Martin W. Hetzer - US grants

DESCRIPTION (provided by applicant): Reformation of the nuclear envelope (NE) around the segregated chromosomes is a key event at the end of mitosis. Defects in this key process may result in alteration of gene expression patterns and genomic instability. We have generated new information about the two major regulators of nuclear assembly, the GTPase Ran and the AAA-ATPase p97. Our specific aims are: 1. To understand how Importin b, a major RanGTP binding protein, regulates NE fusion and to identify the molecular targets with which it interacts. We will use a 2-color fusion assay and transmission electron microscopy (TEM) to characterize how Importin b inhibits the fusion of chromatin bound vesicles. The dynamics of NE tubule formation and reorganization will be visualized by live imaging on chromatin-coated glass slides. We will use biochemical fractionation methods and affinity chromatography to identify the binding partner(s) of Importin b. 2. To characterize NE membrane sealing and to identify the Ufd1/Np14 regulated NE fusion machinery. To understand p97/Ufd1/Np14-dependent formation of a closed NE, we will use TEM, a novel nuclear exclusion assay, and real time microscopy. To identify additional proteins that interact with Ufd1/Np14 and participate in NE sealing, we will use a recombinant form of Ufd1/Np14 complex as matrix for affinity chromatography. 3. To characterize a second GTPgS-sensitive step in NE formation. We will use the 2-color fusion assay and TEM to characterize this membrane fusion event. We will analyze a membrane-associated GTPase activity on chromatin-bound vesicles. To identify the nature of the additional GTPase(s) we will perform an RNAi screen in C. elegans to test if GTPases known to mediate intracellular membrane fusion (e.g. Rab GTPases) are involved in NE formation. As an alternative approach we will use photo-affinity methods, overlay assays and proteomic approaches to identify the second GTPase.

DESCRIPTION (provided by applicant): Nuclear pore complexes (NPCs) form the only sites for entry and exit from the nucleus. This includes the diffusion of small molecules and the selective, active transport of large proteins and RNA. Proper NPC biogenesis is critical for cell division, differentiation, and responses to changes in metabolic activities. Understanding NPC assembly at the molecular level will be key for designing strategies to inhibit cell growth and gene expression, for example in oncogenesis. However, the molecular pathway for de novo insertion of NPCs into the intact nuclear envelope (NE) is largely undefined. The most outstanding question in the field involves delineating how nuclear pore formation is executed and regulated. The pathway for coordinating interactions between at least 30 different NPC proteins (Nups) and multiple pore membrane proteins (Poms) is unknown. This project aims to reveal the molecular biogenesis mechanism for the NPC transport apparatus. Our specific aims will each utilize aggressive yeast S. cerevisiae genetics/biochemistry/cell biology-based approaches directly coupled with state-of-the-art microscopy and biochemistry in metazoan cells and the Xenopus egg extract in vitro assembly system. This innovative merger of strengths in multiple model systems is only possible through the full-fledged collaborative efforts of the two PIs. We speculate that de novo assembly is a step-wise process. Our first specific aim will address early assembly events at the NE and chromatin. Using a novel set of yeast NPC assembly mutants and in vitro Xenopus assays, we will identify the essential Nups and assembly factors that are targeted to chromatin, outer and/or inner nuclear membranes. The targeting mechanism and role in assembly will be directly tested. The second aim will focus on the mechanism for fusion of the outer and inner nuclear membranes, i.e. formation of a pore across the NE, and builds on our recent discovery of ER/NE proteins required for NPC assembly and our novel pore fusion assay in Xenopus extracts. We hypothesize that the highly curved pore membranes are formed by the action of Poms, stabilized by transient association of the reticulons, and maintained by the recruitment of a membrane coat formed by the Nup107-160/Nup84 complex. In the third aim, we will analyze the coordination between sequential steps in the biogenesis process. Assembly intermediates will be examined by scanning electron microscopy and purified from yeast mutant cells arrested at distinct steps. We will also pinpoint the order of metazoan Nup recruitment after membrane hole formation by fluorescence imaging of NPC assembly with single pore resolution in real time. Together, these studies will define the membrane fusion machinery and the sequence of self-assembly steps for NPC biogenesis in intact NEs. PUBLIC HEALTH RELEVANCE: Transport of proteins and RNA between the nucleus and cytoplasm is essential for all aspects of normal cell function, and requires large protein machines (nuclear pore complexes) to allow cargo movement. Trafficking is perturbed in some disease states, such as cancer and viral infections, and there are also genetically heritable diseases that are linked to changes in the genes encoding transport factors and nuclear pore complexes. Knowledge of how nuclear pore complexes are assembled and function will be important for finding therapeutic strategies to regulate nuclear transport in disease states and moderate viral proliferation/pathogenesis.

DESCRIPTION (provided by applicant): Eukaryotic gene expression is regulated at multiple levels in the cell nucleus, from histone modification and chromatin compaction to the synthesis, processing and export of mRNA. The precise execution of transcriptional programs during cell differentiation and development relies on faithful reproduction of a specific chromatin state through mitosis. Failure to maintain these epigenetic programs through multiple cell divisions commonly leads to pathological conditions such as cancer. Thus, understanding how chromatin organization is established and propagated will enhance the understanding of how disease-causing errors in gene expression can occur and be prevented. The central hypothesis guiding this proposal is that the nuclear pore component Nup98 plays an essential role in transcriptional activation of developmentally regulated genes. Biochemical, genetic, genomic and cell biological methods will be used to investigate the potential interaction between Nup98 and the members of the trithorax protein complex, a well-known chromatin regulator and mediator of developmental active gene memory. First, the molecular composition of the intranuclear Nup98 complex will be established, its recruitment mechanism to chromatin identified and thus provide key functional insights into the link between Nup98 and epigenetic memory. Second, the molecular function of Nup98 at genomic target sites will be investigated. For instance, the consequences of Nup98 knockdown and over-expression will be analyzed by chromatin immune- precipitation to investigate which binding partners, histone modifications and RNA polymerase II phosphorylation events depend on Nup98. In addition, a potential effect of Nup98 on mRNA processing and export will be determined by RNA fluorescence in situ hybridization and analysis of recruitment of mRNA processing and nuclear export factors. Furthermore, Nup98 recruitment in developing tissues that activate targets genes as well as genetic effects of Nup98 on established Trx mutant phenotypes will be analyzed. Finally, a potential role of Nup98 in long-range gene interactions and organization of active chromatin domains will be investigated with genome-wide techniques. These studies will provide important information about the role of Nup98 in transcriptional initiation and establishment of active chromatin. Third, the interaction between Nup98 and mammalian MLL and Wdr5 may prove to be important in understanding Acute Myelogenous Leukemia (AML) and development-associated roles of these proteins. Proposed is a comprehensive genome-wide analysis of Nup98 binding in human cell lines and in mouse hematopoietic cell lines. The latter have been transformed by a known leukemia-inducing gene fusion Nup98- NSD1 and will be compared relative to normal hematopoietic cells. These approaches have the potential to reveal the gene regulatory role of mammalian Nup98 and provide insight into its leukemia-causing potential.

? DESCRIPTION (provided by applicant): As proteins age they are more likely to acquire molecular damage. To combat the functional decline of the proteome, most cellular proteins are rapidly turned over. In this way, potentially impaired polypeptides are constantly replaced with newly translated copies. Proteins with slower rates of turnover are therefore at greater risk of suffering a deleterious modification. Recently, long-lived proteins (LLPs) were discovered in post-mitotic cells of the rat central nervous system. Strikingly, these LLPs are associated with chromatin, the nuclear envelope, and the plasma membrane, all of which are cellular compartments that coordinate a myriad of regulatory functions. LLPs are therefore constantly exposed to potentially harmful metabolites. It is unknown how the functional integrity of these proteins is maintained over such a long time period, and whether the longevity of these proteins plays a specific role in long-lived cells. Thus, proposed experiments will test the specific hypothesis that LLPs regulate biological processes over extremely long time frames and that their functional decline drives cellular and organismal aging. This research proposal is designed to study the biology of LLPs, primarily focusing on nucleoporins and histones, and how they relate to post-mitotic tissue function. First, quantitative mass spectrometry and multi- isotope imaging mass spectrometry will be used to measure cell, organelle, and protein turnover rates in post-mitotic tissues in rats. Second, biochemical, cell biological, and genome-wide approaches will be used to determine the molecular properties of long-lived nucleoporins and histones. Efforts will be directed toward identifying and characterizing protein-repair pathways and protective post-translational modifications that are responsible for LLP longevity. In addition, it will be determined whether long-lived nucleoporins and histones serve as molecular timers to regulate nuclear pore function and gene expression, respectively. Finally, LLPs will be depleted in post-mitotic cells using a novel protein extraction and degradation system to determine how LLP impairment affects cellular function and aging. These analyses will focus on aberrant intracellular trafficking and gene regulation. Proposed studies promise to establish novel links between protein longevity and aging, and provide new molecular targets for understanding and potentially treating age-associated degenerative disorders.

Project Summary/Abstract The genome is housed within the nucleus, where transcription is regulated by architectural features that direct DNA packaging and epigenetic modifications. How genome organization and modification are controlled to allow proper gene expression and normal organismal development is an ongoing area of inquiry. Recent discoveries highlight the important role of the nuclear pore complex (NPC), which regulates transport of biomolecules between the cytosol and nucleus, in transcription control. The NPC protein Nup98 has the ability to move on and off the NPC structure to associate with specific sites in the genome inside the nucleus. Research has shown that this interaction promotes gene activation in developing tissues and that its disruption causes leukemia, hinting at the functional importance of Nup98-directed gene activation for normal development. A recently identified variant of another nucleoporin protein termed sPom121 (?short Pom121), which has completely lost the ability to associate with the NPC but interacts with Nup98 and other nucleoporins within the nucleus to regulate genes. sPom121 recruits additional nucleoporin proteins, and likely other unknown proteins, to structures within the nucleoplasm that we refer to as chromatin-associated nucleoporin complexes, or CNCs. Building on recent findings on the overlapping functions of Nup98 and sPom121 to dissect the roles of CNCs in gene regulation in mammals. Specifically, it will be determined what chromatin modifying enzymes Nup98 associates with to mediate gene activation, and determine how those interactions and gene targets are de-regulated in leukemia. sPom121 specifically marks intranuclear sites of genome regulation (CNCs) but does not associate with the NPC to identify nucleoporin-associated proteins that function in gene regulation. Studies will provide new information about Nup98 as a transcription regulator in hematopoietic cells and have the potential to establish CNCs as a novel feature of gene regulation and genome organization.

Abstract In type 1 diabetes (T1D) insulin producing ?-cells of the pancreatic islets of Langerhans are lost and secretion of the glucose-raising hormone glucagon from ?-cells is dysregulated, contributing to hyperglycemia and impaired counter-regulation. Recent studies demonstrate appreciable heterogeneity within the ?-cell and ?-cell populations both in vitro and in situ. Emerging single-cell approaches have established ?-cell sub-groups that differ in their Ca2+ signaling and transcriptomic profiles and may represent ?pacemaker? cells or replication niches. Evidence is also accumulating, including preliminary data in the present application, to suggest that the pancreatic ?-cells are both heterogeneous and malleable ? the altered function of human ?-cells in type 1 diabetes (T1D) is consistent with a shift towards a ?-cell phenotype. This could contribute to the dysregulation of glucagon secretion. Others have shown the persistence of ?resistant? or surviving ?-cells in T1D, both within islets and throughout the pancreas, although the nature and function of these remain unclear. Understanding the variability and malleability of human islet cell function, and the relationship of this to components of the islet microenvironment such as vasculature or nerves, is important since this may provide avenues for correction of glucagon secretory dysfunction, protection of ?-cells, or the regeneration of ?-cell mass. The present proposal will combine in-depth transcriptomic, proteomic, functional phenotyping on a cell-by-cell basis to understand the underlying regulation of islet cell functional heterogeneity and will map these in situ in relation to other islet cells types and components of the local environment. The Aims are to (1) examine human islet cell functional phenotypes, and the linkage of phenotypic variability to single-cell gene expression; (2) map the markers that define islet cell heterogeneity and sub-populations within the 3D islet microenvironment in health and T1D using approaches that span a range of resolutions and scales; and (3) link islet cell function, single-cell gene expression, single-cell metabolism, and single-cell proteomics in situ to understand islet cell pathophysiology. Integration of an in-house human islet isolation program, multi-dimensional cell imaging expertise, and single-cell dual functional and transcriptomic profiling using electrophysiology (Patch-Seq) on isolated cells and in situ using live human pancreas slices will help accomplish the goal of obtaining a high resolution understanding of islet cells within the local tissue architecture in health and diabetes.

Advanced Biophotonics Core Shared Resource - Project Summary/Abstract The Advanced Biophotonics Core (ABC) provides imaging and analysis instrumentation coupled with technical and collaborative support staff for advanced light and electron microscopy of biological systems. Cancer Center members use the facility for high-throughput imaging assays, high-resolution imaging of live cell and tissue dynamics, super-resolution microscopy, large 3D volume imaging of tissues, electron microscopy analysis of subcellular morphology and protein distribution, and automated computational image processing, visualization, and analysis. The ABC Core is also actively pursuing and developing new cutting-edge imaging and analysis methodologies to better serve the needs of Cancer Center researchers, such as cryo-correlative light and electron microscopy, light-sheet imaging of cleared and expanded tissues, and machine-learning based processing, segmentation, and analysis of light and electron microscope images. The ABC Core is committed to providing Cancer Center members: 1) access to light and electron microscopes, specialized sample preparation reagents and technologies, and computational hardware and software for analysis and visualization, 2) free one-on-one training on all microscopes, as well as image processing and analysis software, 3) consulting and collaborative support for experimental design and implementation of imaging and analysis experiments, 4) sample preparation for electron microscopy, tissue clearing, and expansion microscopy, 5) workshops and demos with advanced microscope and software technologies, 6) weekly open- door imaging boot camp on advanced imaging and image processing techniques, and 7) a monthly Biophotonics scientific seminar series followed by town-hall style discussions with ABC Core staff and users.

PROJECT SUMMARY ? Heterogeneity of Aging Core In line with the pioneering work of Nathan Shock, it is clear that aged tissues accumulate cellular heterogeneity or mosaicism. This heterogeneity is likely a cause of aging due to impairments in both intercellular interactions and the coordination of tissue function. The Heterogeneity of Aging Core (Heterogeneity Core) within the San Diego Nathan Shock Center of Excellence in Basic Biology of Aging (SD-NSC) will enable investigators to probe the heterogeneity of aging over a broad range of scales (from molecules to organelles to single cells and tissues) by providing access to a diverse suite of state-of-the-art instrumentation and analytical technologies, as well as experienced Core staff. The Core will provide specific resources for studying key processes implicated in aging and disease at high resolution, including single-cell next-generation sequencing platforms, high-resolution imaging systems, and mass spectrometry approaches to measure proteomic and metabolomics signatures of aging in cells and tissues, with an emphasis on cell-cell heterogeneity and heterogeneity across tissues. These technologies are rapidly evolving and will continue to do so over the coming years. Utilizing established Core resources with proven track records for staying current with evolving technologies is the most effective way to ensure new innovations in analytical technologies are available to the greatest breadth of aging researchers. The Heterogeneity Core provides specific support for researchers in the aging field by: 1) providing access to specific scientific services, advice, and expertise, 2) developing and disseminating novel methods for correlative data acquisitions, and 3) running on-site and virtual training sessions. The Heterogeneity Core is a critical component in the pipeline of research resources that our SD-NSC will create. The value of this Core is bolstered by the generation of age-equivalent induced cell types and organoids by the Human Cell Models of Aging Core, and novel machine-learning capabilities in the Integrative Models of Aging Core. Together we will provide researchers in the basic biology of aging field with the resources necessary to make key discoveries into the mechanisms by which we age. The Heterogeneity Core will enable studies into the cell-cell and tissue heterogeneity of aging and, ultimately, the contributions and mechanisms by which heterogeneity causes the degeneration and dysfunction that characterizes aging.