Rebecca Heald - US grants

Bipolar spindle assembly is required for the correct segregation of duplicated chromosomes during cell division. While spindle assembly has been studied for many years, the principles and mechanisms governing it remain unclear. The general goal of the project is to elucidate the principles that underlie spindle assembly and function, as well as to identify and study the roles of individual proteins. In the long term, this approach will allow the reconstitution of spindle assembly from purified components. Since uncontrolled cell division is at the heart of the cancer problem, a molecular understanding of spindle assembly and function could lead to new approaches for cancer therapy. To study spindle assembly mechanisms we have developed an in vitro system utilizing DNA linked to beads as artificial chromosomes, which function physiologically in cytoplasmic extracts prepared from Xenopus eggs. In mitotic extracts, DNA beads induce the formation of bipolar spindles in the absence of centrosomes and kinetochores. This assay has revealed that spindle assembly around chromatin proceeds in two phases. First microtubule nucleation and growth is induced around chromatin, then the stabilized microtubules are reorganized into a bipolar spindle by microtubule-based motors. The specific aims of the proposal address the molecular mechanisms behind these two steps. The general strategies used take advantage of the open nature of extracts, which allows specific inactivation of individual components; while the use of DNA beads facilitates magnetic isolation of chromatin proteins for functional and biochemical analyses. The aims are: (1) To examine the role of different microtubule-based motors in the bipolar organization of microtubules by specific inactivation through immunodepletion, or addition of specific antibodies or dominant negative constructs. (2) To explore the mechanism of specific microtubule growth around chromatin by gamma-tubulin inactivation and reconstitution experiments. (3) To examine the requirements for functionally active DNA beads by correlating chromatin assembly with the ability of beads to induce spindle assembly. (4) To identify chromatin factors important for microtubule stabilization through biochemical analysis of proteins and enzymatic activities bound to DNA beads. (5) To reconstitute aspects of anaphase chromosome segregation using beads coupled with centromeric DNA in order to develop an assay for kinetochore function and to identify DNA sequences and proteins required.

DESCRIPTION (provided by applicant): Of central importance to the process of cell division is the accurate transmission of a complete set of chromosomes to each daughter cell, which in eukaryotes is achieved through the function of the mitotic spindle. The goal of this research proposal is to elucidate the role of the highly conserved GTPase Ran in cell division. The current model suggests that RanGTP functions as a spatial marker that signals the position of the genome in eukaryotic cells. During mitosis, RanGTP is thought to promote spindle assembly by stimulating microtubule polymerization and organization in the vicinity of chromosomes. This "local effect" results from the chromosomal localization of RCC1, the guanine nucleotide exchange factor that generates RanGTP, which binds to transport factors causing them to release cargoes required for spindle assembly. However, the nature of the cargoes, their mitotic function and the molecular mechanisms underlying the RCC1-Ran-microtubule signaling cascade are still poorly understood. A major experimental approach described in this proposal takes advantage of complex cellular extracts prepared from eggs of the African frog Xenopus/aevis that can be studied using biochemical and functional assays. This provides an excellent model system for fractionation and reconstitution experiments to identify and characterize downstream factors. In addition, we propose to examine conservation of the mitotic Ran pathway in vivo using somatic cells. Our aims are: (1) To identify and characterize intermediates in the RanGTP-microtubule cascade. (2) To use fluorescent sensors of the Ran nucleotide state to visualize and characterize the RanGTP gradient in extracts and in molecularly-defined reactions. (3) To determine the existence and role of RanGTP/cargo gradients in vivo using cultured mammalian cell lines combined with microinjection and RNA interference techniques. Proper spindle assembly and function is required to maintain genome stability, and defects in this process are associated with birth defects and cancer. The identification and characterization of factors in the mitotic Ran pathway is fundamental to our understanding of mitotic and meiotic spindle assembly in eukaryotic cells, and supports our long term goal of reconstituting the complex morphogenetic events of cell division using purified components.

DESCRIPTION (provided by applicant): Bipolar spindle assembly is required for the correct segregation of duplicated chromosomes during cell division. While spindle assembly has been studied for many years, the mechanisms governing it remain unclear. The general goal of the project is to elucidate the principles that underlie spindle assembly and function, as well as to identify and study the roles of individual proteins. In the long term, this approach will allow the reconstitution of spindle assembly from purified components. Since uncontrolled cell division is at the heart of the cancer problem, a molecular understanding of spindle assembly and function could lead to new approaches for cancer therapy. To study spindle assembly mechanisms we use assays based on cytoplasmic extracts prepared from eggs of the frog Xenopus laevis that can recapitulate mitotic spindle assembly and anaphase chromosome segregation in vitro. The key role of chromatin in this system is illustrated by the ability of plasmid DNA-coated beads to induce the formation of bipolar spindles in the absence of centrosomes and kinetochores. However, anaphase chromosome segregation requires kinetochore function, which can be studied using sperm chromosomes. Our specific aims focus on elucidating fundamental aspects of chromosome function and microtubule organization during mitosis. The general strategies take advantage of the open nature of extracts, which allows specific inactivation of individual components, biochemical analysis of protein activities and interactions, and time-lapse video microscopy to study chromosome and microtubule morphogenesis at high resolution. Our aims are: (1) To elucidate the role of higher order chromatin architecture in chromosome functions by interfering with the major condensation machinery, the condensin complex. (2) To study the roles of the mitotic chromosome-associated kinases Plxl and Aurora B in regulating spindle assembly and function, and identify their substrates. (3) To characterize the centromeric DNA and protein components of the Xenopus kinetochore, and elucidate their functions and assembly mechanisms.

ABSTRACT A fundamental problem in cell and organism biology is to understand how intracellular structures are properly scaled to carry out their essential functions. I propose to explore the phenomenon of organelle size, documenting the changes that occur during development and tumorigenesis, and investigating the underlying molecular mechanisms. My laboratory will undertake a systematic analysis of cell and organelle scaling during embryonic development, evaluating nuclei, spindles and other compartments in Xenopus laevis, as the ~1 millimeter diameter egg rapidly cleaves to form smaller blastomeres, which by the 15th division are reduced to 40 microns across. Which structures have constant dimensions, and which change their size as cells become smaller? Other model organisms and cancer cells will be compared to generate a survey of organelle scaling in normal and abnormal cell growth states. Cytoplasmic egg and embryo extracts of X. laevis and the related, smaller frog X. tropicalis will be used to monitor nuclear, spindle and cellular compartment scaling in vitro. This approach is prompted by our observation that meiotic extracts prepared from X. tropicalis eggs generate spindles that are ~30% shorter than those in X. laevis reactions using the same chromosome source, and mixing experiments have revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors. We will determine which organelles in addition to the spindle are scaled in X. laevis and X. tropicalis extracts, and use activity-based assays to identify the factors responsible for the observed differences. Candidate factors will be tested for their roles in organelle scaling during development and cancer progression, and computational approaches applied to model our observations. These studies will provide novel insight into how cell/organelle scaling contributes to intracellular morphogenesis and cell division, processes essential for viability and development, and defective in human diseases including cancer.

DESCRIPTION (provided by applicant): Of central importance to the process of cell division is the accurate transmission of a complete set of chromosomes to each daughter cell, which in eukaryotes is achieved through the function of the mitotic spindle. The goal of this research proposal is to elucidate the role of the highly conserved GTPase Ran in cell division. The current model suggests that RanGTP functions as a spatial marker that signals the position of the genome in eukaryotic cells. During mitosis, RanGTP is thought to promote spindle assembly by stimulating microtubule polymerization and organization in the vicinity of chromosomes. This "local effect" results from the chromosomal localization of RCC1, the guanine nucleotide exchange factor that generates RanGTP, which binds to transport factors causing them to release cargoes required for spindle assembly. However, the nature of the cargoes, their mitotic function and the molecular mechanisms underlying the RCC1-Ran-microtubule signaling cascade are still poorly understood. A major experimental approach described in this proposal takes advantage of complex cellular extracts prepared from eggs of the African frog Xenopus laevis that can be studied using biochemical and functional assays. This provides an excellent model system to characterize the components and function of mitotic cargoes, to characterize mitotic gradients, and to develop novel reconstitution assays. Our aims are: (1) To elucidate the function of a Ran-regulated cargo, Rae1 that exists in a complex requiring RNA for its activity, and investigate spindle-associated RNAs. (2) To use fluorescent sensors to visualize and characterize RCC1-induced gradients throughout the cell cycle in extracts and in a variety of cell types. (3) To reconstitute RanGTP gradients and spindle assembly using RCC1-coated beads. (4) To use a newly developed small molecule inhibitor of the Ran pathway to further dissect the functions of RanGTP during cell division. In all eukaryotes, assembly and function of the spindle apparatus is essential to accurately distribute the genetic information during cell division, and errors in this process are associated with birth defects and cancer. The identification and characterization of factors in the mitotic Ran pathway is not only fundamental to our understanding of mitotic and meiotic spindle assembly, but may also provide important new insight into our understanding of human diseases.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Membrane-bound organelles show a characteristic spatial organization and their regulated positioning and movement are key to fundamental processes like mitosis and cell polarization. Disruption of polarity and abnormal cellular organization are linked to the development and progression of cancer. Linker proteins between cytoskeletal elements and organelles are critical for determining cellular organization. We are using a Xenopus egg extract-based proteomics approach to identify novel potential linkers.

DESCRIPTION (provided by applicant): Investigating Mechanisms of Intracellular Scaling Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, placing variable metabolic and functional demands on organelles and internal structures. A fundamental question is how essential subcellular components such as the nucleus, mitotic spindle and chromosomes are regulated to accommodate cell size differences. Xenopus frogs offer two physiological contexts in which we can investigate this question. First, we can compare Xenopus laevis to the smaller, related species Xenopus tropicalis, which lays smaller eggs and has proportionally smaller cells throughout development. Second, we can compare different stages of Xenopus laevis embryogenesis, as the ~1 millimeter diameter egg rapidly cleaves to form smaller blastomeres, which by the 15th division are reduced to 40 microns across. A unique aspect of our approach is to prepare cytoplasmic extracts from eggs and embryos that recapitulate organelle scaling in vitro, which we can use to identify molecular differences that underlie size changes. Our first specific aim focuses on the mitotic spindle, and we take advantage of computer simulations to identify parameters of microtubule dynamics and organization that could contribute to spindle size and morphology changes between species and during development. This aim also develops novel methods to examine extrinsic scaling mechanisms by physically confining spindle assembly reactions inside different sized droplets, which will reveal whether there are size thresholds that scale the spindle externally or alter assembly pathways. Aim 2 investigates how the size of mitotic chromosomes is altered during development to coordinate with spindle length so that complete segregation occurs. In Aim 3, we begin addressing the importance of organelle scaling by examining the consequences of altering nuclear size during development in Xenopus laevis. These experiments will provide insight into how scaling occurs and contributes to intracellular morphogenesis and cell division, processes essential for viability and development, and defective in human diseases including cancer.

DESCRIPTION (provided by applicant): Of central importance to the intracellular organization of all eukaryotes is the accurate transport of macromolecules between the nucleus and the cytoplasm, which is achieved through the function of the nuclear pore complex (NPC). The NPC is one of the largest macromolecular assemblies in the cell perforating the double membrane of the nuclear envelope. It is not known how NPCs form and insert into the nuclear membrane, and overall, the structure of the NPC remains poorly understood. NPCs function in the bidirectional transport of a very large set of diverse cargos. This includes the nuclear export of messenger RNAs, which is essential for the expression of every eukaryotic gene. Despite this fundamental significance, the pathway by which messenger RNAs directionally translocate through the NPC is ill defined. In addition to its role in nucleocytoplasmic transport, the NPC also functions in the regulation of multiple nuclear processes including the regulation of gene expression and the three-dimensional organization of the genome. The objectives of this research proposal are to elucidate the structure and assembly mechanism of the NPC and to characterize the roles of the NPC in messenger RNA export and in the organization of chromatin. Our Specific Aims are: (1) To understand how mRNAs are directionally transported across the NPC. (2) To characterize the structure and biogenesis of NPCs. (3) To elucidate the function of the NPC in genome organization. We employ a combination of innovative biochemical, genetic and state-of-the-art single molecule imaging approaches in the single cellular eukaryote Saccharomyces cerevisiae to address these three specific aims. Yeast provides an outstanding model system to characterize the components and function of the NPC, to study NPC transport events in living cells, and to develop novel reconstitution assays. The NPC is a highly conserved structure, and mechanistic insights obtained from our studies will be directly relevant to all eukaryotes, including humans. Because NPC components are mutated in many diseases (e.g. cancer, heart diseases, or developmental disorders) and NPC function is disrupted by many viruses during infection to promote viral replication, our studies are also directly relevant for our understanding of human disease.

? DESCRIPTION (provided by applicant): Research in my laboratory is supported by two highly productive R01s and has 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 set 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 varies 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 basis and significance of variation in astral microtubule morphology at spindle poles. 2) What activities are sufficient to establish the mechanochemical core of the spindle? Whereas the functions of many individual spindle factors have been studied extensively, reconstituting the spindle from purified components remains a holy grail as the key to a complete understanding of the process. We will extend our bead-based spindle assembly system to define the chromatin-associated activities sufficient for spindle self-organization. 3) What is the role of RNA in kinetochore assembly? Transcription of centromeric sequences appears to be a conserved mechanism required for kinetochore formation, but the fate and mitotic function of nascent transcripts is unclear. We will examine centromeric transcription and RNA processing during mitotic progression using a novel in vitro assay and elucidate its role in spindle assembly. Together, these projects elucidate mitotic mechanisms and advance the field toward a systems-level understanding of the spindle. 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 components of the cells. 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 contrl 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? The determinants of mitotic chromosome architecture are poorly understood, and a major challenge in addressing this question is to establish live chromosome labeling methods. We will utilize our new CRISPR-based imaging technique to test the role of candidate factors in chromosome scaling during development. 2) Is there a scaling mechanism that senses the cell surface area-to-volume ratio? Accumulating evidence suggests that cells sense surface area-to-volume as a direct readout for size, and that this information is used to scale subcellular structures. We hypothesize that importin ?, an abundant regulator of spindle and nuclear size that also associates with the plasma membrane and is depleted from the cytoplasm of small cells relative to large cells, acts as a cell size sensor. We will use our size-tunable microfluidi droplet system to test this hypothesis. 3) How is size regulated at the cellular and organism levels? The relative contribution of maternal cytoplasmic factors versus genome content and expression to cell and organism size is unclear. The close phylogenetic relationship between the two Xenopus species used in our lab enables us to generate hybrid frogs of intermediate size and evaluate the role of genome size and content on size relationships. Together, our projects utilizing in vitro and in vivo approaches are identifying cellular and molecular mechanisms underlying biological size control and scaling. 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, biophysical assays, proteomics, RNA sequencing, microfluidics and computational modeling to create new and innovative approaches. 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.

Abstract Xenopus is a widely-used model organism for basic biomedical discovery. This application requests funds to support the 17th International Xenopus Conference. Held every two years since 1984, this biennial meeting?s prime objective is to provide a forum for information exchange and interaction amongst researchers using Xenopus as a model organism for biomedical research. The last iteration of this meeting (in the United States) was attended by over 350 researchers from all over the world. In addition to serving as a platform for information exchange, this meeting will also provide 1) a venue for introducing younger scientists to more established members of the field, 2) an opportunity for the Xenopus community to learn about the latest technologies and to coordinate its infrastructure, and 3) an opportunity to introduce this powerful model organism to students from under-represented groups. This meeting will have a significant impact because Xenopus is widely used in developmental biology, cell biology, molecular biology, and neurobiology, and many of the most exciting discoveries now are coming from the interfaces between these disciplines. By bringing together researchers with divergent research interested, but a common model system, the meeting should foster outstanding interdisciplinary thinking. Modern biology demands that we be well-informed, broad-based, organized, and collaborative in our approaches, and the International Xenopus Conference will facilitate exactly that style of science.