A 3D biomimetic model of lymphatics reveals cell-cell junction tightening and lymphedema via a cytokine-induced ROCK2/JAM-A complex.

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

'Chip'-ing away at morphogenesis - application of organ-on-chip technologies to study tissue morphogenesis.

Emergent cell behaviors that drive tissue morphogenesis are the integrated product of instructions from gene regulatory networks, mechanics and signals from the local tissue microenvironment. How these discrete inputs intersect to coordinate diverse morphogenic events is a critical area of interest. Organ-on-chip technology has revolutionized the ability to construct and manipulate miniaturized human tissues with organotypic three-dimensional architectures in vitro. Applications of organ-on-chip platforms have increasingly transitioned from proof-of-concept tissue engineering to discovery biology, furthering our understanding of molecular and mechanical mechanisms that operate across biological scales to orchestrate tissue morphogenesis. Here, we provide the biological framework to harness organ-on-chip systems to study tissue morphogenesis, and we highlight recent examples where organ-on-chips and associated microphysiological systems have enabled new mechanistic insight in diverse morphogenic settings. We further highlight the use of organ-on-chip platforms as emerging test beds for cell and developmental biology.

A non-canonical Notch complex regulates adherens junctions and vascular barrier function.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.

Conversation before crossing: dissecting metastatic tumor-vascular interactions in microphysiological systems.

Tumor metastasis via the circulation requires crossing the vascular barrier twice: first, during intravasation when tumor cells disseminate from the primary site through proximal vasculature, and second, during extravasation, when tumor cells exit the circulation to form distant metastatic seeds. During these key metastatic events, chemomechanical signaling between tumor cells and endothelial cells elicits reciprocal changes in cell morphology and behavior that are necessary to breach the vessel wall. Existing experimental systems have provided a limited understanding of the diverse mechanisms underlying tumor-endothelial interactions during intravasation and extravasation. Recent advances in microphysiological systems have revolutionized the ability to generate miniaturized human tissues with tailored three-dimensional architectures, physiological cell interfaces, and precise chemical and physical microenvironments. By doing so, microphysiological systems enable experimental access to complex morphogenic processes associated with human tumor progression with unprecedented resolution and biological control. Here, we discuss recent examples in which microphysiological systems have been leveraged to reveal new mechanistic insight into cellular and molecular control systems operating at the tumor-endothelial interface during intravasation and extravasation.

Ubiquitylation of phosphatidylinositol 4-phosphate 5-kinase type I γ by HECTD1 regulates focal adhesion dynamics and cell migration.

Phosphatidylinositol 4-phosphate 5-kinase type I γ (PIPKIγ90) binds talin and localizes at focal adhesions (FAs). Phosphatidylinositol (4,5)-bisphosphate (PIP2) generated by PIPKIγ90 is essential for FA formation and cell migration. On the other hand, PIPKIγ90 and the ß-integrin tail compete for overlapping binding sites on talin. Enhanced PIPKIγ90-talin interaction suppresses talin binding to the ß-integrin. It is unknown how PIPKIγ90 is removed from the PIPKIγ90-talin complex after on-site PIP2 production during cell migration. Here we show that PIPKIγ90 is a substrate for HECTD1, an E3 ubiquitin ligase regulating cell migration. HECTD1 ubiquitinated PIPKIγ90 at lysine 97 and resulted in PIPKIγ90 degradation. Expression of the mutant PIPKIγ90(K97R) enhanced PIP2 and PIP3 production, inhibited FA assembly and disassembly and inhibited cancer cell migration, invasion and metastasis. Interestingly, mutation at tryptophan 647 abolished the inhibition of PIPKIγ90(K97R) on FA dynamics and partially rescued cancer cell migration and invasion. Thus, cycling PIPKIγ90 ubiquitylation by HECTD1 and consequent degradation remove PIPKIγ90 from talin after on-site PIP2 production, providing an essential regulatory mechanism for FA dynamics and cell migration.

Micro-environmental control of cell migration--myosin IIA is required for efficient migration in fibrillar environments through control of cell adhesion dynamics.

Recent evidence suggests that organization of the extracellular matrix (ECM) into aligned fibrils or fibril-like ECM topographies promotes rapid migration in fibroblasts. However, the mechanisms of cell migration that are altered by these changes in micro-environmental topography remain unknown. Here, using 1D fibrillar migration as a model system for oriented fibrillar 3D matrices, we find that fibroblast leading-edge dynamics are enhanced by 1D fibrillar micropatterns and demonstrate a dependence on the spatial positioning of cell adhesions. Although 1D, 2D and 3D matrix adhesions have similar assembly kinetics, both 1D and 3D adhesions are stabilized for prolonged periods, whereas both paxillin and vinculin show slower turnover rates in 1D adhesions. Moreover, actin in 1D adhesions undergoes slower retrograde flow than the actin that is present in 2D lamellipodia. These data suggest an increase in mechanical coupling between adhesions and protrusive machinery. Experimental reduction of contractility resulted in the loss of 1D adhesion structure and stability, with scattered small and unstable adhesions, and an uncoupling of adhesion protein-integrin stability. Genetic ablation of myosin IIA (MIIA) or myosin IIB (MIIB) isoforms revealed that MIIA is required for efficient migration in restricted environments as well as adhesion maturation, whereas MIIB helps to stabilize adhesions beneath the cell body. These data suggest that restricted cell environments, such as 1D patterns, require cellular contraction through MIIA to enhance adhesion stability and coupling to integrins behind the leading edge. This increase in mechanical coupling allows for greater leading-edge protrusion and rapid cell migration.

Regulation of cell adhesion and migration by cell-derived matrices.

Three-dimensional in vitro extracellular matrix models provide a physiological alternative to regular two-dimensional cell culture, though they lack the full diversity of molecular composition and physical properties of whole-animal systems. Cell-derived matrices are extracellular matrices that are the product of matrix secretion and assembly by cells cultured at high density in vitro. After the removal of the cells that produced the matrix, an assembled matrix scaffold is left that closely mimics native stromal fiber organization and molecular content. Cell-derived matrices have been shown to impart in vivo-like responses to cells cultured in these matrices. In this review, we focus on mechanisms through which the distinct molecular and topographical composition of cell-derived matrices directs cellular behavior, specifically through regulation of cell-matrix adhesions and subsequent contributions to the process of cell migration.

Quantitative Comparison of Monomeric StayGold Variants Using Protein Nanocages in Living Cells.

To standardize comparison of fluorescent proteins and independently determine which monomeric StayGold variant is best for live microscopy, we analyzed fluorescent protein tagged I3-01 peptides that self-assemble into stable sixty subunit dodecahedrons inside live cells. We find mStayGold is 3-fold brighter and 3-fold more photostable compared with EGFP and superior to other monomeric variants in mammalian cytoplasm. In addition, analysis of intracellular nanocage diffusion confirms the monomeric nature of mStayGold.

Arp2/3 complex activity enables nuclear YAP for naïve pluripotency of human embryonic stem cells.

Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

Doublecortin reinforces microtubules to promote growth cone advance in soft environments.

Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, the molecular function of DCX in neuron morphogenesis remains unknown and controversial. We demonstrate that DCX function is intimately linked to its microtubule-binding activity. By using human induced pluripotent stem cell (hiPSC)- derived cortical i 3 Neurons genome engineered to express mEmerald-tagged DCX from the endogenous locus, we find that DCX-MT interactions become highly polarized very early during neuron morphogenesis. DCX becomes enriched only on straight microtubules in advancing growth cones with approximately 120 DCX molecules bound per micrometer of growth cone microtubule. At a similar saturation, microtubule-bound DCX molecules begin to impede lysosome transport, and thus can potentially control growth cone organelle entry. In addition, by comparing control, DCX-mEmerald and knockout DCX -/Y i 3 Neurons, we find that DCX stabilizes microtubules in the growth cone peripheral domain by reducing the microtubule catastrophe frequency and the depolymerization rate. DCX -/Y i 3 Neuron morphogenesis was inhibited in soft microenvironments that mimic the viscoelasticity of brain tissue and DCX -/Y neurites failed to grow toward brain-derived neurotrophic factor (BDNF) gradients. Together with high resolution traction force microscopy data, we propose a model in which DCX-decorated, rigid growth cone microtubules provide intracellular mechanical resistance to actomyosin generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated forces in the growth cone periphery may be insufficient for productive growth cone advance. These data provide a new mechanistic understanding of how DCX mutations cause lissencephaly-spectrum brain malformations by impacting growth cone dynamics during neuron morphogenesis in physiological environments.

Proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage in prenatal human brain.

Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45+ cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45+ cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain's innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants.

Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion.

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here we employ a 3D organotypic model of a ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from loss of a transcription-independent Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover a Notch1-FAM83H interaction underlies stabilization of adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation, and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion.

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here, we employ a 3D model of a human ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from the loss of a transcription-independent, Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover that a Notch1-FAM83H interaction underlies control of epithelial adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

A convolutional neural network STIFMap reveals associations between stromal stiffness and EMT in breast cancer.

Intratumor heterogeneity associates with poor patient outcome. Stromal stiffening also accompanies cancer. Whether cancers demonstrate stiffness heterogeneity, and if this is linked to tumor cell heterogeneity remains unclear. We developed a method to measure the stiffness heterogeneity in human breast tumors that quantifies the stromal stiffness each cell experiences and permits visual registration with biomarkers of tumor progression. We present Spatially Transformed Inferential Force Map (STIFMap) which exploits computer vision to precisely automate atomic force microscopy (AFM) indentation combined with a trained convolutional neural network to predict stromal elasticity with micron-resolution using collagen morphological features and ground truth AFM data. We registered high-elasticity regions within human breast tumors colocalizing with markers of mechanical activation and an epithelial-to-mesenchymal transition (EMT). The findings highlight the utility of STIFMap to assess mechanical heterogeneity of human tumors across length scales from single cells to whole tissues and implicates stromal stiffness in tumor cell heterogeneity.

Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation.

Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA-driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA-driven VMs.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling.

Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

Monte Carlo analysis of neck linker extension in kinesin molecular motors.

Kinesin stepping is thought to involve both concerted conformational changes and diffusive movement, but the relative roles played by these two processes are not clear. The neck linker docking model is widely accepted in the field, but the remainder of the step--diffusion of the tethered head to the next binding site--is often assumed to occur rapidly with little mechanical resistance. Here, we investigate the effect of tethering by the neck linker on the diffusive movement of the kinesin head, and focus on the predicted behavior of motors with naturally or artificially extended neck linker domains. The kinesin chemomechanical cycle was modeled using a discrete-state Markov chain to describe chemical transitions. Brownian dynamics were used to model the tethered diffusion of the free head, incorporating resistive forces from the neck linker and a position-dependent microtubule binding rate. The Brownian dynamics and chemomechanical cycle were coupled to model processive runs consisting of many 8 nm steps. Three mechanical models of the neck linker were investigated: Constant Stiffness (a simple spring), Increasing Stiffness (analogous to a Worm-Like Chain), and Reflecting (negligible stiffness up to a limiting contour length). Motor velocities and run lengths from simulated paths were compared to experimental results from Kinesin-1 and a mutant containing an extended neck linker domain. When tethered by an increasingly stiff spring, the head is predicted to spend an unrealistically short amount of time within the binding zone, and extending the neck is predicted to increase both the velocity and processivity, contrary to experiments. These results suggest that the Worm-Like Chain is not an adequate model for the flexible neck linker domain. The model can be reconciled with experimental data if the neck linker is either much more compliant or much stiffer than generally assumed, or if weak kinesin-microtubule interactions stabilize the diffusing head near its binding site.

3D mesenchymal cell migration is driven by anterior cellular contraction that generates an extracellular matrix prestrain.

We describe a cellular contractile mechanism employed by fibroblasts and mesenchymal cancer cells to migrate in 3D collagen gels. During 3D spreading, fibroblasts strongly deform the matrix. They protrude, polarize, and initiate migration in the direction of highest extracellular matrix (ECM) deformation (prestrain). This prestrain is maintained through anterior cellular contractions behind the leading edge prior to protrusion, coordinating a distinct 3D migration cycle that varies between cell types. Myosin IIA is required for strain polarization, generating anterior contractions, and maintaining prestrain for efficient directional cell migration. Local matrix severing disrupts the matrix prestrain, suppressing directional protrusion. We show that epithelial cancer and endothelial cells rarely demonstrate the sustained prestrain or anterior contractions. We propose that mesenchymal cells sense ECM stiffness in 3D and generate their own matrix prestrain. This requires myosin IIA to generate polarized periodic anterior contractions for maintaining a 3D migration cycle.

Uncovering mutation-specific morphogenic phenotypes and paracrine-mediated vessel dysfunction in a biomimetic vascularized mammary duct platform.

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.