The TGF-beta signals Vg1 and Nodal form heterodimers to induce the vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal. Here we investigate the mechanisms underlying Vg1 retention, processing, secretion and signaling in zebrafish. First, using a newly devised Synthetic Processing (SynPro) system, we find that Vg1 can be processed by intraor extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. Our results establish SynPro as a new in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active Vg1-Nodal heterodimers. These observations suggest two strategies for rapid mesendoderm induction: chaperone-binding motifs help store Vg1 as an inactive but ready-toheterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intraand extracellularly.

Myocardial fiber orientation is closely related to the functions of the heart. The development of imaging tools for depicting myocardial fiber orientation is important. We developed a polarized hyperspectral imaging microscope (PHSIM) for cardiac fiber orientation imaging, which is capable of polarimetric imaging and hyperspectral imaging. Polarimetric imaging is realized by the integration of two polarizers. Hyperspectral imaging is realized by snapscan Preliminary imaging experiments were implemented on an unstained paraffin embedded tissue slides of a chicken heart. We also set up a Monte Carlo simulation program based on the cylinder optical model to simulate the cardiac fiber structure of the sample and the optical setup of the PHSIM system, in which we can calculate the system output light intensity related to cardiac fiber orientation. According to the imaging and simulation results, there exists a variation of intensity of acquired images with the polar angles from the maximum to the minimum under different wavelengths, which should relate to the orientation of cardiac fibers. In addition, there is a shift of the polar angle where the maximum intensity appears when a rotation of the sample happened both in the simulation and imaging experiments. Further work is required for imaging more types of myocardial tissues at different parts and the design of a complete quantitative model to describe the relations among polar angles, wavelengths, and cardiac fiber orientations.

The TGF-beta signals Vg1 (Dvr1/Gdf3) and Nodal form heterodimers to induce vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal, but the mechanisms underlying Vg1 biogenesis are largely elusive. Here, we clarify the mechanisms underlying Vg1 retention, processing, secretion, and signaling and introduce a Synthetic Processing (SynPro) system that enables the programmed cleavage of ER-resident and extracellular proteins. First, we find that Vg1 can be processed by intra- or extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. These observations suggest two mechanisms for rapid mesendoderm induction: Chaperone-binding motifs help store Vg1 as an inactive but ready-to-heterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intra- and extracellularly. These results establish SynPro as an in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active TGF-beta heterodimers.

Tissues such as brain, muscle, and bone differ greatly not only in their biological functions but also in their mechanical properties. Brain is far softer than muscle while bone is the stiffest tissue. Stiffness of extracellular microenvironments affects fundamental cell biological processes such as polarization and DNA replication, which affect nuclear size, shape, and levels of nuclear proteins such as the lamins that modulate gene expression. Reductionist approaches have helped dissect the effects of matrix mechanics away from confounding biochemical signals. Here, we summarize materials and methods for synthesizing and characterizing soft and stiff synthetic hydrogels widely used for mechanobiological studies. Such gels are also easily made to mimic the mechanical heterogeneity of fibrotic tissues. We further describe a nano-thin collagen fiber system, which enables control of anisotropy in addition to stiffness. With the different systems, we illustrate the effects of matrix mechanics on nuclear size, shape, and proteins including the lamins.

Synergistic cues from extracellular matrix and soluble factors are often obscure in differentiation. Here the rigidity of cross-linked collagen synergizes with retinoids in the osteogenesis of human marrow mesenchymal stem cells (MSCs). Collagen nanofilms serve as a model matrix that MSCs can easily deform unless the film is enzymatically cross-linked, which promotes the spreading of cells and the stiffening of nuclei as both actomyosin assembly and nucleoskeletal lamin-A increase. Expression of lamin-A is known to be controlled by retinoic acid receptor (RAR) transcription factors, but soft matrix prevents any response to any retinoids. Rigid matrix is needed to induce rapid nuclear accumulation of the RARG isoform and for RARG-specific antagonist to increase or maintain expression of lamin-A as well as for RARG-agonist to repress expression. A progerin allele of lamin-A is regulated in the same manner in iPSC-derived MSCs. Rigid matrices are further required for eventual expression of osteogenic markers, and RARG-antagonist strongly drives lamin-A-dependent osteogenesis on rigid substrates, with pretreated xenografts calcifying in vivo to a similar extent as native bone. Proteomics-detected targets of mechanosensitive lamin-A and retinoids underscore the convergent synergy of insoluble and soluble cues in differentiation.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.

Integration of soluble factors and physical properties of extracellular matrix is likely key to stem cell differentiation but the extent to which those pathways overlap remains unclear. Here motivated by the micromechanics of osteogenic niche we looked at the synergy between matrix stimuli and pharmacological perturbation of Retinoic Acid (RA) pathway on primary and iPSC-derived mesenchymal stem cells (MSCs) including iPSC- derived cells from progeria patients towards osteogenesis. Retinoic acid receptor RARG transcription factor is known to regulate nucleoskeletal protein Lamin-A. A cell-by-cell analysis showed that rigid matrix favor higher LMNA and correlates with increased nuclear-to-cytoplasmic ratio of RARG. We found that the Progerin allele of lamin-A is similarly regulated by specific RARG agonist/antagonists. A mechanochemical gene circuit in which tension on lamin-A ultimately favors RARG activity describes well the experimentally observed trend. Scatter-plots of single-cell analyses also show that some cells on stiff substrates fall within the response envelopes of cells cultured on soft substrates and that shared sub- population of non-responding cells we also found that don’t respond to RA agonist or antagonist regulation of Lamin-A. RA antagonist drove lamin-A dependent upregulation of osteogenic markers on rigid substrates and pretreated xenografts showed bone-level calcification suggesting a synergistic effect of soluble and insoluble factors on subpopulation of stem cells that are highly mechanoresponsive.

Molecular engineering and synthetic biology approaches have been applied (1) to replace the intracellular domain (NICD) of Notch with synthetic transcription factors 1 that bind to genes other than natural Notch targets; (2) to replace human Notch extracellular domain (NECD) with recognition motifs against novel targets (e.g. cognate receptor ligands such as Delta versus single-chain variable fragments raised against a specific antigen). The project aims to interrogate and engineer the fate of a receiver cell to surface-receptor levels of a sender cell by harnessing contact-mediated Notch-Delta signaling and RNA-guided CRISPR genome engineering. We have developed Notch1-Cas9 chimeric receptors that, upon binding Delta ligand to the extra-cellular Notch domain, cleave and release Cas9 to manipulate gene expression of transgenic reporter constructs - and - of endogenous targets in mammalian cells. First, we describe the challenges and solutions in the construction of Notch-Cas9, which led to the discovery of a naturally occurring sequence in bacterial Cas9 that is being co-opted as a nuclear localization signal (NLS) when expressed in mammalian cells. To our knowledge, this is the first reported evidence of a cognate NLS in bacterial Cas9. Second, we expand the toolkit with not only Delta-dependent gene activation, but also with gene editing in two Cas9 orthologs (Streptococcus pyogenes and Staphylococcus aureus Cas9). The toolkit performs well in reporter-gene activation and editing assays. Finally, we demonstrate activation of and phenotypic changes associated with endogenous genes encoding for regulators of the cell-division cycle, CDKN1B and CASP8; and editing of CXCR4 and CD47, receptors relevant to immune trafficking and tolerance. This Notch-Cas9 fusion technology opens up novel biological behaviors in response to cell-cell contact and can be utilized to interrogate or manipulate existing signaling pathways. Citation Format: P C Dave P. Dingal, Nathan Kipniss, Yuchen Gao, Lei S. Qi. Conversion of extracellular signals to programmable genome manipulation via CRISPRouter [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B075.

Scarring is a long-lasting problem in higher animals, and reductionist approaches could aid in developing treatments. Here, we show that copolymerization of collagen I with polyacrylamide produces minimal matrix models of scars (MMMS), in which fractal-fibre bundles segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally-as in scars-while allowing separate control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs): cells spread and polarize quickly, increasing nucleoskeletal lamin-A yet expressing the 'scar marker' smooth muscle actin (SMA) more slowly. Surprisingly, expression responses to MMMS exhibit less cell-to-cell noise than homogeneously stiff gels. Such differences from bulk-average responses arise because a strong SMA repressor, NKX2.5, slowly exits the nucleus on rigid matrices. NKX2.5 overexpression overrides rigid phenotypes, inhibiting SMA and cell spreading, whereas cytoplasm-localized NKX2.5 mutants degrade in well-spread cells. MSCs thus form a 'mechanical memory' of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state.

489-Pos Board B269 Matrix and Soluble Factor Pathways to Lineage Specification Irena L. Ivanovska, Joe Swift, Kyle Spinler, Dave Dingal, Dennis E. Discher. University of Pennsylvania, Philadelphia, PA, USA. Integration of soluble factors and physical properties of extracellular matrix is likely key to stem cell differentiation but the extent to which those pathways overlap remains unclear. Here motivated by the micromechanics of osteogenic niche we looked at the synergy between matrix stimuli and pharmacological perturbation of Retinoic Acid (RA) pathway on primary and iPSC-derived mesenchymal stem cells (MSCs) including iPSCderived cells from progeria patients towards osteogenesis. Retinoic acid receptor RARG transcription factor is known to regulate nucleoskeletal protein Lamin-A. A cell-by-cell analysis showed that rigid matrix favor higher LMNA and correlates with increased nuclear-to-cytoplasmic ratio of RARG. We found that the Progerin allele of lamin-A is similarly regulated by specific RARG agonist/antagonists. A mechanochemical gene circuit in which tension on lamin-A ultimately favors RARG activity describes well the experimentally observed trend. Scatter-plots of single-cell analyses also show that some cells on stiff substrates fall within the response envelopes of cells cultured on soft substrates and that shared subpopulation of non-responding cells we also found that don’t respond to RA agonist or antagonist regulation of Lamin-A. RA antagonist drove laminA dependent upregulation of osteogenic markers on rigid substrates and pretreated xenografts showed bone-level calcification suggesting a synergistic effect of soluble and insoluble factors on subpopulation of stem cells that are highly mechanoresponsive.

Stem cell differentiation is regulated by both soluble factors and the physical properties of extracellular matrix, but the extent to which differentiation pathways are distinct or overlap is often unclear. Here, the micromechanical stiffness of the collagenous bone surface together with broad compositional correlations with collagen-I across many soft tissues suggests enzymatic cross-linking of matrix correlates with nucleoskeletal protein lamin-A, with a retinoid receptor RARG, and with induction toward osteogenesis. Collagen films just 2 nm thick on mica were stiffened or not by transglutaminase cross-linking and used as minimal culture substrates for Mesenchymal stem cells (MSCs). Cells pulling on pristine nano-films visibly deformed and aligned with the collagen fibrils, but on cross-linked films, cells spread isotropically as if adhering to a substrate of greater effective stiffness. Cell nuclei also spread and stiffened, with an increase of lamin-A, nuclear localization of RARG, and upregulation of key early and late osteogenic factors. RARG antagonists also increased lamin-A, and enhanced osteogenesis on rigid substrates in vitro as well as in xenografts of MSCs in mice. A model of the underlying Mechanochemical Gene Circuit couples the sensitivity of stem cells to both insoluble and soluble factors, while a proteomic comparison underscores both differences and overlaps in differentiation pathways.

Mechanotransduction pathways convert forces that stress and strain structures within cells into gene expression levels that impact development, homeostasis, and disease. The levels of some key structural proteins in the nucleus, cytoskeleton, or extracellular matrix have been recently reported to scale with tissue- and cell-level forces or mechanical properties such as stiffness, and so the mathematics of mechanotransduction becomes important to understand. Here, we show that if a given structural protein positively regulates its own gene expression, then stresses need only inhibit degradation of that protein to achieve stable, mechanosensitive gene expression. This basic use-it-or-lose-it module is illustrated by application to meshworks of nuclear lamin A, minifilaments of myosin II, and extracellular matrix collagen fibers—all of which possess filamentous coiled-coil/supercoiled structures. Past experiments not only suggest that tension suppresses protein degradation mediated and/or initiated by various enzymes but also that transcript levels vary with protein levels because key transcription factors are regulated by these structural proteins. Coupling between modules occurs within single cells and between cells in tissue, as illustrated during embryonic heart development where cardiac fibroblasts make collagen that cardiomyocytes contract. With few additional assumptions, the basic module has sufficient physics to control key structural genes in both development and disease.

Tissue microenvironments are characterized not only in terms of chemical composition but also by collective properties such as stiffness, which influences the contractility of a cell, its adherent morphology, and even differentiation. The nucleoskeletal protein lamin-A,C increases with matrix stiffness, confers nuclear mechanical properties, and influences differentiation of mesenchymal stem cells (MSCs), whereas B-type lamins remain relatively constant. Here we show in single-cell analyses that matrix stiffness couples to myosin-II activity to promote lamin-A,C dephosphorylation at Ser22, which regulates turnover, lamina physical properties, and actomyosin expression. Lamin-A,C phosphorylation is low in interphase versus dividing cells, and its levels rise with states of nuclear rounding in which myosin-II generates little to no tension. Phosphorylated lamin-A,C localizes to nucleoplasm, and phosphorylation is enriched on lamin-A,C fragments and is suppressed by a cyclin-dependent kinase (CDK) inhibitor. Lamin-A,C knockdown in primary MSCs suppresses transcripts predominantly among actomyosin genes, especially in the serum response factor (SRF) pathway. Levels of myosin-IIA thus parallel levels of lamin-A,C, with phosphosite mutants revealing a key role for phosphoregulation. In modeling the system as a parsimonious gene circuit, we show that tension-dependent stabilization of lamin-A,C and myosin-IIA can suitably couple nuclear and cell morphology downstream of matrix mechanics.

Harnessing pluripotent stem cells for functional regeneration of various solid tissues, for modeling of disease, or for drug testing and development has been hampered by a poor understanding of the microenvironmental factors that direct stem cell fate. Unlike bone marrow reconstitution with hematopoietic stem cells, which has succeeded for decades in the clinic, implantation of embryonic or induced pluripotent stem (iPS) cells is not clinically tenable because it causes teratomas—tumor tissues resembling more than one germ layer (1, 2); lineage differentiation before transplantation is currently believed to be necessary for therapeutic applications. In vitro, incomplete differentiation and complex (and potentially expensive) differentiation protocols have prevented human embryonic stem cells and iPS cells [collectively referred to as human pluripotent stem cells (hPSCs)] from reaching their full potential for both pharmaceutical applications and basic and translational research. There is clearly an urgent need for the development of simple, reproducible, and scalable technologies that enhance hPSC differentiation along specific and well-defined pathways. In PNAS, Wrighton et al. (3) begin to unravel the many signaling pathways that drive hPSCs into the three primary germ layers. Remarkably, simple differences in insoluble cues are shown to drive cells toward an ectodermal or mesendodermal fate in a context-specific soluble milieu. They identify the balance between protein kinase B (Akt)/integrin-linked kinase (ILK) and Smad pathways as being the downstream determinant of cell fate (Fig. 1A).

Early in embryogenesis, the heart begins its rhythmic contractions as a tube that helps perfuse the nascent vasculature, but the embryonic heart soon changes shape and mechanical properties, like many other developing organs. A key question in the field is whether stresses in development impact the underlying gene circuits and, if so, how? Here, we attempt to address this question as we review the mechanical maturation of heart - and, to a limited extent, lung and blood - with a focus on a few key abundant structural proteins whose expression dynamics have been suggested to be directly sensitive to mechanical stress. In heart maturation, proliferating fibroblasts deposit increasing amounts of collagenous matrix in parallel with cardiomyocytes expressing more sarcomeric proteins that increase the contractile stress and strength of the tissue, which in turn pumps more blood at higher stress throughout the developing vasculature. Feedback of beating cardiomyocytes on the expression of matrix by fibroblasts seems a reasonable model, with both synthesis and turnover of matrix and contractile elements achieving a suitable balance. Based on emerging evidence for coiled-coil biopolymers that are tension-stabilized against degradation, a minimal network model of a dynamic cell-matrix interaction is proposed. This same concept is extended to nuclear mechanics as regulated by stress on the nuclear structural proteins called lamins, which are examined in part because of the prominence of mutations in these coiled-coil proteins in diseases of the heart, amongst other organs/tissues. Variations in lamin levels during development and across adult tissues are to some extent known and appear to correlate with extracellular matrix mechanics, which we illustrate across heart, lung, and blood development. The formal perspective here on the mechanochemistry of tissue development and homeostasis could provide a useful framework for 'big data' quantitative biology, particularly of stress-sensitive differentiation, maturation, and disease processes.

Materials-based control of stem cell fate is beginning to be rigorously combined with traditional soluble-factor approaches to better understand the cells' behaviour and maximize their potential for therapy.