A Novel, Matrix-Specific GEF/GAP Interaction Regulates Rho GTPase Crosstalk Critical for 3D Collagen Migration

Differential activation of the Rho family GTPases, Cdc42, Rac1, and RhoA, helps to govern the distinct morphological and migratory phenotypes downstream of adhesion to different extracellular matrix (ECM) proteins. However, it is not known how specific GTPase-dependent signaling pathways are activated in response to different ECM ligands. We hypothesized that adhesion to different ECM molecules, such as collagen and fibronectin, will trigger selective regulation of guanine nucleotide exchange factors (GEFs) to regulate the appropriate matrix-specific cell migratory response. We utilized an affinity precipitation-based mass spectrometry screen to isolate active GEFs from primary human fibroblasts migrating in collagen, fibronectin, or ECM-free environments. Among the GEFs identified, we found that βPix, a Rac1/Cdc42 GEF, was robustly activated only during migration in collagen matrices. Knockdown of βPix led to a collagen-specific migration defect characterized by rapid, spatially-deregulated protrusions, rounded morphology, the absence of stable leading and trailing edges, and robust contraction of the adjacent collagen matrix. In contrast to fibroblasts migrating on fibronectin, βPix in cells migrating in collagen did not localize to focal adhesions, but instead transiently accumulated on the membrane adjacent to areas of cellular protrusion as determined by live cell imaging, immunofluorescence staining, and biochemical iii fractionation. Mechanistically, we found that βPix is critical for efficient migration in fibrillar collagen environments by restraining RhoA signaling. Live FRET imaging and RNAi knockdown established this suppression occurs through a mechanism of Rho GTPase crosstalk between Cdc42 and RhoA that is regulated by a collagen-specific functional interaction between βPix and the GTPase activating protein (GAP) srGAP1. Additionally, we identified that binding of α2β1 integrin to fibrillar collagen leads, through PP2A, to loss of phosphorylation at T526 on βPix and promotes association with srGAP1. We conclude that ECM-dependent regulation of a specific GEF is a fundamental mechanism of migration in different microenvironments. Our results reveal a conserved, matrix-specific pathway controlling migration involving a GEF/GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.Doctor of Philosoph

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

Rho GEFs and GAPs: Emerging integrators of extracellular matrix signaling

Investigating cell migration in 3D settings has revealed that specific extracellular matrix environments require differential activities of the Rho GTPases for efficient migration. However, it is largely unknown how the activities of specific Rho GTPases are modulated to direct cell migration in response to different extracellular matrix cues. We have recently reported that extracellular matrix-dependent regulation of a specific Rho GEF is a fundamental mechanism governing cell migration in different microenvironments, providing a direct mechanism for extracellular matrix-specific regulation of Rho GTPase activity directing cell motility. We discovered that the Rho GEF βPix has a unique function during cell migration in fibrillar collagen environments by restraining RhoA signaling through a conserved signaling axis involving Cdc42 and the Rho GAP srGAP1. In this Commentary, we expand upon this new pathway and discuss potential mechanotransductive and therapeutic applications. Additionally, we speculate on a generalized role for Rho GEFs and GAPs in providing localized, context-dependent responses to the cellular microenvironment during cell migration and other cellular processes

Forces and mechanotransduction in 3D vascular biology

The effects of hemodynamic and interstitial mechanical forces on endothelial biology in vivo have been appreciated for over half a century, regulating vessel network development, homeostatic function, and progression of vascular disease. Investigations using cultures of endothelial cells on two-dimensional (2D) substrates have elucidated important mechanisms by which microenvironmental stresses are sensed and transduced into chemical signaling responses. However recent studies in vivo and in three-dimensional (3D) in vitro models of vascular beds have enabled the investigation of forces and cellular behaviors previously not possible in traditional 2D culture systems. These studies support a developing paradigm that the 3D chemo-mechanical architecture of the vascular niche impacts how endothelial cells both sense and respond to microenvironmental forces. We present evolving concepts in endothelial force sensing and mechanical signaling and highlight recent insights gained from in vivo and 3D in vitro vascular models

Rho GEFs and GAPs: Emerging integrators of extracellular matrix signaling

Investigating cell migration in 3D settings has revealed that specific extracellular matrix environments require differential activities of the Rho GTPases for efficient migration. However, it is largely unknown how the activities of specific Rho GTPases are modulated to direct cell migration in response to different extracellular matrix cues. We have recently reported that extracellular matrix-dependent regulation of a specific Rho GEF is a fundamental mechanism governing cell migration in different microenvironments, providing a direct mechanism for extracellular matrix-specific regulation of Rho GTPase activity directing cell motility. We discovered that the Rho GEF βPix has a unique function during cell migration in fibrillar collagen environments by restraining RhoA signaling through a conserved signaling axis involving Cdc42 and the Rho GAP srGAP1. In this Commentary, we expand upon this new pathway and discuss potential mechanotransductive and therapeutic applications. Additionally, we speculate on a generalized role for Rho GEFs and GAPs in providing localized, context-dependent responses to the cellular microenvironment during cell migration and other cellular processes

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

Microfabricated blood vessels for modeling the vascular transport barrier

The vascular endothelium forms the inner lining of blood vessels and actively regulates vascular permeability in response to chemical and physical stimuli. Understanding the molecular pathways and mechanisms that regulate the permeability of blood vessels is of critical importance for developing therapies for cardiovascular dysfunction and disease. Recently, we developed a novel microfluidic human engineered microvessel (hEMV) platform to enable controlled blood flow through a human endothelial lumen within a physiologic 3D extracellular matrix (ECM) into which pericytes and other stromal cells can be introduced to recapitulate tissue-specific microvascular physiology. This protocol describes how to design and fabricate the silicon hEMV device master molds (takes ~1 week) and elastomeric substrates (takes 3 d); how to seed, culture, and apply calibrated fluid shear stress to hEMVs (takes 1-7 d); and how to assess vascular barrier function (takes 1 d) and perform immunofluorescence imaging (takes 3 d)