Orion D. Weiner, Ph.D. - US grants

DESCRIPTION (provided by applicant): Neutrophils are innate immune cells that use directed migration to hunt and kill bacteria. This directed migration depends on several fundamental signaling capabilities. Neutrophils can migrate up chemotactic gradients spanning several orders of magnitude, requiring signaling adaptation so that cells respond to relative changes rather than steady-state concentrations of ligand. Neutrophils generate a consistent internal polarity that does not depend on the steepness of the external gradient, requiring positive feedback to amplify subtle signaling asymmetries and long-range inhibition so that protrusions can compete with one another to generate a dominant leading edge. Through genetic and pharmacological loss-of-function experiments, we know many of the core components required for chemotaxis. However, there are still fundamental gaps in our understanding of the how these signaling components interact to generate cell polarity and movement. Because the overall process of polarity is highly complex, we have developed tools to isolate and dissect individual steps in the signaling cascade to better understand the overall signaling circuit. In the last gran period, we developed a general approach for quantitative optogenetic control of intracellular signaling in mammalian cells. This system gives us unprecedented spatial and temporal control of a wide range of intracellular signals and will enable us to dissect the logic of signal processing in a manner that has not been possible with conventional tools. Quantitative control of intracellular signals has played a fundamental role in uncovering the logic of action potentials and bacterial chemotaxis, and we envision that our optogenetic tools will be similarly transformative for understanding cell polarity in neutrophils. Our specific aims are to understand the sensory adaptation that accounts for the remarkable dynamic range of chemotaxis (Aim 1) and to dissect the positive feedback loops (Aim 2) and long-range inhibition (Aim 3) that make neutrophil polarity possible.

DESCRIPTION (provided by applicant): Genetically-encodable optical reporters, such as Green Fluorescent Protein, have revolutionized the observation and measurement of cellular states. In principle, it would be equally revolutionary to be able to control and precisely manipulate diverse cellular processes using light. Most light-regulated proteins, however, either require engineering of precisely tethered semi-synthetic chromophores or control specific functions, such as channel opening. Thus our ability to use light to specifically perturb biological systems is impeded by the lack of a generic, genetically-encodable light-sensitive protein equivalent to GFP. We have recently demonstrated the use of a new genetically encoded light-control protein-protein interaction switch, derived from the phytochrome signaling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this system can in principle be generically used to control diverse functions. We have shown that this system can be used to precisely and reversibly translocate target proteins to the membrane with micrometer spatial resolution and second time resolution. By translocating Rho family GTPases and their upstream activators, which control the actin cytoskeleton, we can use light to precisely reshape and direct the cell morphology of mammalian cells. We have demonstrated that this system works in yeast, Dictyostelium, and mammalian cells. Here we propose to develop the phytochrome system as a plug-and-play module that can be used for light-gated control of labeled molecules in space and time. We aim to optimize the light control instrumentation and to develop a molecular toolkit of single cell light control modules in a number of model systems. This highly flexible light-gated protein-protein interaction will provide remote control dials that will enable a new generation of perturbative, quantitative experiments in cell biology.

DESCRIPTION (provided by applicant): Loss-of-function approaches such as gene knockout/knockdown have been widely used to identify the proteins that are necessary to organize various cellular behaviors. However common approaches, such as genetics or RNAi, change the cellular environment permanently. Over days and weeks compensatory changes can accumulate, alter the cell's physiology, and confound conclusions. Current approaches that act on faster (seconds) timescales have limitations in their specificity, are often irreversible, hrd to titrate, or difficult to apply to a wide range of target proteins. Moreover, in cases where most components of a pathway have been identified, questions shift towards timing At what stage is my protein involved? and level Is cell behavior sensitive to the concentration of my protein? Answers to such questions require the ability to trigger, set, reverse, and monitor the extent of protein inactivation with high precision - an area where traditional methods fail, and new tools are required. Towards this end, we are developing an optogenetics based loss-of-function method termed DeLIGHT (Depletion with LIGHT), as a much-needed tool for the growing field of single-cell biochemistry. We have recently made breakthroughs in developing a light-induced protein interaction module and will here combine this tool with an 'anchor away' strategy to reversibly sequester proteins at inert intracellular locations. Our technique is generalizable, acute, and reversible, enabling significantly greater control over protein activity than existing inactivation methods. DeLight makes it possible to measure single-cell responses at user-defined intermediate concentrations of a protein of interest, where the extent of inactivation can be visually monitored and set by the user in an interactive fashion. We propose to establish and optimize this approach in 3 model systems - Dictyostelium, budding yeast, and mammalian cells to test the modularity of this system, demonstrate its versatility, and provide tools for immediate application by the cell biology community.

DESCRIPTION (provided by applicant): T cells rely on the exquisite sensitivity, specificity, and speed of T cell receptor (TCR) triggering to properly distinguish pathogenic from non-pathogenic peptides. How T cells factor the spatial and temporal dynamics of extracellular peptides into appropriate TCR triggering and cellular activation is not well understood, largely because the field has lacked tools to specifically manipulate independent parameters of ligand presentation to the TCR. In previous work, we have developed the Phytochrome/PIF photoreversible protein-protein interaction module to for micron-level spatial control and second level temporal control of intracellular signaling. In this study, we are adapting this module for photoreversible activation f TCR signaling. By combining this optogenetic system with assays of TCR engagement, TCR triggering, and cellular activation, we will probe the fundamental question of how TCR ligands are decoded. On a whole-cell level, we are investigating the role of spatial and temporal dynamics of ligand presentation for overall T cell activation and polarization (Aim 1). On a molecular level, we are investigating the role of ligand kinetics in triggering the TCR (Aim 2).

PROJECT SUMMARY/ABSTRACT Eukaryotic genome structure fluctuates across both 3-dimensional space and time and must be precisely orchestrated to achieve regulated gene expression. This structure underlies regulatory interactions between distal control elements (i.e. enhancers) and the genes that they target. While many thousands of enhancers have now been annotated and their interacting genic partners identified, we have a poor understanding of the molecular nature of these interactions, their regulation, and their significance in the cell. Chromosome conformation capture (3C) techniques have accelerated study of chromosomal organization but provide only population-averaged snapshots with poor temporal resolution and so fail to describe chromosomal interactions in their relevant context. Elucidating the molecular basis of enhancer-gene interactions in living, single cells is paramount to understanding transcriptional control. To address this need we are building new tools to visualize and manipulate chromosomal interactions in living cells. This proposal aims to dissect enhancer function by probing the dynamics and control of a model chromosomal interaction: the Sox2 gene and its distal, essential Sox2 Control Region (SCR). Sox2 encodes a tissue-specific transcription factor (TF) involved in pluripotency and reprogramming, making its transcriptional control of broad interest. In mouse embryonic stem cells (ESCs), Sox2 expression is established through the function of the SCR, located >100 kb away. To probe the dynamics of Sox2's interaction with its enhancer in living ESCs, we are combining techniques for marking DNA with advances in genome editing. To determine how these dynamics inform gene expression, we are developing tools to acutely and specifically manipulate the stability of the Sox2/SCR interaction and assay the consequences on Sox2 gene expression in individual living cells. These studies should broadly inform our understanding of transcriptional control by enhancer elements and should be generalizable to a wide range of other genomic contexts.

? DESCRIPTION (provided by applicant): My lab is broadly interested in how signaling networks generate complex cell behaviors such as polarity and motility. Understanding how these complex networks function requires tools to isolate and interrogate individual steps in the signaling cascade. We have invested significant effort developing novel optogenetic tools that enable us to activate or inhibit intracellular signals in a manner that is rapid, reversible, titratable, and can be manipulated in space and time. Initially we developed these tools to understand the regulation of polarity and motility during neutrophil chemotaxis. We are particularly interested in the molecular basis of the sensory adaptation that accounts for the remarkable dynamic range of chemotaxis and how cells convert small asymmetries in external agonist to large asymmetries in actin assembly for efficient directional movement. More recently, we have expanded our optogenetic toolkit to probe other signaling networks, such as how T cells discriminate between chemically similar ligands and commit to all-or-none activation. We continue to develop the technology to bring a wider range of signals and systems under optogenetic control, such as our analysis of pattern formation during zebrafish development.

DESCRIPTION (provided by applicant): This is an application for renewal of the Integrative Program in Complex Biological Systems (ipCBS), a graduate training program providing a specialization for students that are members of the Biophysics and Bioinformatics group. The ipCBS is a comprehensive interdisciplinary program specifically designed around the understanding and engineering of complex biological systems (systems biology). At a fundamental level, this program seeks to solve the sociological and linguistic problems associated with training scientists to be simultaneously conversant in the multiple languages of biology, mathematics, physics, and engineering. This program was built on an entirely new foundation focused on observation, modeling, and manipulation of complex systems. Over the past grant period, the ipCBS has continued to develop and evolve the curriculum, especially in the area of hands-on project based, experiential learning. As a result, a purpose-built Graduate Teaching Laboratory was constructed as a direct outcome of this training program, and a pair of required courses Dynamical Systems, conducted in the spirit of Cold Spring Harbor / Woods Hole, have become a keystone of the graduate first year experience. The key points of the ipCBS are as follows: Long term objective: To provide critical training necessary for a research environment driven by team-based interdisciplinary approaches to biomedical science and human health and dominated by large-scale and high complexity data. Approach: The ipCBS holds four core principles as the basis for graduate training. 1) Focus on the basic principles of biological organization, from the molecular to the cellular level. 2) Focus on complex systems from an interdisciplinary team-based perspective. 3) Focus on breaking down the language/sociology barrier. 4) Focus on mentoring.

Summary Cell migration is essential for immune surveillance, wound healing, and the development of multicellular organisms. Cancer cells inappropriately engage the migration program to spread throughout the body. The objective of this meeting is to bring together an international group of junior and senior scientists who share an interest in directed cell movement to present their latest discoveries, foster new collaborations, and facilitate new ways of thinking about the process. Our invited speakers are selected from topics ranging from cancer metastasis to leukocyte guidance to embryonic development to neuronal pathfinding to biochemical reconstitutions to wounding and regeneration and will emphasize novel, unpublished results directly related to how cells regulate their movement. Bringing together this diversity of cell types and approaches facilitates the identification of general principles of directed movement beyond the details of the individual systems. Our program of 26 invited speakers includes a mixture of established cell migration labs, rising stars in the field, and experts from other disciplines who have something new to say about the underlying machinery and logic of cell movement. Young investigators will be encouraged to present and discuss their findings at the GRC in a supportive environment through the invitation of 20 additional speakers, selected from submitted abstracts, as well as flash talks and participation in the two poster sessions. In a first for this meeting, the GRC will include a ?Power Hour?, which is an informal gathering open to all participants that is designed to help address the challenges women face in science and support the professional growth of women in our communities by providing an open forum for discussion and mentoring. Throughout the conference, interactions between senior and young investigators will be promoted in both formal and informal settings. To further promote the participation and development of graduate students and postdoctoral fellows, the meeting will be preceded by a GRS that is organized by trainees, with 2 invited keynote speakers, 11 trainee talks selected from submitted abstracts, two poster sessions, and an ask-the-PI panel discussion, where we cover the joys and challenges of running an independent research group and field questions from the participants. Not only does the GRS give an opportunity for more trainees to orally present their work and network with one another, but we have found from previous meetings that this activates the trainees to be more involved in the GRC. Due to the central role cell migration plays in many physiological and pathophysiological contexts and because of the wide range of systems covered at this meeting, this conference closely aligns with the missions of several NIH institutes, including NCI, NHLBI, NIAID, NICHD, and NINDS.

This project seeks to understand how cells control their shape and movement using synthetic biology tools. Cell movement is essential for single cells to hunt and mate and for the correct development and function of multicellular organisms. Sheet-like protrusions called lamellipodia are the engines that power and guide this motility. However, the rules of their formation are not understood. Similar to ant colonies where no individual is ?in charge?, and the overall behaviors of a colony depends on simple rules of local interaction between ants, many aspects of cell biology are dominated by local rules of interaction between proteins. This project seeks to define these rules of protein-protein interactions by constructing lamellipodia from synthetic, designer proteins whose patterns of interaction can be built to order. This work is a collaboration between an expert in cell shape/movement (Orion Weiner at the University of California-San Francisco) and an expert in protein design (Dek Woolfson at the University of Bristol). STEM workforce diversity will be enhanced through multiple synergistic activities. (1) reforming the grad school admissions process to make it more inclusive; (2) research opportunities for high school students from underrepresented groups and hands-on science demonstrations for teachers and elementary/middle/high-school students; (3) engagement of a wider audience by presentations to the general public through the Exploratorium, Science Festival Discovery Days, and Science Cafés; (4) Engagement of policy makers and local and national industries to advocate for the use of designed proteins and systems in biotechnology; (5) interdisciplinary training for three postdoctoral fellows and five high school students. This work will strengthen links between US and UK labs and between the NSF and the BBSRC.

Weiner?s lab recently discerned the nanoscale organization of a key actin nucleator that suggests a self-organizing template for lamellipodia formation. These templates are not sufficiently understood to manipulate directly their biophysical parameters (such as linearity vs. curvature, rigidity, dynamics, etc.) and to probe fully the relation between actin nucleator oligomerization and cell shape and function. A powerful alternative to the typical genetic and biochemical approaches would be to build actin regulators from scratch, so that the molecular logic of cell shape and movement can be probed systematically. To do this, protein engineering and de novo protein design (Woolfson lab) will be used to generate synthetic proteins that assemble with defined geometries at membranes to nucleate actin, guided by our knowledge of the native system. These synthetic systems will be tested for their ability to support lamellipodial formation in vivo using cells defective in lamellipodia formation and ultimately in complete reconstitutions. These studies will advance protein design towards in-cell application, and, in turn, they will help define the molecular logic of cell shape and movement.

This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.

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.

PROJECT SUMMARY / ABSTRACT This is an application for renewal of the Integrative Program in Complex Biological Systems (ipCBS), a graduate training program providing a specialization for students that are members of the Biophysics and Bioinformatics group. The ipCBS is a comprehensive interdisciplinary program specifically designed around the engineering of complex biological systems (?synthetic biology?). At a fundamental level, this program seeks to solve the sociological and linguistic problems associated with training scientists and engineers to be simultaneously conversant in the multiple languages of biology, mathematics, physics, and engineering. A key feature of the ipCBS is the fact that UCSF is medical campus, so that our students can exploit opportunities to explore and engineer biological complexity in the context of health and disease. Over the past grant period, the ipCBS has continued to develop and evolve the curriculum, especially in the area of hands-on project based, experiential learning. As a result, a purpose-built Graduate Teaching Laboratory was constructed as a direct outcome of this training program, and projects-based courses conducted in the spirit of Cold Spring Harbor / Woods Hole, have become a keystone of the graduate first year experience. The key points of the ipCBS are as follows: ? Long term objective: To provide critical training necessary to lead innovation in a synthetic and systems biology research environment driven by team-based interdisciplinary approaches to biomedical science and human health and dominated by large-scale and high complexity data characteristic of precision medicine. ? Approach: The ipCBS holds four core principles as the basis for graduate training. 1) Focus on the ?design principles? of biological organization, from the molecular to the cellular level. 2) Focus on the challenge of engineering synthetic circuitry within complex systems from an interdisciplinary team-based perspective. 3) Focus on breaking down the language/sociology barrier. 4) Focus on mentoring.