Sanjay Kumar - US grants

Actomyosin stress fiber bundles, or ?stress fibers,? are cable-like structures that run along the base of a living cell and enable the cell to generate tractional forces against its environment, a functionality that is critical to cell structure control and migration. This program seeks to investigate the role played by stress fibers in the regulation of cell and tissue structure and mechanics. There are two key program goals. The first goal is to elucidate the relationship between extracellular matrix rigidity and stress fiber elasticity. Key approaches here include fabrication of cell culture substrates with defined mechanical and biochemical properties, and use of femtosecond laser ablation and other advanced optical imaging methods to probe individual stress fibers in living cells. The second goal is to investigate the role played by individual stress fibers in controlling the architecture and mechanics of multicellular structures, both in two-dimensional and three-dimensional culture.
This program will contribute to our fundamental understanding of mechanisms that living cells use to define and modify their structure, behaviors that are central both to normal tissue development and disease. This program will also provide mechanistic insight into the increasingly-appreciated role played by mechanical force in controlling cell physiology, which in turn may contribute to more rational design strategies for cell and tissue engineering systems. In addition, there are two key educational goals of this program, including development of a graduate-level course and organization of a regional research symposium featuring faculty and students from predominantly undergraduate institutions, both in the subject area of cell and tissue mechanotransduction.

09: Physiology and Integrative Systems Cellular Mechanobiology: Biophysics and Therapeutics Sanjay Kumar, MD, PhD University of California, Berkeley One of the most important lessons from cellular physiology in the past decade is that living cells sense, process, and physiologically respond to specific physical stimuli in their environment, including the geometry, dimensionality, and rigidity of the extracellular matrix (ECM). This has spawned an intense effort to understand how mechanical cues manifest themselves in the context of problems ranging from stem cell engineering to tumor growth to scar formation, which has collectively led to the genesis of a completely new field: Mechanobiology. Yet, despite this recent flurry of activity, the field of mechanobiology continues to suffer from two limitations which threaten to restrict its long-term progress: mechanistic disagreements about how cells sense and process mechanical cues, and uncertainty about whether mechanobiological relationships observed in vitro also operate in a more clinically relevant setting. Here I propose to advance the field of mechanobiology by addressing both of these issues, organizing my research around three questions: (1) How are intracellular and extracellular mechanical stimuli applied to microscale portions of a living cell chemically and physically communicated to the rest of the cell, and how do these signals physically trigger changes in gene programs?; (2) How does the regulation of specific genes, gene networks, and signaling pathways differentially depend on physical cues from the ECM, such as ECM rigidity, geometry, and dimensionality, and can cells be genetically engineered to alter their responses to these cues?; (3) Can targeting mechanobiological interactions between cells and the ECM influence tissue physiology and pathology in vivo? By directly tackling these questions, we will strengthen the mechanistic foundations of this nascent field and facilitate the creation of cellular engineering and therapeutic strategies which leverage its principles.

The objective of this Faculty Early Career Development (CAREER) Program award is to use a combination of biophotonic, genetic, and computational tools to investigate the mechanics of actomyosin stress fibers and the underlying signaling mechanisms that control these properties. Stress fibers are micrometer-scale, contractile structural cables that traverse across living cells and enable cells to generate tractional forces against their surroundings, a process critical to locomotion and maintenance of tissue architecture. Studies conducted under this award will apply molecular biological tools to manipulate the concentrations and activities of specific molecules within stress fibers and use laser nanosurgery and related microtechnologies to investigate the contributions of these molecules to stress fiber function. In parallel, a multi-scale computational model will be developed to relate the activities of these molecular components to stress fiber mechanics and overall cellular mechanobiological properties.

If successful, these studies would add significantly to the field's understanding of the molecular regulation of cytoskeletal contractility and represent a tight integration of experiment and computation. The knowledge gained from these efforts will also enhance the field's ability to manipulate cell and tissue structure and function by revealing "design principles" for cell structure and mechanics. This in turn may help drive the evolution of systems for tissue engineering, regenerative medicine, and other bio-interfacial technologies. The educational plan focuses on course development in cellular bioengineering at the undergraduate and graduate level as well as the creation of partnerships between the awardee's institution and two non-PhD-focused bioengineering programs for BS- and MS-level curriculum development. Both initiatives will involve extensive sharing of course material and discussions of how to effectively incorporate cellular bioengineering concepts into undergraduate and pre-professional training.

The goals of this NSF-NCI joint research grant are to establish the influence of extracellular matrix (ECM) stiffness and microarchitecture on tumor cell invasion and to elucidate the molecular mechanisms that underlie this regulation. While the strong influence of ECM stiffness in governing tumor cell migration has been well established in traditional two-dimensional culture paradigms, understanding this phenomenon in three-dimensional (3D) ECMs that more closely mimic tissue has proven considerably more challenging. In part, this is because perturbations that change 3D ECM stiffness often concurrently change microscale matrix parameters that also critically regulate cell migration, such as pore size, fiber architecture, and local material deformability. By combining a new microscale culture platform that enables orthogonal separation of ECM stiffness and pore geometry with gene targeting approaches and computational modeling, this project will explore the contributions of these two parameters to the invasion of tumor cells.

The studies will initially focus on glioblastoma multiforme (GBM), a highly malignant brain tumor that routinely kills patients within two years of diagnosis. Successful completion of the work will provide new biophysical, mechanistic insight into the progression of GBM, and the resulting experimental and computational platforms may be readily applied to probe the invasion and metastasis of other tumor types. The project will also feature an educational plan in which undergraduates will be mentored and actively included in research efforts. Finally, a learning module for elementary school students on cell motility and the ECM will be created and taught through a local program that places practicing scientists and engineers in area public schools to enhance science education.

PROJECT SUMMARY/ABSTRACT Mechanical forces within the material microenvironment are increasingly recognized as important regulators of stem cell self-renewal and differentiation. Over the past decade we have been exploring these concepts in the context of adult hippocampal neural stem cells (NSCs), which generate neurons throughout adult life and play key roles in learning, memory, and disease processes. In our first period of R01 support, we have shown that extracellular matrix (ECM) stiffness cues can act through Rho GTPase- and myosin-dependent contractility to influence lineage commitment within a defined temporal window. Moreover, manipulation of these stiffness- sensing pathways in vivo can control hippocampal neurogenesis in a manner that is predictable from culture studies. More recently, we have created reversibly-stiffening oligonucleotide-crosslinked materials and applied this technology to narrow this window to 12-36 h and to begin elucidating key signals that are activated during this period to induce lineage commitment. In this renewal application, we now propose to build upon these advances by tackling two key questions of high general interest within the stem cell field: First, do the mechanoregulatory signaling relationships observed in simplified 2D systems hold in more complex 3D microenvironments, particularly ones with dynamic mechanical properties analogous to those encountered in vivo? Second, precisely how do the signals triggered by mechanical inputs (e.g. Rho GTPase-dependent myosin contraction) interface with the signals canonically understood to regulate NSC neurogenesis? In Aim 1, we will investigate mechanosensitive lineage commitment in 3D by applying new click-crosslinked hyaluronic acid hydrogels with tunable stiffness. We will also innovate upon these materials by incorporating reversible oligonucleotide-based crosslinks that allow variable degrees of stress relaxation, and then use these materials to ask if we can shift the time window of mechanosensitive lineage commitment. In Aim 2, we will investigate integration of mechanotransductive signaling and canonical pro-neurogenic signaling in the control of NSC neurogenesis. Specifically, we will test the hypothesis that mechanosensitive lineage commitment is controlled by a master signaling circuit involving YAP, angiomotin, and b-catenin. We will also apply genome-wide CRISPR gain/loss-of-function screens to identify additional candidates, which we will then characterize and incorporate into this regulatory framework. Successful completion of these studies will not only dramatically improve the field?s understanding of how mechanical signals influence NSC lineage commitment but offer a new intellectual roadmap and set of tools that will be broadly applicable to all stem cell types.

DESCRIPTION (provided by applicant): It is now widely acknowledged that cell behavior is highly sensitive to mechanical crosstalk with the extracellular matrix (ECM). While many powerful methods have been developed to control this communication through manipulation of the ECM, there are few tools available for the direct, cell-intrinsic control of cellular mechanotransductive signaling. In this proposal we advance and apply a genetic strategy we recently developed in which we control cell-ECM mechanical signaling through inducible expression of mechanotransductive genes. We have shown that this method enables graded and dynamic control of cortical stiffness, traction force generation, cell migration speed, and ECM remodeling. We have also shown that this approach vastly outperforms traditional pharmacologic strategies in terms of dose-response relationship, target availability, toxicity, and duration of action. We now propose to develop a second generation of this strategy and leverage it to address two unmet needs in the field of cell mechanobiology: First, we will place two genes under the control of promoters that can be induced or suppressed by two different small molecules, thereby enabling orthogonal control over two mechanotransductive genes. We will use this capability to construct a phase diagram of cell mechanical properties that quantitatively maps how the myosin activators Rho- associated kinase and myosin light chain kinase contribute to mechanobiological phenotype. Second, we will apply this strategy to quantitatively control how ECM mechanical properties regulate two important cell behaviors: cell motility speed and neural stem cell neurogenesis. In successful, this will enable us to decouple mechanically-triggered cell behaviors from the inputs themselves, thus potentially offering a way to rewire cell-matrix crosstalk to achieve desired phenotypic endpoints in arbitrarily specified microenvironments. This could offer a new and very powerful way to engineer cell behavior at cell-material interfaces in vitro and in vivo. Taken together, these studies will provide key proof of-principle for this approach as a tool for both quantitative cell biological discovery and cell ad tissue engineering/regenerative medicine applications.

DESCRIPTION (provided by applicant): Tumor invasion and metastasis are strongly regulated by biophysical interactions between tumor cells and the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility has been extensively studied in two-dimensional (2D) culture, extension of these concepts to three- dimensional (3D) microenvironments characteristic of most tissues has proven extremely challenging given that manipulations normally used to vary ECM stiffness (e.g., variation of matrix and crosslink density) often concurrently alter matrix pore size (confinement), which can create steric barriers that regulate invasion speed independently of mechanics. To address this challenge, we have developed a novel matrix platform based on microfabrication of channels of defined wall stiffness and geometry that allows orthogonal variation of ECM stiffness and channel width. We have used this platform to characterize the regulation of glioblastoma cell invasion by ECM stiffness and confinement, which has led us to discover that stiff, narrow pores maximize cell invasion as a consequence of enhanced polarization of traction forces. As evidenced by this and other novel findings, this platform offers the best of both worlds with respect to experimental 2D and 3D cell migration paradigms, in that it retains the throughput, standardization, and screening power of the former while capturing key biophysical regulatory elements of the latter. With the support of this IMAT R21 award, we now propose to develop this platform as a microfluidic technology for high-throughput molecular screening and analysis. We will organize our research around three specific aims: (1) To develop an enclosed microfluidic device for the directed migration of tumor cells through channels of defined geometry and stiffness; (2) To use the platform to screen small molecule libraries for agents that slow migration in a stiffness- and confinement-dependent fashion; and (3) To relate invasion speed to gene expression in primary glioblastoma tumor initiating cells through comparative proteomic analysis. The proposed studies will address an unmet need for platforms capable of rapidly identifying drugs and genes that underlie physical microenvironmental control of tumor invasion. Ours is one of the first systematic efforts to study the roles of ECM stiffness and pore size (confinement) in regulating tumor cell invasion in 3D and to apply high-throughput molecular screening approaches to a problem in cell-ECM mechanobiology. By integrating mechanobiology, tumor stem cell biology, microfluidics, and proteomics, our work will create a valuable new discovery tool that is likely to open significant new translational opportunities for clinical oncology.

PROJECT SUMMARY/ABSTRACT Stem cell differentiation is intimately regulated by the extracellular matrix (ECM) surrounding cells. While substantial research has identified extracellular signals and their intracellular transducers, there remains an incomplete knowledge of these signals regulate the broad genetic networks that cause lineage commitment. One potentially interesting candidate for this regulation is microRNAs (miRNAs), which are known to exert profound influence on gene networks in many contexts. In this proposal, we seek to ask whether miRNAs serve as a networking bridge between ECM-mediated signals and the stem cell lineage commitment. One of the critical bottlenecks in understanding ECM-mediated signals is the low-throughput nature of current state-of- the-art technologies to study mechanobiology in vitro. We have recently developed a novel technology that condenses hundreds of experiments conducted with current technologies into a single experiment. We now propose to combine this technology with high-throughput analysis of microRNA expression (miRNA-seq) and use this platform to identify ECM-sensitive miRNA networks involved in stem cell differentiation. First, we will combine and optimize our high-throughput mechanobiology platform with laser capture microdissection and miRNA-seq to produce a list of miRNA candidates whose expression is correlated with changes in matrix stiffness and/or ligand density. Second, we will perform gain- and loss-of-function studies to confirm that the miRNAs candidates functionally contribute to ECM-mediated stem cell differentiation. If successful, this proposal will develop a very powerful high-throughput technique to study other ECM-mediated phenomenon in other cells. Furthermore, this proposal will develop a deeper understanding of the relationship between ECM- signals and gene-network regulation, which would in turn yield a new set of molecular targets that could be leveraged to facilitate successful stem cell engineering for therapeutic translation.

PROJECT SUMMARY/ABSTRACT Actomyosin stress fibers (SFs) enable cells to tense the extracellular matrix (ECM), a process key to cell shape determination, polarity, motility, and tissue morphogenesis. SFs within motile cells have been broadly classified into three specialized ?subtypes? (dorsal fibers, transverse arcs, and ventral fibers) that differ in their antero-posterior location and network connectivity. In addition to driving normal tissue development and homeostasis, SFs and analogous contractile structures contribute to the invasion of tumors within tissue, a notable example of which is the perivascular infiltration of the deadly brain tumor glioblastoma multiforme (GBM). It has been hypothesized that dorsal fibers, transverse arcs, and ventral fibers tense each other and the ECM in very specific ways to govern cell shape, polarity, and motility. However, this paradigm suffers from several critical limitations. For example, it has not been directly demonstrated that each SF subtype generates tension as commonly assumed, which in turn derives from a lack of direct measurement of SF mechanical properties in living cells. Additionally, while these subtypes are broadly understood to vary in the molecular motors they contain (i.e. myosin II isoforms), we know virtually nothing about how these molecular-scale differences create the contractility differences across SF subtypes. Finally, and perhaps most importantly, it is unclear whether this subtype classification is relevant to the persistent migration of cells within tissue, particularly in disease states driven by aberrant cell migration. In this proposal we address all three of these critical open questions by combining single-cell biophotonic technologies, traditional cell and molecular biology approaches, engineered culture systems, and ex vivo tissue models. A key enabling tool for these studies (which our team has pioneered over the past decade) is femtosecond laser nanosurgery (FLN), which enables us to selectively cut single SFs in living cells, thereby allowing us to deduce both the mechanical loads borne by that SF and its structural contributions to the rest of the cell. In Aim 1, we will apply FLN to selectively incise SFs from each canonical subtype to map these mechanical properties and structural contributions. We will also combine FLN with single-cell micropatterning and fluorescence-based readouts of molecular tension to determine how single SFs distribute tension throughout the cell and contribute to EGF-dependent polarization and motility. In Aim 2, we will investigate how the stoichiometry and mechanochemical properties of specific myosin II isoforms collaborate to determine the mechanical properties of the entire SF. In Aim 3, we will combine these approaches with a microfluidic model we developed with a brain-slice paradigm to determine how specific SF subtypes and the myosin isoforms therein contribute to perivascular invasion in GBM. To our knowledge, Aim 3 studies will represent the first measurements of SF mechanics and function in mammalian tissue. In summary, this project will marry innovative single-cell and culture technologies to address major open questions surrounding the microscale, biophysical mechanisms of cell shape shape, polarity, and motility.

PROJECT SUMMARY/ABSTRACT There is a dire need for new technologies to address the obesity epidemic and its associated sequellae, including Type II Diabetes. Increasing caloric output through expansion and activation of brown adipose tissue (BAT), which ?burns? metabolic fuels to produce heat, is garnering increasing interest as a novel mechanism to trigger weight loss in adults. However, the technological translation of this approach, including the engineering of biomaterial platforms to support BAT in vitro and in vivo, has been limited by a poor understanding of how cues from the physical microenvironment regulate BAT activation. Our preliminary data hint at a novel and unexpected model in which beta-adrenergic (ß-AR) stimulation triggers BAT activation through a myosin- and YAP/TAZ-dependent mechanotransductive signaling network, ultimately enhancing expression of the heat- generating mitochondrial protein UCP1. This model has profound implications, because it would suggest that incorporation of mechanical cues within the microenvironment could be leveraged to activate BAT and promote caloric output as a strategy to combat obesity. Thus, the goal of this proposal is to critically test the hypothesis that ß-AR and mechanotransductive signaling collude to stimulate BAT activation and enhanced cellular respiration. We have three aims: (1) To dissect the mechanisms through which actomyosin tension acutely activates BAT; (2) To determine how mechanical activation of YAP/TAZ regulates expression of UCP1; and (3) To investigate the role of mechanosensitive YAP/TAZ-dependent signals in white/beige adipose fate determination. In addition to detailed dissection of signaling events, our approach features an innovative combination of engineered materials, mechanical stimulation, advanced mouse genetic models, inducible expression of myosin-activating proteins, and measurements of cell and tissue mechanics. Successful completion of this work would substantially advance our mechanistic understanding of BAT activation while informing the design of materials technologies to stimulate BAT activation to reduce obesity.