University Of California Berkeley

NIH Research Projects · FY 2025 · 2018-05

Project Abstract Our lab is dedicated to the mechanistic understanding of macromolecular function through the visualization of structure, dynamics, and regulatory interactions. Towards that goal, we use cryo-EM, together with biochemical and biophysical assays. Our areas of study are centered on the characterization of the regulatory molecular mechanisms governing the function of microtubules and of human transcription/epigenetic complexes. Microtubules (MTs) are essential polymers in eukaryotic cells s built of -tubulin dimers. Dynamic instability, the switching between growing and shrinking phases due to the coupling of the assembly process to the exchange and hydrolysis of GTP in -tubulin, is an essential property for MT function. Many MT cellular partners modulate MT dynamics or utilize it to carry out specific functions. In the past. In the last 5 years, we confirmed and complemented our past studies, which used non-hydrolyzable GTP analogs to characterize conformational changes in MTs that accompany GTP hydrolysis, now using of GTP-hydrolysis tubulin mutants. We also defined the mode of binding and action of cellular factors that regulate MT assembly, dynamics and organization in the cell and described the effect of tubulin acetylation on MT structure and function. We will continue this work with the central theme of adding complexity to our studies in order to bring us closer to the regulated function of microtubule cellular systems as we also add techniques complementary to our major tool, cryo-EM. Transcriptional regulation of gene expression is critical for growth and survival, and of obvious significance to human health. Its initiation involves RNA polymerase II (Pol II) together with TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Regulation is achieved by sequence-specific activators or repressors, co-factors, and chromatin remodeling/modifying complexes. During the last funding period we defined the structures of human TFIID[17] and TFIIH[18], as well as other large transcriptional coactivators like the human SAGA[19], and the yeast NuA4 (in preparation) and RSC complexes[20]. We will now characterize the human TIP60 complex, the binding of transcriptional co-activators to chromatin substrates, and pursue the dynamic visualization of TFIID engagement with promoter DNA to gain further knowledge of how these complexes work in the nucleus to regulate gene expression. Polycomb repressive complex 2 (PRC2) is an epigenetic gene silencer that methylates lysine 27 of histone H3 and is essential for cellular differentiation and development. After obtaining the structure of human PRC2 with its cofactors JARID2 and AEBP2 and its interaction with a di-nucleosome, we have now defined how PRC2 recognizes mono-ubiquitylated nucleosome, the substrate created by the other major Polycomb complex, PRC1. We will now further characterize the regulatory landscape of PRC2 looking into other histone modifications, different forms of PRC2 (i.e., other cofactors), the role of PRC2 auto-methylation, and binding of PRC2 to RNA.

NIH Research Projects · FY 2026 · 2018-05

The traditional structure-function paradigm has provided significant insights for well-folded proteins in which structures can be easily and rapidly revealed by X-ray crystallography beamlines and NMR. However approximately one third of the human proteome are comprised of intrinsically disordered proteins and regions that do not adopt a dominant well-folded structure, and therefore remain “unseen” by traditional structural biology methods. Current experimental and computational approaches to structural descriptions of disordered proteins, while often valuable, still lack predictive power, particularly for dynamic complexes of IDPs, as well as lack of insight into the relationships between IDP structural ensembles and function. We made significant progress in the last grant cycle in the following four directions: (1) Generating and quantifying the utility of experimental data types for IDP monomer ensembles; (2) Applying atomistic and coarse-grained physical models and machine learned sampling methods for generating monomer ensembles; (3) Advancing new Bayesian models for IDP monomer ensemble selection; (4) development of highly novel machine learning (ML) methodology for ensemble generation and selection; (5) Creating software and monomer ensemble data and placing them in the hands of practitioners. Many of these results serve as preliminary studies for this renewal and are described in more detail in proposed research. But to fully address the biological activity of IDPs we propose to adapt these computational methods further and develop new integrative biology tools that will be more selective for dynamic associations of IDPs within both discrete dynamic complexes and biological condensates and for post-translational modifications (PTMs) that create relevant IDP functional states. Building on strong preliminary data from our experimental collaborators, we will record NMR, SAXS and single molecule fluorescence data on phosphorylated and non-phosphorylated 4E-binding protein 2 (np-4E-BP2, 5p-4E-BP2) and its dynamic complex with the eukaryotic translation initiation factor (eIF4E); tropoelastin and mixed and condensed-phase elastin fragments; and mixed and condensed-phase CAPRIN1 C-terminal IDR, including novel NMR experiments that probe electrostatic potentials (ESPs), 3-color smFRET, and fluorescence correlation spectroscopy (FCS). These studies will illuminate mechanisms of translational regulation and elasticity, and provide insights into pathological states, including autism spectrum disorder and cardiovascular disease.

NIH Research Projects · FY 2026 · 2018-05

Much like their eukaryotic counterparts, numerous bacterial species use lipid-bounded organelles to execute essential, and at times toxic, biochemical reactions in a compartmentalized fashion. Despite their prevalence and importance to the health and survival of many organisms, relatively little is understood regarding the formation, function, and diversity of bacterial organelles. To advance the mechanistic study of lipid-bounded bacterial organelles, my group has developed two distinct model systems: magnetosomes of magnetotactic bacteria and the ferrosome compartments of diverse anaerobic microbes. Magnetosomes are lipid-bilayer invaginations of the cell membrane with a unique protein content, within which nanometer-sized iron-based magnetic crystals are produced. Individual magnetosomes are arranged into a chain with the help of an actin-like cytoskeleton, thus allowing magnetotactic bacteria to use geomagnetic fields as a simple guide for low oxygen environments. The cell biological features of magnetosomes make them ideal for understanding the evolution and molecular basis of organelle biogenesis and biomineralization in bacteria. The magnetic and physical properties of magnetosomes make them attractive targets for the development of biomedical applications including their use as contrast agents for magnetic resonance imaging, as drug delivery vehicles and as a medium for hyperthermic killing of tumor cells. More recently, my group has discovered a novel iron-accumulating lipid-bounded organelle named the ferrosome. Ferrosomes are formed through the action of a small number of genes and are found in diverse bacteria including resident members of the gut microbiome and opportunistic pathogens. The research program outlined in this proposal will leverage the expertise and existing knowledge within my group to explore three general areas of magnetosome and ferrosome biology. First, we will study the molecular components, biochemical activities, and cellular pathways that define the cell biological characteristics of bacterial organelles. Our current focus is to understand the mechanisms of membrane biogenesis, protein sorting, and subcellular arrangement for magnetosomes and ferrosomes. Second, we are interested in the biochemical output and cellular function of magnetosomes and ferrosomes. Using comprehensive genetic, chemical, and physiological assays we aim to understand how these organelles are integrated into the essential functions of their host organisms. Third, we look to exploit the natural diversity of magnetosome- and ferrosome-forming organisms to understand the common and unique evolutionary paths of organelle formation in bacteria. The combination of these approaches will shed light on the molecular blueprint and evolutionary diversity of bacterial compartments. In the process, we hope to devise more rational paths for synthetic re- engineering of magnetosomes and ferrosomes to deploy them more effectively in applied settings.

NIH Research Projects · FY 2026 · 2018-04

Project Summary/Abstract Our goal is to understand early vertebrate development at the molecular level. We study the problem in the frog Xenopus, whose abundant eggs are large and readily manipulated by microinjection and microsurgery. The embryos are large enough to produce material for biochemical and proteomic analysis, and are also ideal for imaging whole embryo morphogenesis, and confocal imaging of explants. During previous grant periods, we have identified potent signaling and signal transduction activities that contribute to embryonic development, neural induction and anterior-posterior patterning of the neural plate. In parallel we improved genome assemblies and annotation for X. tropicalis and X. laevis, enabling a systems level approach. These, and new assemblies for other frogs, not only provide the resources necessary for hypothesis driven research for the community, but also delivered new insights into genome structures, recombination and evolution. In the next grant period, we focus on the genes that control cell shape changes, and tissue level movements in the embryo, focusing on gastrulation and neurulation. Based on the premise that Rho GTP Exchange Factors (GEFs) are often deployed selectively to mediate specific cell behaviors, we will continue our analysis of members of the Plekhg family, among which three of seven show specific morphogenetic defects after CRISPR mediated knockdown, and extend the analysis to other GEFs or GTPase Activating Proteins (GAPs) that show early morphogenetic defects. The results of these experiments will provide basic understanding of normal vertebrate morphogenesis, and insights into the ways that different cellular behaviors are controlled.

NIH Research Projects · FY 2026 · 2017-12

Project Summary/Abstract White adipose tissue has a central role in maintaining whole-body metabolic health. Thus, it is critical to understand how adipocyte develops and how it remodels in response to high caloric diet. Accumulating evidence indicates that epigenetics plays a critical role in orchestrating these processes, however, the mechanisms by which it does so remain largely unknown. In the last funding cycle, we have demonstrated how the enzymatic effectors of DNA methylation regulates multiple aspects of adipose biology, including insulin sensitivity and WAT browning. The goal of this proposal is to better understand how epigenetics controls adipose tissue development and remodeling under chronic nutrition and its impact on metabolism, focusing specifically on TET3, an active DNA demethylase. Consistent with our initial discovery finding an increased Tet3 expression during adipogenesis of naïve APCs, we noted a pro-adipogenic function for TET3 using primary APCs. Moreover, reduced WAT development was observed in mice deficient for Tet3 in APC cells, but not Tet1- or Tet2, nor deficient for Tet3 in mature adipocytes. WAT from Pdgfra-Tet3 KO mice on a high fat diet (HFD) had reduced adipose mass and adipocyte number, with decreased signs of fibro-inflammation, suggesting that Tet3 deficiency prevents unhealthy adipose expansion. Further, these KO mice on HFD dramatically improved insulin resistance and glucose intolerance compared to WT mice. In line with these morphological and metabolic changes in KO mice, our transcription and DNA methylation profiling studies revealed that TET3 targets a defined set of key target genes, including many genes involved in extracellular remodeling (ECM). Based on these novel and exciting preliminary findings, we hypothesize that TET3 is a critical epigenetic regulator of adipocyte development and remodeling by regulating important target genes. To test our hypothesis, Aim1 will firmly establish the role of TET3 function in adipogenesis using various Tet3 conditional knockout and lineage tracing mouse models. Aim2 will elucidate transcriptional and epigenetic basis of TET3 function by the use of transcriptomic and epigenomic approaches. Aim 3 will investigate the TET3 targets critical for adipogenesis and remodeling, and Aim 4 will examine the metabolic consequence of reduced adipogenesis from Tet3 deficiency. Successful completion of the proposed studies will provide novel therapeutic targets for metabolic disorders including obesity and type 2 diabetes.

NIH Research Projects · FY 2026 · 2017-09

ABSTRACT CHAMACOS is a longitudinal cohort study of Latino mothers and children from a California farmworker community who have been followed for more than 20 years to assess the impact of environmental exposures and social determinants on children’s health and development. CHAMACOS mothers were enrolled during pregnancy in 1999-2000; they and their children have been seen regularly since then. Over the past two decades, we have amassed a rich trove of behavioral, health, and exposure data from this cohort. Our active biorepository houses 330,000 biological (e.g. blood, urine, breastmilk, hair, saliva, teeth) and environmental samples (e.g. dust, allergens) that we continue to mine for new insights in environmental health. We also hold two decades’ worth of data on children’s neurodevelopment, lung function, physical growth, pubertal timing, cardiometabolic health, and psychosocial functioning and mental health into young adulthood. The cohort currently consists of almost 600 mothers (1st generation) and their 20-22 year old young adult children (2nd generation), some of whom have begun to have children themselves (3rd generation). We propose to begin data collection on the 3rd generation of participants, providing an unprecedented opportunity to assess, under future funding, multigenerational epigenetic effects of environmental exposure and social determinants in a well-characterized Latino cohort. Under the previous R24, we continued our strong community engagement that is vital to retention of the cohort; facilitated new research partnerships; improved our data sharing capabilities, protocols, and transparency through a web-portal; and maintained a strong Biorepository that is poised to address new research questions. In the next phase of this project, under the U24, we will expand on this important work by enriching our cohort with additional data on social determinants of health; laying the groundwork to enroll the 3rd generation of CHAMACOS participants; and promoting scientific and workforce diversity through collaborations with Hispanic-Serving Institutions and students and clinicians from Latino farmworker communities. We will expand resource sharing by making our data publicly available through an open-access data repository and promote sharing of biological and environmental samples through our web portal. Additionally, as the CHAMACOS founders move into retirement, we will transfer the leadership of the cohort to a new generation of investigators to ensure its continued, long-term success. This cohort represents a wealth of resources that should be maintained, enriched, and shared for future research opportunities. It has an engaged Latino participant population that allows scientific insight into an underserved population and provides opportunities to engage young Latino students, clinicians, and junior faculty to build the pipeline of future environmental health researchers.

NIH Research Projects · FY 2025 · 2017-09

ABSTRACT Leukemia is the most common pediatric cancer affecting more than 40,000 children worldwide each year. During the last decades, childhood leukemia incidence has increased in the US by ~35% overall, with an even larger rise among LatinX. Similar trends are also observed in several Latin American countries. This rapid increase points to the critical role of environmental factors in the development childhood leukemia, possibly in combination with genetic factors. Despite improved prognosis of childhood leukemia overall, there are major differences by subtype, region, racial/ethnic group, and socio-economic status, and leukemia survivors remain at risk for serious lifelong complications. Altogether, these observations highlight the need to support more research and prevention to reduce leukemia burden and disparity. The overall objective of this U24 competing renewal is to expand the support of existing population studies (here case-control design) to accelerate childhood leukemia environmental research and prevention, with a focus on LatinX populations. In the first R24 cycle (2017-22), we maintained and enhanced the resources and data sharing of two NIEHS-funded studies with large numbers of LatinX children in California, United States and Guatemala. To further address childhood leukemia disparity in LatinX, while increasing diversity of the research team, we now propose to (i) include additional childhood leukemia studies with low-resources in Mexico and Costa Rica, (ii) coordinate research translation/prevention among participating countries, and (iii) collaborate with LatinX researchers and health workers in California and Latin America. The four participating studies have enrolled 8,480 childhood leukemia cases and 4,462 controls, and have collected a wealth of environmental/genetic data and biospecimens providing the most comprehensive resources in LatinX populations worldwide. Individually or as part of the Childhood Cancer and Leukemia Consortium (CLIC), these studies have produced seminal findings, documenting the prenatal origin of childhood leukemia and identifying many factors that contribute to the increased or decreased risks of the disease including chemical exposures, diet/vitamins, breastfeeding, immune response, birthweight and genetic factors. The maintenance of these resources and the enhancement of data sharing procedures are needed to efficiently expand ongoing etiologic and tumor-biology studies of childhood leukemia, especially myeloid subtypes which are more common in LatinX, and uncover sources of disparity in leukemia risk and outcomes. To achieve our goals, we plan to (1) use an interoperable management system REDCap to provide unifying support for management, harmonization, storage and sharing of study resources; (2) enrich case-control studies by conducting linkages to population-level databases on exposures to potential carcinogenic agents/mixtures and social/built environments; (3) facilitate broader data use nationally and internationally with the CLIC Consortium, and (4) expand community engagement with the public, lay health workers, and health professionals in the US and Latin American countries to increase awareness about preventable risk factors of childhood leukemia.

NIH Research Projects · FY 2025 · 2017-09

ABSTRACT CHAMACOS is a longitudinal cohort study of Latino mothers and children from a California farmworker community who have been followed for more than 20 years to assess the impact of environmental exposures and social determinants on children’s health and development. CHAMACOS mothers were enrolled during pregnancy in 1999-2000; they and their children have been seen regularly since then. Over the past two decades, we have amassed a rich trove of behavioral, health, and exposure data from this cohort. Our active biorepository houses 330,000 biological (e.g. blood, urine, breastmilk, hair, saliva, teeth) and environmental samples (e.g. dust, allergens) that we continue to mine for new insights in environmental health. We also hold two decades’ worth of data on children’s neurodevelopment, lung function, physical growth, pubertal timing, cardiometabolic health, and psychosocial functioning and mental health into young adulthood. The cohort currently consists of almost 600 mothers (1st generation) and their 20-22 year old young adult children (2nd generation), some of whom have begun to have children themselves (3rd generation). We propose to begin data collection on the 3rd generation of participants, providing an unprecedented opportunity to assess, under future funding, multigenerational epigenetic effects of environmental exposure and social determinants in a well-characterized Latino cohort. Under the previous R24, we continued our strong community engagement that is vital to retention of the cohort; facilitated new research partnerships; improved our data sharing capabilities, protocols, and transparency through a web-portal; and maintained a strong Biorepository that is poised to address new research questions. In the next phase of this project, under the U24, we will expand on this important work by enriching our cohort with additional data on social determinants of health; laying the groundwork to enroll the 3rd generation of CHAMACOS participants; and promoting scientific and workforce diversity through collaborations with Hispanic-Serving Institutions and students and clinicians from Latino farmworker communities. We will expand resource sharing by making our data publicly available through an open-access data repository and promote sharing of biological and environmental samples through our web portal. Additionally, as the CHAMACOS founders move into retirement, we will transfer the leadership of the cohort to a new generation of investigators to ensure its continued, long-term success. This cohort represents a wealth of resources that should be maintained, enriched, and shared for future research opportunities. It has an engaged Latino participant population that allows scientific insight into an underserved population and provides opportunities to engage young Latino students, clinicians, and junior faculty to build the pipeline of future environmental health researchers.

NIH Research Projects · FY 2026 · 2017-09

ABSTRACT Leukemia is the most common pediatric cancer affecting more than 40,000 children worldwide each year. During the last decades, childhood leukemia incidence has increased in the US by ~35% overall, with an even larger rise among LatinX. Similar trends are also observed in several Latin American countries. This rapid increase points to the critical role of environmental factors in the development childhood leukemia, possibly in combination with genetic factors. Despite improved prognosis of childhood leukemia overall, there are major differences by subtype, region, racial/ethnic group, and socio-economic status, and leukemia survivors remain at risk for serious lifelong complications. Altogether, these observations highlight the need to support more research and prevention to reduce leukemia burden and disparity. The overall objective of this U24 competing renewal is to expand the support of existing population studies (here case-control design) to accelerate childhood leukemia environmental research and prevention, with a focus on LatinX populations. In the first R24 cycle (2017-22), we maintained and enhanced the resources and data sharing of two NIEHS-funded studies with large numbers of LatinX children in California, United States and Guatemala. To further address childhood leukemia disparity in LatinX, while increasing diversity of the research team, we now propose to (i) include additional childhood leukemia studies with low-resources in Mexico and Costa Rica, (ii) coordinate research translation/prevention among participating countries, and (iii) collaborate with LatinX researchers and health workers in California and Latin America. The four participating studies have enrolled 8,480 childhood leukemia cases and 4,462 controls, and have collected a wealth of environmental/genetic data and biospecimens providing the most comprehensive resources in LatinX populations worldwide. Individually or as part of the Childhood Cancer and Leukemia Consortium (CLIC), these studies have produced seminal findings, documenting the prenatal origin of childhood leukemia and identifying many factors that contribute to the increased or decreased risks of the disease including chemical exposures, diet/vitamins, breastfeeding, immune response, birthweight and genetic factors. The maintenance of these resources and the enhancement of data sharing procedures are needed to efficiently expand ongoing etiologic and tumor-biology studies of childhood leukemia, especially myeloid subtypes which are more common in LatinX, and uncover sources of disparity in leukemia risk and outcomes. To achieve our goals, we plan to (1) use an interoperable management system REDCap to provide unifying support for management, harmonization, storage and sharing of study resources; (2) enrich case-control studies by conducting linkages to population-level databases on exposures to potential carcinogenic agents/mixtures and social/built environments; (3) facilitate broader data use nationally and internationally with the CLIC Consortium, and (4) expand community engagement with the public, lay health workers, and health professionals in the US and Latin American countries to increase awareness about preventable risk factors of childhood leukemia.

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY/ABSTRACT Actomyosin stress fibers (SFs) enable cells to tense the extracellular matrix (ECM), a process key to cell shape determination, motility, and morphogenesis. Over the past 15+ years, including the past period of R01 support, we have made significant contributions to the field’s understanding of SF mechanics and contributions to cell structure. Our work is particularly notable for the use of femtosecond laser nanosurgery (FLN), which has enabled us to show that the three canonical SF subtypes – dorsal fibers, transverse arcs, and ventral fibers – collectively enforce a front-back tension gradient that underlies two-dimensional (2D) mesenchymal migration. We also showed that the SF network architecture can mechanically reinforce individual SFs, which has significant implications for symmetry breakage during directed migration and force propagation through cell monolayers. With this intellectual foundation in place, our renewal application turns to two important questions: How is polarization of tension in the SF network encoded by molecular signals classically understood to establish front-back polarity? And how does our knowledge of 2D SF networks translate to confined migration geometries like those found in tissue? We will address these questions through two specific aims, both of which build upon publications from this award. In Specific Aim 1, we will investigate mechanistic contributions of cofilin-1 to establishment and maintenance of SF front-back tension polarization during migration. We hypothesize that cofilin-1 establishes front-back polarization of SF tension by promoting the assembly and contractile maturation of transverse arcs. By combining biophysical, engineering, and cell biological tools, we will identify key molecular and force-based signals that modulate recruitment of cofilin-1 to developing transverse arcs. In an innovative new collaboration with Dr. Bruce Goode (Brandeis) we will reconstitute actin bundles in microfluidic devices and quantify the relationship between tensile force and cofilin- 1 engagement. In Specific Aim 2, we will dissect contributions of SF networks to migration in confined geometries where the ECM imposes axial cues and sterically precludes elaboration of 2D SF networks. We hypothesize that increasing confinement redirects SF assembly from the 2D dorsal fiber-transverse arc-ventral fiber assembly pathway towards de novo parallelized SF assembly. We will combine microengineered culture platforms, single-cell mechanical tools, and superresolution imaging to probe confinement-induced changes in SF assembly, architecture, and mechanics. Aim 2 will leverage two established, productive collaborations: With Dr. Ulrich Schwarz (U. Heidelberg), we will develop multiscale computational models that relate SF network architecture and mechanics to cell migration in confined spaces. With neurosurgeon Dr. Manish Aghi (UCSF), we will test the clinical value of our observations by asking if confined migration of glioblastoma stem cells is retrospectively predictive of in vivo invasion patterns. Our studies will create unprecedented new insight into how SFs contribute to migration, with innovative methodology and close connection to human disease.

NIH Research Projects · FY 2026 · 2017-08

Project Summary Although aging, germline mutations, and family history of cancer are significant risks for breast cancer, it is still unclear why one person’s breast cells are more susceptible to this disease than another person’s. Aging changes cells and tissues such that they become more susceptible to cancer initiation. Our published data show that breast epithelial cells from young women who have high-risk germline mutations show accelerated aging with intermediate filament distribution, biological clock acceleration, and stromal immune cell milieu changes comparable to those of women who are 20–40 years older. In our currently funded R01EB024989, we discovered that the mechanical properties of normal breast epithelial cells, as measured by our mechano-Node Pore Sensing (mechano-NPS) platform, differ among younger and older women and that normal epithelial cells from genetically high-risk women who carry germline BRCA1, BRCA2, or PALB2 variants are mechanically “older” than their chronological age. We hypothesized that mechano-NPS can detect disease states based on the emergent mechanical properties that arise from the underlying molecular networks that define lineage and disease states. In this competitive renewal application, we extend this hypothesis to include detection of cancer susceptibility or risk, which is so far not detectable with genetic screening. We will innovate mechano-NPS and advance an in silico model of our device to increase the number of physical parameters it can measure, thereby providing a more complete portrait of single human mammary epithelial cells (HMECS) (Aim 1). We will build a machine learning cancer susceptibility detection system based on measuring mechanical properties of different primary HMEC (young, old, high-risk, family history of breast cancer, etc.) (Aim 2). Finally, we will dissect the molecular mechanisms of mechanical states measured by our advanced mechano-NPS platform (Aim 3). In the last funding period, we successfully designed, built, and validated the first-generation mechano-NPS platform at UC Berkeley and showed it to be portable and robust by building a second platform at City of Hope. The impact of our competitive renewal application will be far more reaching. Clinically useful genetic testing relies on a handful of known monogenic risk traits, but we hypothesize that emergent mechanical properties, measured from just a few hundred cells, are a characteristic of the biology that underlies cancer susceptible states, even those that are polygenic or epigenetic in nature and are passed within a family but that so far have defied definition.

NIH Research Projects · FY 2025 · 2017-08

The development of single-cell genomics technologies has been a driving force in biomedical research over the past decade because of the throughput, sensitivity, and precision with which such techniques can dissect complex biological systems. A primary example of the impact of these tools is the rapid translation of single-cell RNA sequencing (scRNAseq) for comprehensively cataloging cellular states for the Human Cell Atlas project. Technology that combine microfluidic platforms with DNA barcoding strategies have drastically increased the throughput and accessibility of scRNAseq so that to-date, the Human Cell Atlas consortium has logged over 67 million cells, enabling unprecedented insight into the cellular diversity in healthy and diseased organs and tissues. However, while the transcriptome provides a comprehensive and quantitative proxy for cellular state, proteomic measurements provide a more direct understanding of cellular function, and epigenetic measurements that profile methylation, histone modifications, and protein-DNA interactions, provide a more complete picture of the regulatory mechanisms that maintain cell state or drive cellular transitions. Furthermore, cell morphology and the spatial distribution of proteins and chemicals within the cell can reveal important cellular phenotypes that can only be characterized by microscopy. Finally, the relative positions of cells within tissues and organs are necessary for a more complete understanding of the cellular interactions that lead to functional tissues and organs. This research program focuses on the development of technology to facilitate multimodal precision measurements in single cells and tissues. We use molecular biology tools and DNA sequencing platforms to measure the proteome, transcriptome, and epigenome of single cells and nonlinear optical imaging to characterize chemical composition and morphology of cells. We leverage microfluidic technology to integrate molecular and optical measurements to enable multimodal single-cell measurement. Additionally, this research program aims to develop novel computational approaches for integrated analysis of multimodal single-cell measurements. Our ultimate goal is to develop a tool to make all of these measurements in situ, in order to retain single-cell spatial information and cellular context in a developing tissue or whole organism.

NIH Research Projects · FY 2026 · 2017-06

ABSTRACT Cue-driven behaviors are actions motivated by salient environmental stimuli. Maladaptive changes in cue- driven behaviors are fundamental to several neuropsychiatric disorders, including substance use disorder. Ventral tegmental area (VTA) dopamine (DA) neurons have often been assumed to homogeneously encode reward prediction errors. However, even within the nucleus accumbens (NAc), which is the major projection target of VTA DA neurons, DA release has been implicated in several behavioral functions, including reward, aversion, motivation, and incentive salience. This discrepancy might be, at least in part, due to the different methodologies used to record DA cell body activity versus DA release at the axon terminal level. Additionally, recent investigations suggest that VTA DA neurons might differentially contribute to reward and aversion dependent on their projection target and mediolateral position within the VTA. Here, we propose to further detail and define, in a circuit- and cell-type specific manner, the functional heterogeneity of the mesoaccumbal DA system. We will employ a three-pronged approach that leverages pharmacological manipulations, fiber photometry-based recordings of DA cell body activity and DA release, as well as in vivo electrophysiological recordings of DA neurons using Neuropixels and optogenetics. Our investigations will that takes the precise neuroanatomical position of DA neurons within the VTA and their corresponding NAc projection target into consideration. Experiments will be performed in head fixed mice during pharmacological manipulation or during reward seeking behavior as well as in freely behaving mice performing a two-armed bandit task. This will allow us to test whether DA dynamics mediated by different mechanisms in different locations (i.e., at the level of cell bodies versus terminals) underlie distinct behavioral functions. The primary goals are to (1) investigate how different doses of nicotine modulate DA release in distinct NAc subregions. Additionally, we will provide a systematic understanding of how nicotinic acetylcholine receptor function contributes to nicotine-induced DA release in different NAc subregions. (2) We will study how DA cell body activity and DA release in separate mesoaccumbal subcircuits contributes to reward learning and motivated behavior. (3) We will establish an in vivo electrophysiological approach that utilizes Neuropixels and optogenetics to record VTA DA neurons in a circuit- and cell-type specific manner in head fixed mice performing a reward seeking task. Together, we anticipate that these experiments will provide further evidence for our hypothesis that VTA DA neurons make specific contributions to reward learning and motivation in a projection-defined manner. Delineating the precise functions of separate mesoaccumbal DA subcircuits for reward learning and motivated behavior is a significant step in improving our understanding of their diverse roles in health and disease.

NIH Research Projects · FY 2025 · 2017-05

SUMMARY Understanding how nature builds new traits is a fundamental goal of evolutionary genetics. Unbiased experimental dissection of trait variation from the wild has to date used linkage or association mapping, which are suitable only for crosses between compatible individuals of a given species. In the first funding period of this methods-development R01, PI Brem developed RH-seq, an approach for the unbiased mapping of natural trait variation that can be applied to reproductively isolated species. Our RH-seq projects in invertebrate test cases have put the complex genetics of ancient traits within reach for the first time in the experimental literature. We now want to advance strategies that investigate deeper themes in complex genetics between species—namely whether evolution uses concerted molecular mechanisms across the loci underlying a polygenic adaptation, and how these loci work together to drive phenotype. To test-drive these approaches, in our first Aim we will use an ecologically relevant model system, a thermotolerance divergence between yeast species that last shared an ancestor five million years ago. In our second Aim, we will port our ideas and tools for interspecies genetics to mouse primary cells. The latter will use as a testbed a cell-autonomous, pro- inflammatory aging program called cellular senescence, which we have found to diverge between between sister species of mice. We will develop RH-seq for unbiased genetic mapping of senescence traits, and we will pursue epistatic and molecular mechanisms of the underlying loci as a parallel to our yeast model. Together, our yeast and mouse projects will advance methods for the analysis of polygenic traits as they differ between species, and accelerate the dissection of such ancient characters in systems across Eukarya.

NIH Research Projects · FY 2026 · 2017-04

PROJECT SUMMARY/ABSTRACT The overall goal of our research is to understand the mechanisms that regulate growth: at the level of the individual cell, at the level of organs and at an organismal level. In order to study the regulation of growth, we have studied the imaginal discs of Drosophila, the larval precursors of adult structures such as the wing. Until recently, an important limitation to our work was that we had few tools to study the cellular heterogeneity that is a key feature of growth regulation in vivo. Single-cell transcriptomics allows us to characterize small subsets of cells that have a major impact on growth regulation. By combining this approach with experimental genetics, we can now study a key aspects of growth regulation that involves heterotypic interactions between small subsets of cells. We aim to obtain a better understanding of the genetic regulation of regenerative growth. We have identified a gene regulatory network that is entirely dispensable for normal development but is essential for regenerative growth. Additional genes identified from our single-cell studies point to genes that are specifically expressed in different portions of the regeneration blastema and which likely regulate different aspects of regeneration including proliferation, cell shape and cell-fate plasticity. We will characterize the function of these genes. We have also found that damage and regeneration of one portion of the disc can impact the development of the remainder of the disc and also distant organs. Another goal is to elucidate the mechanistic basis of these long- range phenomena. We will examine the role of ion channels in regulating Hedgehog signaling and the growth and patterning of the of the wing disc. We will determine why cells with a more depolarized membrane potential survive preferentially in the anterior compartment of the wing disc. We will also study the properties of the mechanosensitive channel Piezo in growth regulation. Finally, by combining our single cell atlas of the wing disc with a genetic screen, we have identified many genes encoding poorly-characterized cell-surface proteins and ligand-receptor pairs that likely function during development to regulate cell survival, cell proliferation and planar cell polarity. We will study these genes.

NIH Research Projects · FY 2026 · 2017-01

07 Project Summary/Abstract The long-term goal of this project is to interrogate the dynamics of membrane voltage in the context of intact brains with high spatiotemporal resolution. Optical methods to dissect neuronal activity promise to revolutionize our understanding of the brain at the cellular and circuit level; however, our understanding remains incomplete due, in part, to a lack of tools that can report directly on neuronal activity with sufficient speed and sensitivity. We propose to use the power of molecular design and organic chemistry to develop and apply new optical tools for monitoring membrane potential with unprecedented speed and sensitivity in intact brains and without disruptive capacitive load associated with other classes of voltage indicators. We plan to exploit photoinduced electron transfer (PeT) through molecular wires as a versatile platform for optical voltage sensing. We will build a palette of colors for optical voltage sensing that extends into the near infrared regions of the electromagnetic spectrum; we will create new voltage sensors with exceptionally high two-photon absorption cross sections for use in thick tissue and intact brains; and we will explore methods for genetically targeting and localizing ultra- sensitive fluorescent voltage sensors to neurons of interest. Throughout, development of molecular tools will be closely wed to applications in neurons and tissues, and we will apply these tools to understand how membrane potential dynamics change in both healthy and neurological disease states.

NIH Research Projects · FY 2025 · 2016-09

Waterborne pathogens like Vibrio cholerae pose significant threats to global health. V. cholerae can persist in the aquatic environment, and it can emerge to cause devastating cholera outbreaks in endemic regions and vulnerable areas lacking adequate water and sanitation infrastructure. The host-pathogen interactions that dictate disease outcome and cholera transmission dynamics occur in the context of a complex microbial ecosystem that includes predatory bacterial viruses (phages). Phages profoundly impact the evolution of their bacterial hosts, both through predation, which selects for hosts with defenses that overcome phage killing and through mobilization and dissemination of genetic material. Certain mobile elements called the phage satellites have evolved sophisticated mechanisms to exploit phages for their own selfish spread. Such elements interfere with the replication of the phages they parasitize, and as such, provide their cellular hosts with a means to limit phage predation. Our lab discovered PLEs (for phage-inducible chromosomal island-like elements) in V. cholerae that provide specific and robust defense against ICP1, the dominant lytic phage co-circulating with V. cholerae in cholera endemic regions. Upon infection by ICP1, PLEs excise from the V. cholerae chromosome, replicate to high copy and are assembled into virions to spread the PLE genome to new cells while concurrently abolishing phage production. PLEs are uniquely potent, highly specific, anti-phage barriers that act through multiple mechanisms to ensure that ICP1 does not propagate and spread to neighboring V. cholerae cells. However, few mechanisms of direct interference with ICP1 are known, and none are essential for PLE activity, indicating that additional mechanisms await discovery in this system. This proposal builds on our prior work defining the PLE lifecycle in response to phage infection to gain a mechanistic understanding of how PLEs execute their unusually potent anti-phage activity. Our data indicate that PLE’s most potent anti- phage inhibitors are focused on blocking virion assembly. To understand PLE activity in mechanistic detail, we will pursue the following specific aims: 1) We will define the structural composition of virions and capsid assembly intermediates for ICP1 and PLE 2) We will Interrogate the functions of three PLE-encoded ORFs that are each sufficient to inhibit phage 3) We will determine how a PLE-encoded small RNA perturbs phage gene expression. The proposed studies are expected to reveal novel mechanistic paradigms not previously documented in phage satellites or other anti-phage defense systems. The long-term coevolution of V. cholerae PLE and ICP1 serves as a powerful model system to understand clinically relevant phage defense mechanisms to inform phage therapy efforts and understand the forces driving the evolution of bacterial pathogens.

NIH Research Projects · FY 2026 · 2016-08

PROJECT SUMMARY/ABSTRACT Co-evolution occurs when one species exerts natural selection on another species, and vice versa, resulting in reciprocal adaptation. Host-parasite co-evolution has driven biological diversification, the evolution of immune systems, and the origin of sexual reproduction. Although its genetic and molecular bases are poorly understood, when such knowledge has been gained, it laid the foundation for transformational discoveries such as CRISPR- Cas adaptive immune systems of bacteria, which arose through co-evolution with phages and are now widely used as genome editing tools. Although microbial pathogens (microparasites) like bacteria, viruses, and protozoa have influenced human evolution, parasitic arthropods and worms (macroparasites) are also important agents of natural selection. The same regions of the human genome associated with natural selection by macroparasites are co-associated with auto-immunity risk, implicating human-macroparasite co-evolution in the etiology of these disorders, which are increasing in prevalence. Yet, there is a gap in our knowledge of the genetic and molecular basis of the co-evolutionary mechanisms producing these patterns. This gap is in part because human- macroparasite interactions are largely intractable to study mechanistically. Until remedied, progress toward understanding an elusive but important basic process that profoundly shapes human health is impeded. New model host-macroparasite systems are critical for advancing the field, particularly those that have co-evolved. This research aims to develop and improve such models to study the genetic and molecular mechanisms of co- evolution. In the next phase of EI MIRA support, Drosophila fruit flies are proposed as model hosts of co-evolving, worm-like larvae of parasitoid wasps. Given the tools available, this Drosophila-parasitoid system is a powerful model for studying the genetic and molecular mechanisms of host-macroparasite co-evolution. This research plan focus on the basic biology of a novel innate immune factor expressed in the blood of fruit flies that targets developing parasitoids. This factor is encoded in the fly’s genome by a gene borrowed from bacteria through a process called horizontal gene transfer (Verster et al. 2019; Verster et al. 2023). The horizontally-transferred gene in the fly’s genome encodes cytolethal distending toxin subunit B (CdtB), a bacterial toxin that targets animal cells and is a component of typhoid toxin. CdtB is the first candidate anti-parasitoid humoral toxin known from Drosophila and its expression is carefully regulated by the fly to avoid auto-immunity (Tarnopol et al. 2024). The objectives in the next five years are to: (1) identify regulatory mechanisms that facilitated the domestication in animals of toxins originating from bacteria, (2) dissect how the toxins harm parasitoids without harming hosts, and (3) develop new models and tools in Drosophila and parasitoids to advance mechanistic studies of co- evolution. This research offers a model for illuminating how horizontal gene transfer enhances animal innate immunity and is expected to inform the understanding of how toxins mediate co-evolution, a process that shapes the biology of all organisms and has impacted many elements of human health.

NIH Research Projects · FY 2025 · 2016-07

Project Summary The T32 Neuroscience Training Program (NTP) at Berkeley seeks to provide broad-based neuroscience PhD training, with an emphasis on advanced research methods and quantitative approaches. Our 60 training faculty are from 12 departments and span molecular to cognitive neuroscience, new methods development, and disease-related research. The NTP supports Year 1-2 students from the Neuroscience PhD Program (the largest pool of neuroscience-focused students at Berkeley) plus a smaller number from 3 additional PhD programs who train in our faculty laboratories. Our program provides training across a wide range of neuroscience from molecules to mind. We combine flexible coursework, rigorous research training, quantitative skills, and a major focus on advanced research methods. We have a multi-disciplinary approach to neuroscience that leverages Berkeley’s deep expertise in molecular and cell biology, physical and computational sciences, engineering and psychology. We require coursework in molecular-cellular, circuit-systems, and cognitive neuroscience, plus optional computational neuroscience. We require laboratory rotations and thesis research, an Experimental Boot Camp, and a new Quantitative Boot Camp that teaches programming, data analysis and statistics. Our core competency classes introduce major experimental methods and train students in rigor and reproducibility, experimental design, scientific and grant writing, scientific talk skills, and responsible conduct of research. Seminar series, multi-lab research meetings and journal clubs, and an annual campus-wide retreat provide rich exposure to neuroscience research. A qualifying exam includes an NIH grant-style proposal for thesis research, oral defense of the proposal, and examination on a broad set of foundational questions in neuroscience. Students complete an individual development plan (IDP) at the end of Year 2. A multi-tiered advising system provides extensive scientific and career advising. We strive to provide an inclusive, supportive research climate. We conduct yearly program evaluations to guide improvements. For this renewal, we add important updates—launching our Quantitative Boot Camp, adding more training in experiment design and scientific rigor, reorganizing our advising structure to provide more active student advising and support, adding required mentor training for NTP faculty, and expanding career development resources. We believe this training program will produce scientists who will make important discoveries with cutting-edge technology, and translate these discoveries into solutions for human neurological disease. This training program is followed not just by T32 students, but by all Neuroscience PhD program students, and its activities are open to any neuroscience-oriented student at Berkeley. This gives the NTP critical mass and broad impact, reaching a large number of students and synergizing neuroscience training for students across many PhD programs.

NIH Research Projects · FY 2025 · 2016-07

PROJECT SUMMARY Proposed are complementary studies on the mechanisms and regulation of clathrin-mediated endocytosis (CME) and actin force generation during CME in budding yeast and human stem cells. CME is responsible for uptake of molecules from a cell's environment through the permeability barrier of the plasma membrane and for selective removal of plasma membrane proteins. It is also one of the main routes for COVID-19 to enter cells. Therefore, this process is crucial for determining how cells respond to their surroundings and has heightened translational significance. Many proteins and lipids that mediate CME have been identified and their functions determined biochemically and in living cells. Imaging of fluorescently labeled CME proteins in live cells has revealed the intricate recruitment timing and order for some 60 CME proteins. However, how cargo capture is coordinated with vesicle formation, how correct protein recruitment order and timing are achieved, which events and molecules play critical roles in the pathway, and how forces curve the membrane and drive vesicle scission, are not fully understood. The following key questions will be addressed in budding yeast and human stem cells: 1) How does membrane curvature affect biochemical reaction rates? 2) How does CME become specialized for different cell types during differentiation? 3) How does a checkpoint sense cargo and regulate CME progress? and, 4) How does actin assemble at CME sites and how does its ultrastructure contribute to CME force production and adapt to increased membrane tension? Yeast studies will be empowered by a rich legacy in the lab of elucidating actin assembly and force production mechanisms. Human cell studies will be empowered by over 120 stable human tissue culture and stem cell lines generated using genome editing to express CME and actin cytoskeleton proteins as fluorescent protein fusions at native, endogenous levels. Because CME proteins are highly conserved in structure and function, principles learned from studies of yeast and humans will complement and inform each other. Together, these studies will provide a comprehensive mechanistic understanding that could not be achieved by studies in only one cell type. Because the actin cytoskeleton has been adapted by evolution for diverse, essential activities including cell motility, organelle transport, adhesion, and cell polarity development, what is learned will apply broadly for many cellular processes and will join the growing armamentarium of possible defensive measures against the pandemic.

NIH Research Projects · FY 2025 · 2016-06

NIH R35 GM118121; DNA transposons and alternative pre-mRNA splicing. D. Rio – PI. PROJECT SUMMARY / ABSTRACT DNA transposons and alternative pre-mRNA splicing. D. Rio – PI Mobile genetic elements or transposons are found in the genomes of all organisms. These elements can move via DNA or RNA intermediates. About 50% of the human genome is made up of transposable elements with ~ 2.7% corresponding to DNA-based transposons. Many of these putative transposons or transposase-related genes are uncharacterized. Our previous studies have focused on the P element family of DNA transposons in Drosophila. P element transposase functions as a tetramer, using GTP as a cofactor for transposition. N-terminal domain of the transposase corresponds to a C2CH THAP DNA binding domain, which is a member of a prevalent family of DNA binding domains found exclusively in animal genomes. One THAP gene, called THAP9, is homologous to the Drosophila P element transposase and is present in primates, Xenopus, zebrafish and Ciona, but is absent from rodents. Recent work from our lab has shown that the human and zebrafish THAP9 genes can mobilize the Drosophila and zebrafish P element transposons in human and Drosophila cells. We have also used cryo-EM to solve the structure of the P element transposase strand transfer complex. This proposal is focused on understanding what role the human THAP9 gene may play in human embryonic stem cells and the reaction pathway that the Drosophila P element transposase protein uses to recognize and assemble with the transposon ends, donor DNA, target DNA and GTP/Mg2+ to form an active protein-DNA complex. These studies are aimed at gaining mechanistic insights. Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in metazoans and is a conduit through which genomic sequence is transferred to proteomic information. Most eukaryotic genes are split and have the potential for alternative splicing, dramatically increasing proteomic diversity. Many human and mouse disease gene mutations affect the splicing process. in fact, somatic mutations in splicing factor and spliceosomal genes have been linked to human diseases, such as cancer and the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Our previous work has focused on characterization of the tissue-specific Drosophila P element pre- mRNA exonic splicing silencer element. Recent work from our group has focused on how the action of the RNA binding proteins, PSI and hrp48 and the human RNA binding splicing factors hnRNPA1 and DDX5. We are using this information to identify new Drosophila cellular splicing silencer elements that are controlled by PSI and hrp48. We are also analyzing mutant forms of hnRNPA1 that are linked to ALS to find splicing pattern defects that could be used as biomarkers for the disease or provide clues to have neurons are dying in the disease. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific alternative splicing patterns. The PSI protein also interacts with U1 snRNP and PSI mutant Drosophila strains that abolish this interaction exhibit male courtship behavior defects and altered pre-mRNA splicing of the Drosophila male-specific fruitless pre-mRNA isoforms. We want to investigate how the PSI protein controls fruitless pre-mRNA splicing and how it controls binding of U1 snRNP on the Drosophila transcriptome. U1 snRNP has distinct roles in U1 snRNP binding sites in PCPA (premature cleavage and polyadenylation), splicing at intron 5' splice sites, at cryptic 5' splice sites and at splicing silencers (from our work).

NIH Research Projects · FY 2025 · 2016-06

Project Summary The major goal of this research program is to develop catalytic enantioselective transformations based on transition metal and chiral anion catalysis that will be broadly applicable to the preparation of therapeutically relevant organic molecules. Towards this end, new reactions for the enantioselective construction of C-O, C-N and C-halogen bonds are proposed. Particular emphasis is placed on the development of methods for the selective introduction of fluorine and fluorine-containing functional groups. Additionally, reactions that generate or employ available building blocks, such as alkenes and boronic acids, will be targeted. These methods will leverage new catalyst platforms, based on gold(III) complexes and phosphoric acid-based chiral anions, for the construction of fluorinated building blocks and heterocycles Additionally, the mechanistic underpinnings of these reactions will be studied, with a focus on uncovering the role of non-covalent interactions in achieving selectivity. Thus, we anticipate that this program will provide synthetic chemists and biomedical researchers with additional tools (reactions, catalysts, ligands etc.) for molecular synthesis and for single enantiomer construction, as well as provide principles for further development and application.

NIH Research Projects · FY 2025 · 2016-05

PROJECT SUMMARY Tuberous Sclerosis Complex (TSC) is a multi-system developmental disorder caused by mutations in the TSC1 or TSC2 genes. The protein products of these genes form a complex that is an essential negative regulator of mTORC1 signaling. In the absence of a functional TSC1/2 complex, mTORC1 signaling is deregulated and constitutively active. While the manifestations of TSC can affect several different organ systems, the neurological and psychiatric aspects of the disease are the most burdensome for caregivers and least well understood. These include early-onset epilepsy, varying degrees of intellectual disability, and a high prevalence of autism spectrum disorder and other behavioral conditions. A hallmark pathology of TSC is the presence of cortical tubers, which are focal regions of enlarged, dysplastic neurons and glia in the cortex that form during embryonic development. Cortical tubers can become epileptic foci and in some cases are surgically removed in individuals with intractable seizures. The size and number of cortical tubers is variable between patients and increased cortical tuber load is associated with worse outcomes including more severe epilepsy and cognitive impairment. The goal of this project is to determine the molecular mechanism(s) by which mutations in TSC1 or TSC2 lead to the formation of cortical tuber cells. To do this we will use our recently established human brain organoid models of TSC in which we have engineered loss of function mutations in TSC1 or TSC2. These human brain organoid models robustly reproduce key cellular features of cortical tubers including dysmorphic neurons, reactive astrocytes, and giant/balloon cells. In addition, we have observed a strong bias towards the production of glial-lineage cells at the expense of neurons in TSC brain organoids, which recapitulates observations from patient tuber samples. Here we will define the molecular basis for altered cortical cell development due to TSC1/2 mutations and investigate how the resulting tuber cells impact the function of the surrounding cortical network. In Aim 1 we will explore two potential hypotheses for altered differentiation of TSC1/2 mutant cells in brain organoids: 1) premature activation of astrogenic transcription programs that interfere with normal neurogenesis and/or 2) impaired survival and development of newborn neurons. To test these hypotheses we will use pharmacological, shRNA, and CRISPRi manipulations to test the contribution of candidate pathways. In Aim 2 we will use different strategies to manipulate mTORC1 signaling and specific downstream arms of the pathway to test whether these can prevent or rescue altered cellular development. In Aim 3, we will perform functional analyses to determine how the presence of cortical tuber cells impacts the activity of the surrounding cortical network. Together the results of these aims will generate new insights into the molecular and cellular mechanisms leading to cortical tuber formation and how these cells ultimately impact cortical function.

NIH Research Projects · FY 2025 · 2016-05

The study of budding yeast has provided fundamental insights into the cell biological processes of all eukaryotes. Its study has also made critical contributions to the understanding and treatment of human disorders like diabetes, congenital disabilities, and cancer. My laboratory uses budding yeast to interrogate three areas of cell biology, the higher-order structure of chromosomes, the prevention of chromosome damage and rearrangements, and the mitigation of environmental stress. The basic unit of chromosomes is chromatin, which is composed of the DNA and associated proteins. The organization of chromatin into higher-order structures is essential for high fidelity chromosome segregation, the repair of DNA damage, and the regulation of gene expression. The molecular mechanisms that organize chromatin are major mysteries in cell biology. In this proposal, we study chromatin organization through the analysis of cohesin, a member of the SMC (Structural Maintenance of Chromosomes) family of protein complexes. Cohesin contributes to chromosome organization by tethering together different chromatin regions within a chromosome or between chromosomes. Cohesin also translocates along a chromosome to extrude loops. This proposal interrogates the molecular mechanisms underlying cohesin's tethering and loop-extrusion activities, the regulation of these activities, and their impact on chromosome structure and function in living cells. Cells also maintain genome stability by preventing and repairing chromosome damage. Chromosome damage is often caused by errors in the execution of intrinsic cellular processes. Indeed, during transcription, an RNA transcript can erroneously hybridize with homologous double-stranded DNA sequences on the chromosomes to generates an RNA-DNA hybrid and a displaced single-stranded DNA. This unusual structure, called an R-loop, can cause DNA damage and chromosome rearrangements. Here, we present experiments to understand why only a subset of R-loops in a genome cause DNA damage and how this damage leads to the large chromosome rearrangements that are a common feature of cancer cells. Finally, cellular stress also arises from extrinsic environmental changes. Understanding how some organisms survive extreme environmental changes has provided critical technical and conceptual advances in biology. We study the ability of yeast to survive desiccation. We showed that the expression of a small protein and simple sugar in yeast is necessary and sufficient for yeast to survive desiccation. These two factors prevent the aggregation of model proteins and modulate membranes in vitro. Here, we propose to elucidate the remarkable biological functions of these two factors by identifying the specific cellular proteins and membranes that they protected during desiccation. These studies will provide fundamental insights into protein and membrane homeostasis beyond desiccation and may generate potential novel applications for biomedicine and agriculture.

NIH Research Projects · FY 2025 · 2016-04

PROJECT SUMMARY Mechanisms of Mitosis and Size Control in Xenopus Research in my laboratory is focused on two major areas: Cell division is arguably the most dramatic event in the life of a cell. Chromosomes condense, organelles vesiculate, and the microtubule cytoskeleton rearranges into a bipolar spindle that attaches to chromosomes at their kinetochores and segregates a complete genome to each daughter cell. Although the morphological changes that occur during mitosis were first observed over a century ago, we still do not understand how these dynamic events are orchestrated. Many factors have been identified that contribute to spindle assembly and function, but the molecular and biophysical mechanisms and interactions that ensure mitotic fidelity remain unclear. Our current projects address outstanding questions including 1) What are the molecular underpinnings and functional consequences of different spindle architectures? Spindle size and organization vary dramatically across cell types and organisms, and factors known to affect these parameters are altered in many cancers, but how specific spindle features are established and their effects on chromosome segregation and cell division are poorly understood. We will leverage morphometric and phylogenetic comparisons together with biochemical and functional assays to investigate the dramatic changes in spindle architecture that occur between oocyte meiosis and the mitotic divisions of early development in Xenopus and the sea squirt Ciona intestinalis. We will elucidate the role of specific factors in this transition, and examine the consequences of altering spindle architecture on embryo cell division. 2) What defects in cell division mechanisms underlie speciation? We have observed chromosome mis-segregation in inviable hybrids generated by fertilizing Xenopus tropicalis eggs with X. laevis sperm, and identified incompatibility between a subset of paternal centromeres and maternal cytoplasm as one underlying cause. We will elucidate the molecular basis of inter-species conflicts that impact cell division and contribute to reproductive isolation. 3) What is the molecular basis of mitotic chromosome condensation? We have developed a novel approach using optical tweezers to measure the dynamics of single DNA molecules in real-time in Xenopus egg extracts with high spatial and temporal precision and will use this system to dissect the roles of key factors in driving mitotic chromosome assembly. Absolute and relative size of biological entities varies widely, both within and among species at all levels of organization above the atomic/molecular: the organism, the cells that make up the organism, and the cellular components. How does scaling occur so that everything fits and functions properly? Correct scaling inside cells is crucial for cell function, architecture, and division, but until recently the control systems that a cell uses to regulate the size of its internal structures were virtually unknown. We have established assays to elucidate mechanisms of intracellular scaling between different-sized frog species and during the rapid, reductive cell divisions of early embryogenesis. We are further developing these systems to ask: 1) What scales mitotic chromosome size to cell size? We are testing the hypothesis that a surface area to volume sensor acting on the interphase nucleus and the mitotic spindle also coordinately adjusts mitotic chromosomes to cell size during Xenopus development. 2) What are the connections between genome size, cell size, physiology, and development? Cell size correlates strongly with genome size across evolution, but underlying mechanisms are unknown. We will utilize different ploidy frog embryos to address how altering genome size affects gene expression, and a variety of species including the dodecaploid frog Xenopus longipes to investigate relationships between genome size, cell division mechanisms, development, and physiology. The means to address these fundamental cell biological questions is enabled by powerful experimental systems based on cytoplasmic extracts and functional in vivo assays in vertebrate (Xenopus) embryos. We have established productive collaborations and apply diverse techniques including high-resolution microscopy, single molecule assays, genomics, proteomics, microfluidics and computational modeling to fill important conceptual gaps in an innovative, rigorous, and interdisciplinary manner. Our research will continue to provide novel insight into cell division and size control, processes essential for viability and development, and defective in human diseases including cancer. Although introduced as distinct topics, cell division and size control are intimately linked. We are increasingly focused on how cross-species comparisons can elucidate molecular mechanisms underlying cell division and size control, as well as how biological constraints related to these processes have shaped evolution. Together, these projects uniquely advance our understanding of long-standing questions in biology.