Elizabeth Sztul - US grants

This is a Presidential Young Investigator award.

(Adapted from applicant's abstract): The goal of the research proposed is to further understanding of the mechanisms of membrane traffic. The work will concentrate on the TAP (transcytosis associated protein complex, a recently identified complex of proteins required for fusion of specific membrane partners, but not required for all fusion events. The complex is specifically associated with transcytotic carrier vesicles (TCVs) and is necessary for their fusion with the apical plasma membrane (PM). The data suggests that the complex is involved in membrane recognition events. Currently, only small GTP-binding proteins are known to participate in membrane sorting, and consequently, defining other components that influence this process is of great significance for the elucidation of the mechanisms mediating membrane specificity. Dr. Sztul plans to characterize the subunit structure of the TAP complex and to define the function of distinct subunits. She will analyze the requirements for assembly of functional TAP complex and study the mechanism of its specific attachment to the TCV membrane. She will also analyze the morphology of the complex and correlate distinct functional domains with morphologically defined structures. The function of the complex will be assessed in a modified assay in which targeting and binding can be separated from later stages of fusion. Using the system, interactions of the TAP complex with other molecules known to influence vesicular traffic will be examined. Most of the functional analyses will utilize biochemical and morphological approaches while the characterization of distinct components will use molecular and genetic procedures.

DESCRIPTION (provided by applicant) The goals of this FIRCA application address fundamental cellular processes by exploring molecular mechanisms regulating cargo progression through the secretory pathway. The proposal builds on research interests of collaborating investigators at the University of Alabama at Birmingham and the University of Cordoba, Argentina. The proposal is an extension of a NIH grant RO1 GM62696-06 entitled "Role of p115 in Membrane Traffic". The parent grant addresses basic mechanisms of ER-Golgi traffic by analyzing the role of the transport factor p115. P115 functions in ER-Golgi traffic by participating in protein-proteins interaction with distinct proteins localized in different compartments along the pathway. The experimental goals of the parent grant are to explore p115 function at the Golgi by analyzing tethering interactions of p115 with two Golgi proteins, GM130 and giantin. We now wish to expand the scope of our inquiry by exploring p115-mediated events in pre-Golgi stages of traffic. This FIRCA proposal is based on our recent findings that p115 may act at the level of ER exit sites to generate pre-Golgi transport intermediates. We now plan to test the hypothesis that cargo and p115 are coordinately recruited at ER exit sites and that p115 association is required for the formation of vesicular tubular clusters (VTCs) that transport cargo and p115 to the Golgi. A series of experimental approaches including biochemical, molecular, cellular and imaging technologies will be used to address the following Specific Aims: 1) define how p115 associates with ER exit sites; 2) define how p115 traffics to the Golgi; and 3) define how p115 functions in pre-Golgi transport. Completion of proposed studies will extend our understanding of how p115 is recruited to membranes and moves along the linear secretory pathway, and will provide first ever inquiry into the molecular events by which p115 mediates the formation of transport competent VTCs.

The long term objective of this research is to understand the molecular mechanisms that control cell-cell signaling in developing and adult tissues. Hedgehog (Hh) signaling proteins control cell fates and proliferation during animal development by regulating the specific gene expression. In vertebrates, Hh proteins pattern diverse tissues such as the developing limb, spinal column, and brain. The membrane protein Patched (Ptc) opposes Hh to inactivate specific gene expression. In Drosophila, ptc mutations cause misexpression of Hh target genes and result in abnormal development and cell proliferation. Mutations in a human homolog of ptc, PTCH1 lead to the very common skin tumor, basal cell carcinoma, and to the brain tumor, medulloblastoma. PTCH1 is also mutated in the basal cell nevus syndrome, an inherited disorder characterized by many developmental defects and tumors. The molecular mechanisms of Hh signal reception and transduction are largely unknown. Central to understanding the role of Hh signaling in development and disease is learning how Ptc functions and identifying proteins with which it interacts. Ptc is proposed to bind Hh proteins and to associate with and regulate, Smoothened, (Smo), a membrane protein required for Hh signaling. Ptc also sequesters Hh to limit its range of action. How and where Ptc mediates these important regulatory processes is not known. Ptc may function in vesicle movement as suggested by its sequence similarity to NPC1, a membrane protein implicated in the intracellular trafficking of cholesterol. The proposed studies will identify the critical functional domains of Ptc and characterize the cellular localization of Ptc and its interacting proteins following ligand binding. Using Drosophila, new components of Hh signaling will be identified by a genetic screen involving a specific ptc phenotype.

Protein aggregation and the formation of inclusion bodies are two hallmarks of the cytopathology in neurodegenerative diseases. In diseases such as Parkinson's and Alzheimer's, the deposits are cytoplasmic, while in Huntington's disease and the ataxias, the deposits form predominantly within the nuclei. The inclusions recruit key cellular components, such as chaperones and proteasomes, nuclear matrix components, and specific transcriptional regulators. The aim of this proposal is to explore the hypothesis that the pathology associated with aggregation is related to the sequestration of cellular components involved in critical cellular processes such as protein folding and degradation, and gene expression. To this end, we plan to use a novel model system, based on an artificial protein we call GFP170*. GFP170* lacks polyQ stretches, yet induces cellular responses analogous to those caused by the deposition of polyQ aggregates. Like polyQ proteins, GFP170* forms cytoplasmic and nuclear aggregates, recruits cytoplasmic and nuclear components, alters transcriptional regulation, and kills cells. We propose twin lines of investigation. First, we will examine the physical nature of the sequestered cellular components. GFP-based real-time imaging of live cells will be used to explore the mobility of GFP170* and of cellular components recruited to the aggregates. Such data will inform on the physical stability of the aggregates, and will influence possible models of cytopathology. Second, we will follow the functional consequences of nuclear aggregation on gene expression. In vivo read-out systems and genomic approaches will be used to explore the modulation of transcriptional regulators by aggregating GFP170*. Completion of the proposed studies will establish a firm basis for defining the role of nuclear aggregates in cytopathology.

Intellectual Merit
Membrane traffic mediates secretion and the delivery of proteins and lipids to intracellular organelles and the plasma membrane. The mechanisms regulating this process are largely conserved throughout all eukaryotes and what is learned in one species can reveal events occurring in other species (including humans). In unicellular organisms, membrane traffic sustains cell growth and interactions with the environment. In multicellular organisms, membrane traffic is required for deposition of the extracellular matrix and cell adhesion, the release and uptake of growth factors and nutrients, and the insertion of environment sensing receptor and signal transducers, i.e., all the processes that integrate the developmental and physiological responses of tissues and organs. This project will focus on key regulators of membrane traffic, the ADP-ribosylation factor (ARF) GTPases. Specifically, the mechanisms and molecules that facilitate ARF activation will be identified and characterized. This project studies the molecular mechanisms that regulate membrane trafficking to understand a process essential for cellular and organismal life.

Broader Impacts
The broader impacts of this project are: (1) Training and mentoring of postdoctoral fellows, graduate students and undergraduates in cutting-edge molecular, imaging, biochemical and genetic technologies to probe key questions in cellular physiology. (2) Promoting scientific equity by actively recruiting and mentoring under-represented groups to enter scientific careers. (3) Promoting interdisciplinary synergy by interfacing with structural and biophysical scientists to provide additional cross-field fertilization of technologies and ideas. (4) Promoting global scientific infrastructure by developing and disseminating plasmids, antibodies, recombinant proteins and experimental approaches to labs worldwide. This project has additional societal benefits because it will provide the next generation of academic researchers and teachers, improve the competitiveness of US scientific enterprise and prepare highly trained individuals for non-academic jobs in science and education.

All mammalian cells transport proteins via the secretory pathway to sustain indispensable cell activities such as growth, division and differentiation. Transport is mediated by small vesicles that bud from one compartment, move towards the next compartment, and then fuse in order to deliver their cargo. Vesicular traffic involves a hierarchy of molecular subprocesses linked to one another in time and space. The goal of this project is to model vesicle traffic with the long-term goal of capturing the underlying biophysical principles of this essential cellular process. In addition, this project will support interdisciplinary synergy by providing cross-discipline training opportunities and developing workshops and courses to effectively bridge the gap between biological and physical fields. Importantly, a focus is to advance scientific equity by actively recruiting and mentoring under-represented groups and by participating in programs aimed at increasing representation of minorities and promoting and developing community outreach programs to increase science awareness and literacy.

This project focuses on the formation of vesicles that transport proteins at the ER-Golgi interface. Such vesicles form through the recruitment of the coatomer complex (also called COPI complex) to the nascent bud. The recruitment is mediated by a small GTPase of the ARF (ADP-ribosylation factor) family. Like all GTPases, ARF acts as a molecular switch by cycling between an inactive state when it is bound to a GDP, and an activated state when it is bound to GTP. Only the active form of ARF can recruit coatomer and initiate vesicle formation. The activation of ARF is mediated by a nucleotide exchange factor (GEF) called GBF1. Hence, GBF1 activates ARF, which then recruits coatomer to make COPI vesicle. In addition to coat recruitment, ARF also regulates the sorting of cargo proteins into the nascent bud, and this process requires that ARF continuously cycle from between the GDP and the GTP-bound state. To facilitate the GTP to GDP conversion, a GTPase activating protein (GAP) called ArfGAP1 is required. Hence, the process of vesicle formation requires, at the very minimum, 4 key components: GBF1, ARF, coatomer and ArfGAP1. While we have descriptive knowledge of each molecular subprocess mediated by each component, we lack the knowledge of how these processes are connected in time and space to result in vesicle formation. We also lack even basic understanding of the governing biophysical principles. Thus, the aim of this project is to elucidate this enigma. We propose four Specific Aims: (1) Determine diffusion type and parameters of the 4 key components in cytosol; (2) Determine membrane association processes for the 4 key components; (3) Determine volume and surface densities and define binding constants and reaction rates for the 4 key components; and (4) Develop mathematical models describing the formation of the ternary coating complex. We will measure the dynamic properties of the 4 key components by fluorescence recovery after photobleaching (FRAP) under various experimentally induced conditions to mathematically describe their behavior. The resulting models will be refined through targeted perturbations of the system. Our models will provide the framework for future integration of other participants such as cargo proteins, lipids and a multitude of known regulatory factors that together ensure specificity to vesicular traffic. We stress that the machineries and the mechanisms we will model are highly conserved, and that analogous processes mediate coating of other types of vesicles for transport between different cellular compartments. Thus, our models will be generally applicable and will form an obligatory foundation for understanding the biophysical processes that govern vesicle traffic. A systems understanding of vesicle coating based on the laws of physics is crucial for future manipulation in vivo and for building synthetic organelles and cells.
The funding for this project comes from both the Division of Molecular and Cell Biology and the Division of Mathematical Sciences

Project Summary/Abstract Bi-directional vesicular transport is vital in all eukaryotic cells to deliver proteins to secretory organelles and the cell surface, and for release from the cell. It is estimated that 30% of the mammalian proteomes must traffic the secretory pathway. GBF1 (Golgi localized Brefeldin A-sensitive Factor1) is a key regulator of retrograde traffic from the Golgi to the ER, and GBF1 activity is required for the establishment and maintenance of the secretory pathway. GBF1 belongs to a family of large Guanine nucleotide Exchange Factors (GEFs) and is an enzyme that facilitates GDP/GTP exchange on the ARF subfamily of small Ras-like GTPases. GBF1-mediated ARF activation is required for the formation of retrograde COPI vesicles, and GBF1 represents an upstream regulator of COPI vesicle formation as it dictates the time and site of vesicle formation by restricting ARF activation. Yet, despite the critical importance of GBF1 in cellular homeostasis, we remain ignorant of how GBF1 itself is regulated in cells. Specifically, we do not know how cells signal to GBF1 to ?notify? it of a cellular need for retrograde traffic and what mechanisms ensure that GBF1 initiates COPI vesicle formation only at the right time and the right place. This proposal aims to illuminate this enigma. We will test the hypothesis that cells have mechanisms to inhibit GBF1 activity to prevent spurious ARF activation, but release such inhibition in a time and site-restricted manner in response to a signaling pathway that indicates retrograde traffic demand. Our goal is to define how cells translate their need for ARF activation and membrane transport into space- and time-restricted GBF1 function. We propose 3 specific aims to identify the processes and signaling pathways that regulate GBF1 function in an essential traffic routing in all cells, retrograde COPI traffic that is vital for the homeostasis of the secretory pathway. In Aim 1, we will identify the mechanisms that selectively target GBF1 to Golgi membranes by identifying the intrinsic targeting information within GBF1 and the membrane components that mark membrane sites for GBF1 recruitment. In Aim 2, we will define the mechanisms that regulate GBF1 catalytic activity at the membrane by assessing the role of phosphatidylinositol phosphates (PIPs) in regulating GBF1 catalytic activity. In Aim 3, we will determine the signaling pathways that coordinate GBF1 function with the need for COPI traffic by defining the role of KDEL-R activation and the PKA and SFK pathways on GBF1 membrane association and catalytic activity. GBF1 is ubiquitously expressed and critically important to cell and organismal health; GBF1 depletion from cultured cells causes death and a mouse or Drosophila knockout is embryonic lethal. GBF1 is also important in pathological contexts since it is essential for migration of glioblastoma cells and for replication of human pathogenic enteroviruses. Our studies will provide critical new knowledge of GBF1 regulation in basic cellular physiology and will inform strategies for the design of therapeutic intervention to control GBF1-mediated events in pathological contexts.

One of the major problems in Cell Biology is that detailed knowledge of specific molecular or cellular events is not integrated across time and space, and investigators lack the understanding of how one event relates to or influences another to result in the totality of cellular responses. Understanding how a specific atomic or molecular reaction results in the overall cellular behavior is extremely difficult by experimentation alone, and computational modeling is required to significantly advance our understanding. The main goals of the workshop funded by this award are to promote the discovery of biophysical laws that govern cellular differentiation and homeostasis by promoting the interdisciplinary collaborations between experimental cell biologists and computational modelers. The workshop aims to provide unique opportunities for cell biologists and computational modelers to work together to develop predictive understanding of the cell. The workshop also provides a unique educational experience for researchers who participate. Organizers will work with professional societies to ensure the recruitment of faculty from underrepresented groups as well as graduate students and postdocs and other early career professionals.

This award will provide funding for a 3rd year extension of the extremely successful "Finding your Inner Modeler (FYIM)" workshops supported by the NSF MCB Award 1649160 during 2017 and 2018. The vision for the 3rd year workshop is to use the 2-day format to continue the goals articulated in the original proposal to: 1) stimulate interest in computational modeling among scientists currently not using this approach; 2) promote the formation of interdisciplinary collaborations between cell biologists and computational modelers; and 3) develop an information and guidance network to provide advice to cell biologists and computational scientists on how to start a collaboration, who to contact, and what resources are available to support collaborations.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The project seeks to develop a community of cell biologists working across disciplines to solve complex biological problems using computational approaches. It builds on the success of the three ?Finding Your Inner Modeler? (FYIM) workshops that took place in the summers of 2017-2019. These events introduced computational modeling to traditional cell biologists and promoted interdisciplinary collaborations in systems biology. This RCN project will serve a broader community by promoting collaborations between cell biologists interested in using computational methods in their research with physicists, chemists, engineers, mathematicians, and computer scientists who wish to apply their computational skills to biological problems. Thus, the project will benefit two distinct groups: cell biologists who wish to apply quantitative and modeling tools to their research, and experts in other STEM disciplines who wish to advance the study of important biological questions using their computational skills. Three annual meetings will serve as incubators for interdisciplinary interactions, facilitating crosspollination and leading to synergistic collaborations that advance systems biology. A virtual venue will be developed that will allow remote online participants to interact directly with participants at the in-person meetings, thereby removing most geographical barriers. Standardizing the schedule, locations, and structure of the annual FYIM meetings is expected to encourage a sense of community; an associated FYIM website will provide this community with an online home and communications hub. A 10-person Steering Committee whose members cover a broad range of scientific specialties and geographic locations will work with the PI and two co-PIs to ensure well-attended, high-energy meetings, and utilization of the website. The Broader Impacts of this work involve the training of a diverse group of participants at all academic levels.

The central premise of this project is that computational modeling is required to extract the principles underlying cell processes and structures from what are increasingly large and complex datasets. Emergent properties simply cannot be understood by examining one variable at a time. Yet, many cell biologists lack the expertise to take advantage of computational approaches, while expert modelers in other fields who are interested in working on biological problems lack the opportunities to do so. The three annual FYIM meetings will provide traditional cell biologists with insights into the utility of computational modeling, guidance in how to get started, and the chance to interact with interested modelers from a broad range of disciplines. Unlike most field-specific meetings, the events will bring together scientists and engineers with diverse backgrounds and expertise, thereby promoting productive interdisciplinary collaborations. The ongoing development of the FYIM website will continue during the project period with the goal of creating an active online meeting place and information nexus for the systems biology community at large; in addition to providing a home for meeting registration and information, the website will serve as a prototype for web-based interactive communities. Such communities may become important hubs of scientific communication and attractive alternatives to distant travel within the next decade. Advertising for the meetings and website will target a diverse group of potential participants with respect to their career stage, type and location of home institution, gender, and ethnicity. The diversity of participants is important to achieving the ultimate aim, which is to promote the development of quantitative and computational cell biology into a widely used and high-impact discipline, on par with other interdisciplinary fields such as bioinformatics and genomics.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

All cells contain organelles that are specialized to perform a specific function. The function of these organelles depends on the correct transport of proteins into and out of each organelle. This transport is mediated by small vesicles that carry various cargos around the cell in a highly specific orchestration of movement. Thus, the formation of vesicles that will specifically deliver select proteins, to a specific organelle, is critically important to the normal function of cells. The project seeks to understand how specific vesicles containing specific proteins form in the cells. The aim is to unite molecular/biochemical experiments with computational/arithmetic modeling to understand vesicular traffic with the long-term goal of capturing the underlying biophysical principles of this essential cellular process. Broader Impact activities include the intrinsic merit of the research itself, e.g., the lack of delivery of enzymes to various organelles causes severe diseases. The project will provide cross-discipline training opportunities and develop workshops and courses to effectively bridge the gap between biological and physical fields. Another goal of the project is to advance scientific equity by actively recruiting and mentoring under-represented groups and by participating in programs aimed at increasing representation of minorities and promoting and developing community outreach programs to increase science awareness and literacy.

Protein transport between the compartments of the secretory and endosomal pathways is essential for activities such as growth, division and differentiation. Transport is mediated by vesicles that select cargo from one compartment, and then deliver the cargo to the next compartment. Cargo selection is mediated by coating lattices that assemble on the cytoplasmic aspect of nascent buds in a process involving a hierarchy of subprocesses systematically linked to one another causally or functionally in time and space. The goal is to identify underlying principles that govern coating and to develop a predictive understanding of the emergent properties of the regulatory networks that facilitate coating. The project will focus on the subprocesses that assemble coating lattices for sorting cargo at the trans-Golgi network (TGN). Lattice formation at the TGN proceeds via a complex mechanism, in which an inner core of adaptors assembles first, followed by an outer layer of clathrin. The project seeks to understand the dynamics and the biophysical parameters regulating the formation of adaptor-clathrin (AC) coating modules composed of the tetrameric AP1 complex or three monomeric Golgi-localized -adaptin ARF-binding (GGA1-3) adaptors and clathrin. The adaptors assemble on the membrane by interacting with activated Arf GTPases and the activation of such Arfs at the TGN is mediated by the BIG1 and the BIG2 guanine nucleotide exchange factors. The project will use CRISPR/Cas9-modified knock-out (KO) cell lines to identify the specific Arfs and BIGs that mediate the recruitment of each AC coating module. Adaptor-clathrin (AC) coating will be modeled by mathematically describing the behavior of key components: BIG1, BIG2, Arf1-3, AP1, GGA1-3 and clathrin. The overall coating process is multi-step, multi-component, and variable. Experiments alone cannot deal with this complexity, and computational modeling is needed to illuminate the spatio-temporal parameters of the process. Furthermore, the complexity of AC vesicle formation requires the development of sophisticated mathematical methods to model the behavior of the system. This project combines diverse expertise and ultimately seeks to decipher the general principles of coating module assembly which will define one of the Rules of Life.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.