Frank B. Gertler, Ph.D. - US grants

In response to extracellular stimuli, many types of cells alter their morphology and movement by eliciting rapid and dynamic rearrangement of their actin-based cytoskeleton. Such responses are critical for embryonic development and for the function of many cell types within animals. Furthermore, perturbation in systems coupling environmental cues to regulation of cellular morphology and cell-cell or cell-matrix connections are associated with cancer and many other diseases. While much is known about the ways in which such signals are transmitted across the membrane, the mechanisms which transduce these signals into the mechanical forces necessary to remodel cellular architecture remain largely mysterious. The Mammalian Enabled (Mena) protein is a member of a family of molecules that are thought to link various signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to profilin, a small actin-monomer binding protein that can stimulate actin polymerization. Mena may function to concentrate profilin in structures within cells that require rapid actin polymerization such as the leading edge of motile fibroblasts or the filopodia of neuronal growth cones. The overall goal of the proposed work is to test this hypothesis and to deduce the cellular and developmental requirements for Mena function. The proposal will make use of mice carrying a targeted disruption of the Mena locus. To understand the role of Mena function in development, phenotypes resulting from loss of Mena will be characterized. Cells will be derived from the Mena mutants and analyzed for aberrations of morphology, adhesion or movement. These Mena-deficient cells will also serve as an experimental system in which to conduct a structure-function analysis of the Mena protein.

DESCRIPTION (provided by applicant): During embryonic development, vast numbers of neurons must form connections with their proper targets. Axons are guided to their targets by growth cones, which integrate and convert guidance signals into mechanical forces required for locomotion. Movement arises from dynamic regulation of cytoskeletal polymers and associated proteins. The Ena/VASP protein family functions in various axon guidance pathways and is known to regulate the assembly of actin filaments. Ena/VASP proteins are concentrated in the tips of growth cone filopodia, where the initial response to guidance signals occurs. Several signaling pathways implicated in axon guidance regulate Ena/VASP function. Therefore, Ena/VASP proteins are well positioned to act as key convergence points between signals from guidance pathways and the actin cytoskeleton. We will test the hypothesis that Ena/VASP proteins regulate actin remodeling in response to guidance signals. We propose to examine the role of Ena/VASP in growth cone guidance and translocation using high-resolution light and electron microscopy to analyze cytoskeletal dynamics and geometry. We will also employ genetic manipulation to perturb Ena/VASP function and biochemical approaches to study Ena/VASP's role in signaling pathways. This work should yield valuable insight into how the nervous system develops and how to design methods to repair it after injury.

DESCRIPTION (provided by applicant): Regulation of actin dynamics plays an integral role in many processes important for human health. Morphogenesis during embryonic development involves extensive migration, establishment of cell: cell contacts, regulated secretion and internalization of signaling proteins and numerous other cellular processes dependent upon dynamic actin remodeling. Disease-related processes including metastatic cancer, inflammatory disorders, wound repair and tissue regeneration all involve actin-dependent mechanisms. Cell migration requires coordinated regulation of cellular protrusions, adhesion, contractile forces and rear detachment. Ena/VASP proteins regulate the protrusive step of motility in fibroblasts by controlling the geometry of actin networks within lamellipodia, promoting formation of longer, less branched actin networks. Ena/VASP also likely play key roles in other actin-dependent processes such as phagocytosis, formation of cell: cell junctions and regulation of the vascular endothelium. We hypothesis that Ena/VASP proteins act as key convergence points, integrating specific signals and converting them into dynamic cytoskeletal remodeling. This grant outlines experiments to identify other migration and actin-dependent processes that utilize Ena/VASP function in vivo through analysis of mice lacking two or all three family members. W will also combine cell biological and biochemical approaches to continue our efforts to elucidate mechanisms of Ena/VASP function and regulation. We will also characterize interactions between Ena/VASP and NESH, a member of the Abi family of adaptor molecules that localizes to protruding lamellipodia and filopodia. Interestingly, Abi proteins are present in complexes that regulate the SCAR/WAVE family of Arp2/3 activating proteins, suggesting that NESH could serve as a link between Ena/VASP and SCAR/WAVE function.

DESCRIPTION (provided by applicant): Understanding how a cell moves in response to environmental signals is a fundamental challenge in biology. Mena, VASP and EVL, highly related proteins that comprise the Ena/VASP family, play pivotal roles in movement and shape change1 for a variety of cells, including fibroblasts, endothelial cells, epithelial cells, and neurons;Ena/VASP directly regulates actin filament network assembly, modulate morphology and behavior of membrane protrusions, and influences cell motility. One major goal for the current proposal is to study how Ena/VASP function contributes to chemotactic responses in carcinoma cells. Chemotactic cues trigger second messenger pathways that activate kinases, and phosphorylate Ena/VASP proteins. Specific Mena isoforms play key roles in tumor cell invasion and motility in response to Epidermal Growth Factor Receptor (EGFR) signaling. Thus, we propose that Ena/VASP proteins integrate signals from second messenger pathways to execute required motile responses, and are thus not only well positioned to control chemotactic motility, but are in fact generally involved in systems that require guided motility. The study of Mena tumor invasion-specific isoforms is particularly relevant to understanding how tumor cells acquire the ability to move and respond to a chemotactic cue. A second major goal for the current proposal is to examine the requirements for Ena/VASP function in epidermal morphogenesis and wound repair using newly generated conditional knockout mice. Several lines of evidence indicate that Ena/VASP function is required for epithelial morphogenesis and epithelial sheet fusion. Throughout development, there are many morphogenetic episodes involving fusion of two epithelial sheets or shelves of tissue. The most fully explored of these events are fusion and zippering closed of the neural tube, of the two secondary palates, and of the eyelids as the eyes close part way through gestation;embryos deficient in Ena/VASP exhibit all of these defects. While the forces driving these tissues together have been partially described, very little is known about the genetics or cell biology of the final fusion events for any of these processes;if this final knitting together event fails, the consequences can be devastating, resulting in congenital abnormalities as severe as cleft palate or spina bifda. Epithelial sheet migration and fusion is also required during wound repair, another process we will examine in this proposal. Together, these studies will provide valuable insight into the mechanisms controlling cell motility in both normal development and disease. PUBLIC HEALTH RELEVANCE: Drugs that block the EGFR pathway are widely used to treat a variety of cancers;thus, by understanding how invasion-specific Mena isoforms sensitizes carcinoma cells to EGF and amplifies their response, valuable insight into how certain cancers become resistant to therapies that attenuate EGFR signaling will be gained. Epithelial sheet movements during process such as eyelid closure and wound healing are dependent of EGF and EGF-related factors, thus studies of Ena/VASP in epithelial migration and sheet fusion should prove valuable in developing new ways to enhance treatment of wounds.

DESCRIPTION (provided by applicant): Damage to connections within the adult Central Nervous System (CNS) by injury or disease is often irreparable. To design therapies to repair CNS damage requires a detailed understanding of the cellular mechanisms underlying CNS development. As the brain matures, neurons migrate to their proper positions within the brain and elaborate processes that are guided to their targets to form proper connections. Both initial formation of growth cone-tipped neurites, and subsequent guided locomotion of axonal growth cones, require F-actin and microtubule (MT) dynamics. F-actin:MT interactions likely play key roles in both of these processes. However, the molecular mechanisms that mediate such interactions, and how these interactions drive neuritogenesis and axon guidance, are not understood. Ena/VASP proteins function in growth cone guidance by controlling actin cytoskeleton dynamics. Using a combination of mouse genetics, primary cell culture, live cell imaging and electron microscopy, my lab found that Ena/VASP-deficient cortical neurons fail to form filopodia, finger-like processes comprised of bundled F-actin. Furthermore, we found that cortical neurons devoid of filopodia fail to form neurites, and exhibit altered microtubule dynamics. Restoration of filopodia in Ena/VASP mutant cortical neurons also rescues neurite initiation. Preliminary data indicate that Ena/VASP-deficient sensory neurons form axons, but exhibit striking guidance defects. We will use sensory neuron preparations from Ena/VASP deficient animals to test our working hypothesis is that Ena/VASP- dependent filopodia formation enables interactions between MTs and actin bundles that are required to receive both attractive and repulsive cues. Additional new data indicate that Ena/VASP proteins may act to coordinate F-actin:MT interactions, and that the TRIM9 protein interacts with both Ena/VASP and microtubules;TRIM9 is also implicated in the control of axon navigation. Furthermore, a network of proteins, including Ena/VASP, is likely regulated by Lamellipodin, a molecule that integrates signals generated by cell-surface receptors for axonal guidance factors. Collectively, these data lead us to hypothesize that Ena/VASP proteins participate in protein networks that play key roles in F-actin: MTs interactions, and are in turn linked to signaling pathways controlled by guidance receptors. Our long-term goal is to understand how neurons integrate environmental cues to orchestrate changes in their morphology and movement necessary to establish a functional nervous system. A better understanding of the mechanistic basis of neurite formation and axon guidance will provide fundamental insight into how connections in the nervous system are established and how they are remodeled during plasticity. The results of our research plan should be of great value to the development of therapeutic approaches to repair these connections subsequent to disease or injury. PUBLIC HEALTH RELEVANCE: Damage to the nerves that form connections within the adult brain and spinal column by injury or disease is often irreparable. We seek a comprehensive understanding of how nerve fibers form and are guided to their targets initially during fetal development expecting that this will help us learn to repair damage to the brain.

Research at the Koch Institute combines a wide spectrum of innovafive programs designed to understand tumor development and develop new treatments and diagnostic strategies. Many of the approaches taken, in disciplines ranging from molecular and cell biological to analysis of mouse models and nanotechnology, rely on imaging. In 2005, using non-renewable institufional funds, the Koch Institute established a Microscopy Core Facility that makes centralized, state-of-the art imaging equipment available to Center investigators and other NCI-funded investigators at MIT. CCSG funds are requested to provide confinuing support for this Core. The Microscopy Core curl-ently offers a wide range of imaging platforms including standard light and epifiuoresence to digital deconvolution, spinning disk confocal microscopy, and systems for spectral Karyotyping (SKY) and laser capture microdissection. The Core is run by a highly qualifled Core Manager, who has extensive expertise in a broad range of microscopy platforms and applications including electron microscopy and live cell/organotypic fluorescent 4D imaging. The Core Manager oversees all instrumentafion, provides state-of-the-art training to Center members in both sample preparation and use of the Core's equipment and also conducts specific microscopy techniques for Kl users on a fee-for-service basis. She also collaborates with the Applied Therapeufics & Whole Animal Imaging Core Facility, providing technical support and user training for the whole-animal imaging instrumentafion including luminescence/fluorescence detection and microCT applicafions. The Microscopy Core has proven invaluable to a large fraction of the Kl faculty by allowing them to execute microscopy-dependent projects that were previously impossible given the expense of acquiring and maintaining imaging equipment. In the upcoming grant period, the Kl will confinue to add new equipment to both the Microscopy and Applied Therapeutics & Whole Animal Imaging Core Facilities to further expanding their imaging capabilities. This expansion, as well as high demand for existing services, requires addition of a technical assistant to the Microscopy Core Facility staff. User chargebacks provide partial support the Microscopy Core, but the operational costs are currently undenA/ritten by Institutional funds that will expire in June 2009. Thus, the requested CCSG support is critical for the confinued success of this essenfial Microscopy Core.

The central step in cancer progression that leads to mortality is metastasis, the dissemination of cells from the primary tumor mass to distant organs. Research in the EMT, Migration and Metastasis Networks program is aimed at elucidating the regulatory pathways governing tumor progression to full metastatic disease with the goal of identifying new targets for therapeutic intervention in the treatment of malignant cancers. The proposed research focuses on four general aspects of metastasis: acquisition of a motile phenotype during epithelial to mesenchymal transition (EMT), motility responses to growth factor stimulation, dissemination of metastasis to the CNS and acquisition of resistance to therapy. We will use mathematical modeling to indentify novel regulatory pathways that control cell behavior as tumor cells develop an invasive, metastatic phenotype. Cell behavior during EMT and growth factor elicited motility will be measured quantitatively. The status of signaling pathways, gene expression and alternative splicing wall be interrogated and used to take an integrative systems approach to develop computational, data-driven models that relate these metrics to cell behavior. The models will be used to identify novel regulatory relationships governing the steps in carcinoma metastasis from initial invasion to tumor cell entry into blood or lymphatic vessels. Metastasis often leads to the acquisition of resistance both to conventional chemotherapy and to target treatments, possibly by allowing tumor cells to evade treatment by infiltrating a protected microenvironment such as the central nervous system. Tumor cell targeting of the central nervous system and acquisition of therapy resistant phenotypes will be explored using high-throughput RNAi screening approaches in vivo in a lymphoma model. The research program will employ mammary epithelia cells, breast cancer cells, xenografts and syngeneic tumor models, and lymphoma.

DESCRIPTION (provided by applicant): Cellular structure and function, in healthy and diseased systems, is regulated by the interaction of cells with the underlying and surrounding three-dimensional extra-cellular matrix. These complex biochemical and biomechanical interactions, independently, are well known to regulate tumor progression, invasion and metastasis. For example, the aberrant response of cells to biochemical and biophysical stimuli in metastatic breast cancer is often initiated by engagement of the cytoskeletal machinery. As such, actin interacting proteins are found at the nexus of signaling network crosstalk between biochemical and adhesion-promoting cues. One such example is Mena, a member of the Ena/VASP family of actin regulatory proteins, which has been characterized for aberrant cell-signaling response during invasion and metastasis. However, how the altered signaling network is translated into the mechanical processes, and how are these sub-cellular mechanical processes then converted into whole cell migration in 3D environments remain largely elusive. Here, based on our preliminary data, we hypothesize that increased tumor cell invasiveness in 3D environments, is governed by coupling aberrant molecular level signaling events to molecular, macromolecular and cellular biomechanical processes. Our primary goal in this proposal is to rigorously test our hypothesis by bridging the knowledge gap between in vitro signaling studies at the molecular level, and molecular mechanical and cellular models in 3D, and test the predictions of our models through quantitative experiments in 3D environments. We plan to develop and validate our cellular models using the following three specific aims: Aim I: Develop an integrated subcellular model of cytoskeletal viscoelasticity and intracellular signaling in native like 3D matrices. Aim II: Develop a quantitative model of cell migration, in 3D matrices, utilizing results from the subcellular model of Aim I. Aim III: Validate results of Aims I and II b quantifying how signaling acts cooperatively with cellular mechanics machinery and extracellular matrix properties to regulate cell migration in 3D. All three aims build upon strong preliminary data in both computation and experimental studies and will provide both fundamental insights into the coupling between mechanical and biochemical pathways and integration of information from sub-cellular structures to the cellular level. At the same time, the focus on 3D environments will create new and physiologically relevant knowledge about cellular systems in native like environments. Finally, novel platforms developed through this work will be able to test clinically relevant hypotheses and help in quantitatively understanding complex multi-scale processes during various stages of cancer progression.

DESCRIPTION (provided by applicant): The epithelial-mesenchymal transition (EMT) is a complex cell-biological program that operates during the progression of carcinoma cells to high-grade malignancy, conferring on these cells many of the attributes associated with aggressive tumors, including the ability to disseminate to distant sites and to seed metastatic colonies. This program is orchestrated by a series of pleiotropically acting master transcription factors (EMT- TFs) that organize the complex changes in gene expression causing the replacement of a large cohort of epithelial cell proteins with those associated with the mesenchymal cell state. A major, critical level of control required for expression of the aggressive mesenchymal state is poorly understood however: the precursors of many of the mRNAs whose expression changes during the EMT also undergo alternative splicing (AS) that confer on resulting mature, processed mRNAs altered properties, including changes in stability, protein-coding information, and responsiveness to microRNA-mediated inhibition. The current fragmentary insights into the effects of AS on the execution of the EMT program make it impossible to form a reasonably complete understanding of how this critical cell-biological program is effected. The proposed research will begin by enumerating the hundreds of AS events that occur in response to several alternative mechanisms of inducing an EMT program both in cultured cells and in a living tissue. Having done so, bioinformatics algorithms will be employed to determine the sequences adjacent to involved splice sites. Thereafter, using the known nucleotide-recognizing properties of the large array of already-characterized RNA- binding, splice-regulating proteins, predictions will be made by these algorithms about the identities of the splice-regulators that are likely to b responsible for the observed large-scale shifts in AS occurring during passage through an EMT. This experimental strategy should yield the identities of key regulators of AS that are likely to b as important functionally as the EMT-TFs in executing the EMT program. Experimental tests designed to functionally test the candidacies of these AS factors will be performed. These tests will gauge whether the forced or blocked expression of these factors affect execution of critical components of the EMT program, and whether, as predicted, such imposed changes in AS factor expression affect the production of key EMT-associated proteins, i.e., proteins that play key roles in the expression of the epithelial versus mesenchymal cell phenotypes observed during malignant progression. This work also has the potential to identify novel biomarkers of the EMT program that are applicable, for example, for the detections of stem cells in a variety of epithelial tissues.

A primary focus of the Koch Institute?s research program is to understand the molecular and cellular changes associated with cancer development and progression and develop new treatments and diagnostic approaches. The Microscopy Core provides state-of-the-art imaging in both cells and in the in vivo setting, which are essential tools for these studies. In the current period, the capabilities of this Core have been expanded and enhanced. This includes: moving into a larger, custom-designed space in the new Koch Institute building; the acquisition of new instrumentation; and the recruitment of a world-renowned expert in intravital imaging, Jeffrey Wyckoff, to the Core?s staff. Notably, in the same period, 78% of Center Members used the Microscopy Core, including investigators from all four Programs. Thus, this Shared Resource is essential to the success of the Koch Institute mission. In the upcoming period, this Core will continue to offer a wide range of state-of-the-art imaging technologies to support the research programs of Center Members. To support its expanded staff, the requested budget for Year 44 is increased 45%, compared to the requested and recommended budget in Year 39. The CCSG budgets of other Koch Institute Cores have been reduced or eliminated to more than offset the proposed increase in this, and other, Cores.