored program designed to teach a team science-based approach to problem solving and to emphasize the analysis of intensive or voluminous longitudinal data and high-density, large sample or population level agency databases. Each trainee is supported by a dual-preceptor model in which they are provided with a social sciences faculty mentor and a data science mentor who help to facilitate the trainee's progress through the program. CSSTP trainees are provided with community space at the Berkeley Institute for Data Science (BIDS), a dynamic multi-disciplinary data science research center, where trainees work alongside other data science fellows in residence. After completing their first-year course requirements in their home departments, trainees formally enter the program in their second year of graduate school, devise an individual development plan, and take a core two- semester course in computational social science, team-taught by training faculty. This course introduces students to essential data science methods and tools, including Python and R programming, data management, natural language processing, machine learning, causal inference, and responsible conduct and reproducibility of research. Instructional modes include lectures, in-depth discussion, and small group learning exercises. In the following year, students apply these skills through placements on collaborative health-related research teams or labs on campus and/or with external partners, thus putting skills in advanced analytics into practice through research involving the development and implementation of new methods. Additional training tailored to student needs and interests is provided through elective courses, a weekly computational social science workshop series, and ongoing working groups at BIDS and the Social Science D-Lab, a campus hub for data science training and research for social scientists. CSSTP’s benefits will extend to the greater campus and beyond by stimulating new faculty collaborations and by creating a critical mass of rigorously trained computational social science students who will be competitive and qualified for jobs in rapidly changing and evolving data intensive fields.

NIH Research Projects · FY 2025 · 2020-05

PROJECT SUMMARY This overall goal of this application is to create an infrastructure for supporting and augmenting neuroimaging research in the National Institute of Aging’s Alzheimer’s Disease Centers (ADC) Program. The ADC program is comprised of 30 centers that pursue research goals dedicated to increasing our understanding of Alzheimer’s disease (AD) with a view towards improving diagnosis, care, and treatment of patients. Over the past decade, neuroimaging has played an increasingly important role in this mission, as new approaches to measuring brain structure, function, and biochemistry have produced biomarkers useful in diagnostic, mechanistic, and therapeutic studies. At the same time, methods for standardization of neuroimaging have become accepted, such that, despite the complexity and cost of imaging, we have the ability to combine images obtained at different centers in order to answer important questions about AD that require large datasets. This project will focus on standardizing approaches to magnetic resonance imaging (MRI) and positron emission tomography (PET) images. MR standardization will address structural and functional (task-free) MRI, and PET standardization will address amyloid, tau, and glucose metabolic imaging. We will work with the National Alzheimer’s Coordinating Center (NACC) to develop protocols for securely uploading and de-identifying all images at a NACC image repository, where a searchable database will also be available to researchers. We will develop standards for MR and PET image acquisition, and make these imaging protocols available to the ADC community. Uploaded images will undergo rigorous quality control and processing to permit merging of images obtained on different platforms and, for PET, at different resolutions. Laboratories that have specialized in image processing will produce numerical summary data of brain cerebrovascular pathology, brain volumes, perfusion, diffusion, and cortical thickness, amyloid and tau deposition, and glucose metabolism. These numerical variables will be stored at NACC where it will be linked to other ADC-related data on participants and made available to researchers. Additional aspects of the program will include a website with documentation, search functions, and user help functions. The administrative structure of the project will involve an executive committee with membership from the participating laboratories as well as representation from the NACC, the ADCs, and NIA. Leadership will remain apprised of changes in the neuroimaging landscape, and incorporate new MR and PET measures as they develop with the overall goal of standardizing and disseminating agreed- upon best practices while not stifling the development of new, innovative technical advances.

NIH Research Projects · FY 2026 · 2020-05

PROJECT SUMMARY The goal of this project is to extend our understanding of the cerebellum, and in particular, how this subcortical structure contributes to human cognition. Diverse lines of research provide compelling evidence that the cerebellum is not only involved in sensorimotor control, but also contributes to a range of cognitive functions. For example, the neuroimaging literature has produced maps of the cerebellum that exhibit a stable functional organization, with much of the cerebellar cortex showing hemodynamic changes that cannot be attributed to movement. Moreover, patients with cerebellar disorders exhibit behavioral impairments on tasks assessing cognitive and affective processing. However, our understanding of the functional role of the cerebellum in cognitive domains remains rudimentary: Functional hypotheses have either been largely descriptive or targeted to account for cerebellar function in a relatively narrow, task-specific manner. The research program outlined in this proposal is designed to address this issue, seeking to develop a mechanistic account of cerebellar function. Theoretically, the work will be guided by a novel hypothesis, namely that the cerebellum is essential for processing that requires the continuous transformation of an internal representation, or CoRT(continuous representational transformation). This hypothesis offers a parsimonious account of how the cerebellum supports performance in diverse task domains. In the context of sensorimotor control, CoRT would entail computations required to move a limb from one position to another and to anticipate the sensory consequences of that movement. In other task domains, the continuous transformation of an internal representation may optimize anticipatory behavior; for example, perception frequently involves the internal transformation of the sensory input to account for atypical viewpoints, and social judgments may benefit continuously simulating the intended actions of another individual. The research program will involve the integrated use of behavioral, computational, and neuroimaging studies. One major component of the behavioral work will focus on the performance of individuals with spinocerebellar ataxia (SCA). This work will involve traditional on-site experiments spanning a broad range of task domains to test the CoRT hypothesis, as well as an ambitious on-line testing program. Through an outreach program facilitated by SCA support networks and collaborations with an international team of researchers, the on-line program should produce a unique database to provide well-powered tests of functional hypotheses, and examine relationships between behavioral performance, etiology, and clinical ratings, and relate these measures with region-specific pathology in the cerebellum. A second major component will build on recent neuroimaging work with healthy young adults that has provided a comprehensive functional map of the human cerebellum though the use of a large battery of tasks. This approach will be used to explore constraints on the organization of the functional map by developing models of cortico-cerebellar connectivity and examining changes over the course of learning. As with the neuropsychological studies, the neuroimaging studies will yield a rich database to evaluate different functional hypotheses, as well as establish norms for comparison with atypical populations. !

NIH Research Projects · FY 2026 · 2020-04

Project Summary Cytoplasmic dynein is an AAA+ motor responsible for nearly all minus-end-directed motility and force generation functions along microtubules (MTs). Surprisingly, a single dynein-1 heavy chain gene is responsible for an enormous breadth of cellular activities in intracellular transport, MT organization, and mitosis, in comparison to more than 40 plus-end-directed kinesin motors performing complementary functions on MTs. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for their in-depth structural and biophysical characterization. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. For example, we recently showed that the mammalian dynein complex is a robust motor that rapidly walks along MT tracks and produces forces comparable to kinesins. We also developed a robust mechanistic model for the activation of the dynein transport machinery by accessory proteins, Lis1 and Nde1. Our future goals are to dissect the mechanism of active dynein complexes and determine how dynein activation and motility are regulated across multiple scales using biochemical reconstitution, single-molecule imaging, and cryo-electron microscopy (cryo-EM). Specifically, we will use the recently developed MINFLUX method, single- molecule FRET (smFRET), and protein engineering approaches to directly monitor the conformational dynamics of the dynein motor domain during stepping. These experiments will fill critical gaps in our understanding of how dynein couples ATP hydrolysis to a mechanical step at unprecedented resolution. We will also investigate the mechanism of Lis1/Nde1-mediated activation of the dynein transport machinery using cryo-EM, smFRET, and mutagenesis studies. These studies will reveal how Nde1 recruits Lis1 to autoinhibited dynein and facilitates the opening of dynein for its assembly into functional transport complexes. A large body of evidence shows that dynein is recruited to intracellular cargos side-by-side with kinesins through cargo adaptors. These adaptors may also coordinate motor activity to avoid tug-of-war between the antagonistic motors and determine which direction the cargo moves on MTs. To investigate the mechanism of motor recruitment and coordination on a cargo, we will reconstitute the machinery that transports mitochondria (Miro/TRAK/dynein/dynactin/kinesin) and autophagosomes (HAP1/Huntingtin/dynein/dynactin/kinesin) from purified components. We will also investigate the assembly and regulation of dynein/dynactin by the NuMA adaptor for its mitotic functions. We will characterize the assembly and motility of these complexes in vitro, and use cryo-EM to gain structural insight into the coordination of motor activity on cargo adaptors. The success of our research program will reveal the fundamental mechanochemistry of dynein and how it achieves retrograde transport of intracellular cargos and performs specific functions in mitosis.

NIH Research Projects · FY 2025 · 2020-02

Abstract The Children’s Health and Air Pollution Study (CHAPS), one of the NIEHS/EPA co-sponsored Children’s Environmental Health Centers, has focused on the effects of air pollution on children growing up in the San Joaquin Valley of California, one of the most polluted areas in the country. CHAPS involves the recruitment and follow-up of two age-specific cohorts of children, an infant cohort recruited while in utero and a child cohort recruited at ages 6-8. The primary research goal for both cohorts is to investigate the effects of exposure to ambient air pollution, particularly polycyclic aromatic hydrocarbons (PAHs) and other traffic-related pollutants, on immune and metabolic dysregulation. We have recruited over 200 children in each of the two cohorts and are in the process of following them over a 2-year period. We have collected the following biomarker data: anthropometry; blood pressure (BP); spirometry in the child cohort; assays in blood samples for immune regulation, metabolic function, and systemic inflammation; and a urinary assay for oxidative stress. To date, our research team has a) confirmed a novel epigenetic mechanism by which ambient PAHs and other pollutants contribute to allergic disease in children through hypermethylation of the forkhead box P3 gene (FOXP3), b) confirmed that exposure to ambient PAHs is associated with reduced lung function in children, and c) shown that exposure to traffic-related air pollutants, including ambient PAHs, is associated with BP, hemoglobin A1c (HbA1c), BMI-for-age, and urinary 8-isoprostane. Whether our findings in young children will predict future allergic disease and metabolic syndrome as the children age is an important and currently unanswered question. To address this question we propose in our application for Environmental Epidemiology Cohort (EEC) funding the following specific aims: 1) to retain and continue to follow both CHAPS infant and child cohorts over the next 5 years; 2) to maintain and strengthen data management infrastructure; 3) to maintain and enrich the CHAPS repository of biospecimens; 4) to conduct validation, pilot, and feasibility studies using existing data and samples; and 5) to encourage data sharing. The well-characterized CHAPS cohorts provide the opportunity to study the health effects of traffic-related air pollution from birth through adolescence in a low-income, predominantly Latinx community. The proposed EEC will provide evidence in support of policies to protect the health of such vulnerable communities.

NIH Research Projects · FY 2026 · 2020-02

Project Summary: Our lab works at the interface of chemistry, biology, and medicine. I am drawn to new or complex biological systems whose chemistry and/or mechanism is incompletely understood. New and complex biology is replete with opportunities for fundamental discovery, and history has taught that fundamental discoveries are often accompanied by unexpected therapeutic insights. Efforts on two complex systems comprise my NIGMS portfolio. The first is the organelle interactome, the system of inter-membrane contacts that directly or indirectly control virtually all cell function]. Despite its unquestioned importance, the organelle interactome–especially its dynamics–remains poorly characterized because there are so few useful tools. Visualizing organelle dynamics in live cells for biologically relevant times, especially at resolutions < 100 nm, was impossible, let alone in multiple colors. We developed HIDE probes to fill this void. We discovered that tethering a fluorophore to an organelle membrane via a lipid (rather than a membrane protein) improves imaging time by as much as 50-fold. Even better, as HIDE probes are composed of cell-permeant small molecules, they interrogate cells that are difficult or impossible to modify genetically. We made great progress in the last period developing and applying HIDE probes for imaging. Here we broaden their utility for multi-organelle (>2) HIDE fluorescence lifetime imaging microscopy; to deliver photocatalyst proximity labeling tools; and to sequester and inactivate difficult to target disease-causing proteins. The second portion of my NIGMS portfolio focuses on understanding and improving macromolecule delivery, the single most important bottleneck hindering the development of next-gen biologic therapies. The delivery problem can be summed up in one phrase: inefficient endosomal escape. In previous NIGMS-funded work, we identified a mini-protein (ZF5.3) that is actively endocytosed and escapes the endocytic pathway with unprecedented efficiency. We learned much about ZF5.3 during the last period. Using fluorescence correlation spectroscopy (FCS), a state-of-the-art single molecule method, we learned that ZF5.3 escapes from late endosomes through a previously unrecognized portal (HOPS), and is most efficient when conjugated to cargo that are small or unfold easily. We also discovered that ZF5.3 itself unfolds in late endosomes and unfolding is required for HOPS-mediated endosomal escape. Here we build on these discoveries to develop scaffolds for genuinely cell-permeant protein interaction inhibitors; others that escape earlier along the endocytic pathway and deliver compact gene-editing tools; and probe the mechanism of endosomal escape in vitro and in cellulo.

NIH Research Projects · FY 2026 · 2020-01

Project Summary/Abstract Interpreting vocalizations in communication, such as human speech, necessitates the segmentation of the sound stream into meaningful units and the identification of their corresponding meanings. Although research on the neural basis of human speech perception has demonstrated the entrainment of neural activity in auditory areas by the speech sound envelope, the specific mechanisms underlying neural segmentation and its facilitation of the identification task remain poorly understood. Furthermore, while categorical neural responses to sound classes have been observed, the computational processes and representations enabling the categorization of a complete communication system, for example, encompassing all phonemes in a human language, remain largely unexplored. To bridge these gaps in knowledge, we have developed a songbird model that promises to shed light on these fundamental processes. In previous research, we made a significant breakthrough by uncovering a combinatorial neural code in the avian auditory cortex responsible for identifying all call types within a bird's repertoire. Building upon this discovery, our study aims to investigate how the auditory system generates this ensemble code for identification while concurrently segmenting the sound stream into meaningful segments. In Aim 1, we will examine the neural basis of segmentation using animals actively engaged in listening tasks or natural communication scenarios, allowing us to study the impact of attention on this process. The human vocal communication system also relies on the ability to learn and identify novel sound categories, such as new words or the distinct voice of a speaker. Fascinatingly, our research has demonstrated that songbirds possess a similar capacity to rapidly recognize the voice characteristics of numerous conspecifics. Accomplishing this voice identity task necessitates the formation of auditory memories. In Aim 2, we will explore the neural mechanisms underlying the identification task for these learned vocal categories. Our investigations will provide valuable insights into the roles played by distinct circuits within the auditory cortex in processing semantics and voice. This knowledge is critical for comprehending how dysfunctional auditory processing in multiple mental disorders as well as aging affects speech recognition and, subsequently, other cognitive abilities.

NIH Research Projects · FY 2026 · 2020-01

Abstract. Meiosis is the conserved differentiation program that creates gametes. It fails frequently, with devastating consequences for human fertility and health. As a cell progress through meiotic differentiation, it undergoes irreversible changes in cellular structure and function that are largely driven by gene expression changes. Because the molecular basis for most meiotic transitions remains mysterious, my lab aims to illuminate the gene regulatory circuitry that programs meiotic differentiation. We use budding yeast to study this process because this organism uniquely offers access to the large number of highly synchronous cells that is key to genomic approaches that we routinely employ. Our studies have uncovered major surprises in the genes that meiotic cells express and how they regulate these genes, revealing big gaps in our fundamental understanding of how gene expression works in meiosis, and beyond. These findings have led us to believe that meiosis is a valuable model for identifying and interrogating such broad open questions in gene regulation. Among the surprises we found is the common use of an unconventional mode of gene regulation, involving regulated toggling between a translatable mRNA isoform and one that is 5' extended and poorly translated. A major focus of our future research is to better understand why some of these 5' extended “long undecoded transcript isoforms” (LUTIs) are subject to nonsense mediated decay (NMD), while other are protected from degradation. We will use approaches including machine learning analyses of targeted and protected classes of LUTIs, integrating data including their in vivo structures, to identify NMD transcript cues. This project will not only help us understand the true prevalence of LUTIs in yeast, it aims to leverage this large newly identified class of natural NMD targets to elucidate central principles in NMD targeting in all cellular contexts. We will also use the knowledge gained from our studies of LUTI-based regulation in yeast to determine whether it is a similarly widespread core mode of gene expression regulation in mammalian cells. We also discovered that meiotic cells translate many coding regions were not previously identified. These include hundreds of proteins that are translated starting with non-AUG codons, and thousands that are shorter than the 100 codon cutoff that was used to annotate genomes. We are studying the specific cellular roles of short proteins by performing pooled screens and focusing on directed study of cases in which the short proteins include domains of characterized proteins. Our goal is to reveal the types of functions mediated by this large and poorly studied class of cellular factors that are now known to be commonly in eukaryotes. Finally, MIRA funding enabled development of an entirely new project in my lab, one that studies the way that protein complex members find each other within the complex environment of a cell. This project has the potential to reveal fundamental principles in cellular regulation, enabled by our background in meiosis, the simplicity and power of the yeast system, and our expertise in global gene expression dataset generation and analysis.

NIH Research Projects · FY 2025 · 2019-08

Project Summary / Abstract The U.S. study to PrOtect brain health through lifestyle INTErvention to Reduce risk (U.S. POINTER) is a $35M Alzheimer's Association-sponsored multisite randomized clinical trial investigating the influence of a multidomain lifestyle intervention (exercise, diet, cognitive stimulation, health coaching for risk factor reduction) on 2yr cognitive trajectories in older adults at increased risk of dementia. POINTER was modeled after Finland's FINGER trial that showed a greater cognitive benefit for those assigned to a multidomain lifestyle intervention group versus health education. POINTER will use a similar two-group study design. The health education (“self-guided”) group will receive information and support through twice yearly group meetings to encourage healthy lifestyle practices. The higher-intensity (“structured”) lifestyle intervention group will receive frequent coaching and group meetings to encourage aerobic exercise and cognitive training 4 days/week, adherence to a modified Mediterranean diet, and cardiovascular risk reduction through increased medical monitoring. Based on the successful FINGER results, the structured group is predicted to show the greatest benefits on 2yr cognitive trajectories given the high intensity of the intervention and the potential for synergistic effects across lifestyle domains. Lacking from the POINTER design, however, are neuroimaging measures to investigate intervention effects on underlying Alzheimer's disease (AD) and cerebrovascular pathophysiology. This proposed imaging ancillary study leverages unique resources provided by the parent trial to examine whether changes in lifestyle can protect brain health and alter AD trajectories. The study will assess these intervention effects on changes in AD and cerebrovascular pathophysiology. It will also examine whether these biomarkers at baseline predict cognitive response to the intervention, which has important implications for precision medicine in identifying those most likely to benefit from this approach. AD and cerebrovascular pathophysiology will be assessed with PET imaging (baseline, 2yrs) to measure beta- amyloid and tau burden, and MR imaging (baseline, 1yr, 2yrs) to measure brain morphometry, white matter hyperintensities and microstructural integrity, and cerebral blood flow. An in-depth analysis of lifestyle changes on disease biomarkers has significant implications for public health given the current weight of evidence showing favorable effects of lifestyle on brain health in older adults that overshadow the results of all other pharmacological treatments to date. Moreover, lifestyle modification is an affordable and accessible approach with health benefits that extend beyond brain health. A critical and clinically relevant feature of the POINTER trial is its recruitment approach which targets a community-based and geographically and racially/ethnically diverse sample, ensuring that the intervention will be applicable to a large proportion of older individuals.

NIH Research Projects · FY 2025 · 2019-07

Targeted protein degradation (TPD) has arisen as a powerful therapeutic modality for degrading and eliminating cancer-causing proteins and has enabled potential access into classically undruggable protein targets. Two main TPD approaches currently employed include heterobifunctional Proteolysis Targeting Chimeras (PROTACs) and monovalent molecular glue degraders that both use small-molecules to induce the proximity of E3 ubiquitin ligases with target proteins to ubiquitinate and degrade specific disease targets of interest. However, there are still major bottlenecks in realizing the full potential of TPD platforms. In this proposal, we will overcome bottlenecks in cancer drug discovery by developing innovative next-generation approaches for TPD using covalent chemoproteomic strategies, including expanding upon rational chemical design strategies to discover molecular glue degraders and uncover permissive E3 ligases for TPD applications and covalently targeting and destabilizing undruggable oncogenic transcription factors.

NIH Research Projects · FY 2025 · 2019-07

Project Summary The Genetic Dissection of Cells and Organisms Training Program (GDTP) provides predoctoral trainees from diverse backgrounds with advanced training in classical genetics, quantitative analysis, precision genome engineering, and broader societal challenges that will be ameliorated through genetics. GDTP is the only program at UC Berkeley that provides integrated training across the full range of genetics, from phage to human. GDTP’s 51 training faculty are recognized leaders in basic and biomedical spheres, drawn from the departments of Molecular & Cell Biology (MCB), Plant & Microbial Biology (PMB), and Integrative Biology (IB), and from the interdisciplinary Graduate Group in Microbiology (GGM). This renewal proposal requests support for 16 trainees in their second and third years of PhD training after they commit to genetics as the focus of their graduate research. The GDTP provides long-standing, student-centered training within a flexible but data-driven pedagogical structure that requires a two-year training program to fulfill its goals. All trainees are required to take graduate-level courses in empirical genetics; data analysis and statistics; responsible conduct and reproducibility in research, and laboratory/field safety. The GDTP’s Weekly Genetics Immersion is also required across both years. It includes direct interaction with leading geneticists from both the UC Berkeley campus, our collaborative initiatives with other Bay Area institutes, and outside institutions from around the world; critical evaluation of peer-reviewed papers in genetics; and interweaving of salient topics in ethics and responsible conduct in research. Annual events include a trainee orientation and an Annual Retreat to prepare students for presenting their research results at scientific meetings. The GDTP provides career counseling and a suite of professional development programs in science communication, teaching, grant-writing, and publishing, teamwork, management, and leadership. All trainees will receive extensive individual advising and mentoring tailored to their career objectives using best practices in the context of diversity, equity, inclusion, justice, and belonging. Close tracking of student progress ensures that trainees who are struggling receive appropriate and timely support. GDTP’s evaluation process solicits internal and external input throughout the year, ensuring continuous program improvement. An ambitious recruitment and retention plan—involving the active participation of all training faculty—is employed to ensure full participation and inclusion of trainees from underrepresented minority, disabled, and disadvantaged backgrounds.

NIH Research Projects · FY 2026 · 2019-06

HIV-1 infection of humans is a recently acquired cross-species infection from related primates. In contrast to recent human infection, the HIV-related primate lentiviruses have infected and co- evolved with their primate hosts for millions of years. Understanding how host cells allow for or restrict lentiviral infection allows us to understand how cells can resist infection at the molecular level with important implications for current day immune defenses against HIV. Of particular interest to the work described here, there are known blocks to infection of primate lentiviruses in human cells for which the host genes involved have not been identified. In Aim 1 we will describe the mechanisms through which human cells restrict infection of the primate lentiviruses most closely related to pandemic HIV-1. We will determine how a host protein in human cells that facilitates HIV-1 infection inhibits replication of a virus from chimpanzees, SIVcpz, that has never been observed to infect humans. In Aim 2 we will define how two different restricted lentiviral capsids are inhibited in human cells. We will ask about how integration site differences may impact transcription dynamics and about mechanisms through which cellular chromatin modifications may impact transcription and replication in a capsid-dependent manner. In Aim 3 we will explore the mechanisms through which human cells resist infection by a range of both HIV-1 and closely-related primate lentivirus, SIV, sequences. This work has the potential to discover novel host antiviral genes that limit SIV and HIV strains from replicating in human cells. Previous methodologies for genetic screens to find these important host factors have lacked the depth and power of the HIV-CRISPR screening approach described in this application. Follow-up studies will be focused on furthering our understanding the mechanism of action of genes of interest as well as mechanisms of viral antagonism or escape. Ultimately, manipulation of these factors could be important for approaches aimed at achieving a functional HIV cure.

NIH Research Projects · FY 2026 · 2019-04

Remote-store-and-forward teledermatology has grown exponentially in popularity as an efficient, accurate, and cost-effective way to improve the health and well-being of millions of patients around the world. In remote store- and-forward teledermatology, clinicians are asked to recognize and classify images of skin lesions and make visual judgments about lesion features and malignancy. Dermatologists performing screening in this paradigm can examine hundreds of images, seeing them one after the other. A central underlying assumption of this task is that dermatologists’ percepts and decisions about a current image are completely independent of prior events. Recent results show that this is not true: our perception and decisions are strongly biased by our past visual experience. Although serial dependencies were proposed to be a purposeful mechanism to achieve perceptual stability in natural vision, serial dependencies in remote store-and-forward teledermatology could play a crucial and deleterious role in cancer screening. For example, a malignant lesion could be classified as benign depending on the content of the previously seen image. Given the importance and impact of serial dependencies in dermatological settings, we plan to (1) establish, (2) identify, and (3) mitigate the conditions under which serial effects determine percepts and decisions in a remote store-and-forward teledermatology setting. In Aim 1, based on preliminary and pilot data that indicate a clear detrimental role of serial dependencies in clinical visual judgments, we plan to test the full impact of serial dependencies on lesion recognition, including four common tasks: malignancy classification, lesion symmetry perception, lesion homogeneity perception, and lesion border discrimination. In Aim 2, we plan to identify the specific boundary conditions under which visual serial dependence impacts visual recognition in store-and-forward teledermatology. In Aim 3, we will use the boundary conditions identified in Aim 2 to propose a series of task and stimulus manipulations to mitigate the deleterious effects of visual serial dependence on lesion recognition. As a result of these manipulations, performance should improve in measurable ways (including sensitivity, specificity, accuracy, and d’). Aim 3 is particularly crucial because it will allow us to propose new guidelines that will greatly improve lesion recognition in remote store- and-forward teledermatology screening. Taken together, the proposed studies in Aim 1, 2, and 3 will allow us to establish, identify, and mitigate the deleterious effect of serial dependencies in remote store-and-forward teledermatology tasks, which could have a significant impact on the health and well-being of skin cancer patients around the world.

NIH Research Projects · FY 2026 · 2019-01

PROJECT SUMMARY Epithelia are the core cell type of animals, and constitute the most widespread and ancient mode of tissue architecture. My lab uses a distinctive set of multidisciplinary strategies to study fundamental questions of epithelial biology using Drosophila, leveraging deep evolutionary conservation to uncover general principles applicable across phylogeny. The research proposed in this MIRA renewal tackles basic mechanisms underlying epithelial organ shaping and the total body’s response to epithelial injury. Both goals -- one in which we have a long track record and another stimulated by discoveries during the previous funding period-- build on my laboratory’s 20-year NIGMS-funded research program. In one direction we will investigate how developing epithelia respond not just to intracellular forces, but also to resistance from the extracellular matrix. This understudied question is central to the morphogenetic movements that allow organs to attain the specific forms required for function. Our previous work has shown how an organ can be sculpted by finely patterned mechanical properties of the basement membrane that underlies all epithelia. We will determine the mechanisms that regulate conserved matrix components and modifiers to achieve precise mechanical patterning, and then exploit this knowledge to manipulate tissue shape in a predictable fashion. In the other direction, we will explore the whole-body response to epithelial barrier damage. While the local wound response is well-characterized and conserved across many phyla, animals also have systemic wound responses mediated by signaling factors that communicate with distant organs. The mechanisms and impact of these humoral responses, which include but are not limited to inflammation, are poorly understood compared to the antimicrobial immune response triggered in parallel; they may include unappreciated signaling molecules and exotic forms of signal transmission alongside well-known pathways such as TNF. Leveraging the strengths of Drosophila for discovery biology, we will induce sterile injury in adult epithelial tissues and identify signaling axes that communicate between the organ of insult and responding tissues to promote homeostasis and health. This goal will include a comprehensive analysis of TNF signaling in the adult, and investigation of differences between normal tissue repair and that triggered by chronic inflammation. The proposed experiments tackle these key questions by combining the traditional strengths of Drosophila genetics with advanced imaging and new biochemical techniques, empowered by collaborations. Our results will enhance understanding of conserved mechanisms that generate functional epithelial organs during development, and lay the foundation for addressing disorders driven by epithelial damage and pathology.

NIH Research Projects · FY 2026 · 2019-01

Catalysts and Catalytic Reactions for the Synthesis of Medicinally Relevant Organic Compounds The proposed research focuses on the discovery, development, and mechanistic evaluation of a series of chemical reactions catalyzed by transition-metal complexes that provide new approaches to the synthesis of organic molecules important for human health. Research on these reactions addresses several of the major unmet needs in chemical synthesis. These unmet needs include reactions that occur at C-H bonds with high selectivities and high tolerance for auxiliary functional groups; the creation of catalysts that induce chemical reactions at one of many potential reaction sites in complex structures; catalytic transformations of complex molecules to modulate the structures and properties of biologically active compounds; assembly of aliphatic sub-structures with control of the absolute and relative configurations of stereogenic centers to create more complex three-dimensional architectures; and greater mechanistic understanding of catalytic methods to help select or invent catalysts and reagents that achieve these synthetic goals. This program focuses on the development and mechanistic understanding of catalytic reactions that are some of the most widely used reactions during drug-discovery and production, as well as reactions poised to become the next set of such reactions and classes of catalysts that can lead to new capabilities. These reactions include selective functionalization of C-H bonds with main group reagents to form valuable synthetic intermediates, reactions to form alkyl C-N bonds by addition of N-H bonds across alkenes with unprecedented efficiency, coupling processes to form carbon-heteroatom bonds with organic electrophiles catalyzed by copper and nickel systems, and reactions catalyzed by an unusual class of hybrid structure generated by formally exchanging the metal of natural metalloenzymes with an organometallic unit to create artificial metalloenzymes that form products with site-selectivity and stereoselectivity that would be difficult to achieve with natural enzymes or small-molecule catalysts. In all cases, the proposed research includes detailed mechanistic analysis by kinetic stuides and independent synthesis of catalytic intermediates, as well as the use of these mechanistic data to select or design next-generation systems. A particular focus of these mechanistic studies will be placed on revealing the properties of recently discovered catalysts for the functionalization of primary alkyl C-H bonds and recently discovered copper and nickel intermediates in catalytic cross coupling reactions to form carbon-heteroatom bonds.

NIH Research Projects · FY 2026 · 2019-01

Project Summary/Abstract The goal of this program is to develop new strategies and methods for the synthesis of biologically active secondary metabolites (natural products). A significant portion of our efforts will be dedicated to identifying efficient preparations of these complex molecules. These compounds and their unique derivatives will serve as novel small molecules to combat a range of indications associated with cancer as well as inflammation and pain. Our synthetic studies will lead to new applications of C–H, C–C and C–N bond functionalization methodology in organic synthesis as well as advancing the concept of skeletal editing and its application to the preparation of topologically complex molecules. Finally, working with collaborators, we will gain unique entry into the use of natural products and their derivatives to perturb biological function and ultimately provide promising starting points for new therapeutics.

NIH Research Projects · FY 2025 · 2018-08

Project Summary: Craniofacial abnormalities are the most common form of human birth defect, but their molecular basis remains poorly understood. Highly conserved craniofacial developmental pathways shared across diverse vertebrate species have been shaped by adaptive evolution to produce a tremendous diversity of adaptive craniofacial phenotypes. Fundamental investigation of the genetic basis of these phenotypes will lead to better diagnosis, prevention, and treatment of human birth defects. Indeed, complementary or new information on the genetic basis of many human pathologies can be obtained from naturally occurring organisms, particularly non-model vertebrates, that display analogous divergent phenotypes, known as ‘evolutionary mutant’ models. These natural systems are becoming increasingly tractable for genomic and transgenic approaches and provide an opportunity for ‘evolutionary’ forward genetics. Here I propose to build on my lab’s demonstrated success developing a new vertebrate system studying the genetic basis of highly divergent craniofacial morphology in Caribbean pupfishes. Pupfish exhibit novel craniofacial features not found in other non-model fish systems and are highly tractable for laboratory studies with life histories and eggs comparable to zebrafish. Ongoing gene flow and strong selection for divergent craniofacial features provide an ideal natural ‘experiment’ for fine-mapping candidate variants associated with these traits. Our initial success confirming three new craniofacial genes in Caribbean pupfishes and identifying 27 candidate craniofacial genes found in other vertebrates demonstrates the power and potential of our approach. I hypothesize that fixed mutations between these species control spatiotemporal expression of both known and novel craniofacial genes underlying the highly divergent craniofacial features observed in pupfishes. I propose to investigate the genetic basis of novel adaptive phenotypes in this non-model system using a combination of population genomics, de novo genome assemblies, phenomics, quantitative genetics, transcriptomics, in situ hybridization, gene overexpression and chemical inhibition, and CRISPR-Cas9 genome editing. By integrating candidate gene and variant discovery with functional genetics in a natural system exhibiting diverse craniofacial features the proposed research will demonstrate the feasibility and power of new non-model systems to gain novel insights into the developmental genetics of human diseases.

NIH Research Projects · FY 2026 · 2018-08

PROJECT SUMMARY/ABSTRACT Natural products from bacteria continue to be the frontline defense in the struggle against bacterial infections, and have also found wide use as antifungals, anthelminthics, anti-cancer drugs, and immunosupressants. One group of bacteria, the actinomycetes, has historically been the deepest source of clinically-useful natural products. Over the last decade, numerous reports have demonstrated that natural product biosynthesis often occurs in the context of actinomycete interactions. These include interactions between microbes of different species, and cell-cell coordination within colonies of single actinomycetes. Together, these findings have solidified the idea that induction of natural product biosynthesis is socially driven in these bacteria. Despite the importance of this social aspect, how these interactions unfold at the molecular level and how interactions may best be harnessed for natural products discovery remain open questions. The goals of this study are to understand how inter- and intra- species interactions activate natural product biosynthesis at the molecular and systems levels, and to build framework for translating these insights into natural product discovery. First, this work will examine how the model actinomycete Streptomyces coelicolor activates expression of genes for antibiotic production in the presence of other actinomycetes. This activation requires an unusual and poorly understood signal transduction mechanism found in actinomycetes that shares parallels with eukaryotic systems that rely on G protein activation. Second, this work seeks a systems- level understanding of spatially coordinated antibiotic production within individual S. coelicolor colonies. Knowledge generated from this objective may be employed to someday manipulate cell fates within actinomycete cultures to drive natural products discovery and production. Third, this work leverages actinomycete interactions for the discovery of novel natural products. This research serves as a testbed for putting our knowledge of actinomycete interactions into practice, with an emphasis on discovery of compounds with unusual mechanisms of action. In its entirety, this work will illuminate the social drivers of natural product biosynthesis, and in the long term, provide a foundation for harnessing microbial social cues and genetic regulation to maximize future natural products discovery efforts.

NIH Research Projects · FY 2026 · 2018-05

PROJECT SUMMARY/ABSTRACT The actin cytoskeleton is crucial for cellular properties and behaviors including shape, migration, and division. It is also critical for cell-cell and cell-extracellular matrix connections as well as cell-cell fusion. Because of these numerous essential functions in cell physiology, actin dysfunction is also a common contributor to pathogenesis, for example, in inflammation, cardiovascular disease, cancer metastasis, and microbial infection. Despite many years of study, however, understanding how actin assembly is regulated and harnessed in the cytoplasm and nucleus for intracellular events, cellular behaviors, and cell-cell interactions remains a key outstanding problem in cell biology. In our NIGMS-funded research, my lab has taken a distinctive approach to address this important gap in knowledge, which is to examine the interactions between microbes that do not cause serious human illness and the host cell actin cytoskeleton as a window into actin regulation and function. Our approach leverages the fact that microbes colonize host cells through their ability to target actin, often eliciting amplified cellular responses by mimicking or manipulating host molecules, making them powerful tools for revealing molecular mechanisms of actin regulation and function. This scientific premise is supported by many examples of how studying microbe-host interactions has enhanced our understanding of basic cell biological processes. The research described in this MIRA application makes use of microbes as tools to address three fundamental cell biological questions: (1) How is actin polymerization at membranes regulated and mobilized to drive movement? (2) How and why is actin transported into and polymerized within the nucleus for gene expression, nuclear organization, intranuclear movement, and nuclear envelope dynamics? (3) How is actin polymerization in plasma membrane protrusions harnessed to induce cell-cell fusion? We will investigate these questions using three model microbes that infect cells and mobilize actin in a manner that makes them powerful cell biological tools: Mycobacterium marinum as a tool to understand the regulation of actin assembly at membranes to drive intracellular movement; the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to understand the regulation and function of actin in the nucleus; and Burkholderia thailandensis as a tool to understand the role of actin in cell-cell fusion. By leveraging our expertise in both cell biology and microbiology, and deploying a synergistic combination of microbial and host genetic methods, advanced imaging approaches, and biochemical methods, we are uniquely positioned to advance the field. Our results will enhance our understanding of the mechanisms of actin regulation and may provide new insights into diagnosing, treating, and preventing diseases associated with actin dysfunction.