ABSTRACT
Cell migration is a complex process requiring density and rigidity sensing of the microenvironment to adapt cell migratory speed through focal adhesion and actin cytoskeleton regulation. ICAP-1 (also known as ITGB1BP1), a ß1 integrin partner, is essential for ensuring integrin activation cycle and focal adhesion formation. We show that ICAP-1 is monoubiquitylated by Smurf1, preventing ICAP-1 binding to ß1 integrin. The non-ubiquitylatable form of ICAP-1 modifies ß1 integrin focal adhesion organization and interferes with fibronectin density sensing. ICAP-1 is also required for adapting cell migration in response to substrate stiffness in a ß1-integrin-independent manner. ICAP-1 monoubiquitylation regulates rigidity sensing by increasing MRCKα (also known as CDC42BPA)-dependent cell contractility through myosin phosphorylation independently of substrate rigidity. We provide evidence that ICAP-1 monoubiquitylation helps in switching from ROCK2-mediated to MRCKα-mediated cell contractility. ICAP-1 monoubiquitylation serves as a molecular switch to coordinate extracellular matrix density and rigidity sensing thus acting as a crucial modulator of cell migration and mechanosensing.
Subject(s)
Cell Movement , Extracellular Matrix/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Myotonin-Protein Kinase/metabolism , Ubiquitination , rho-Associated Kinases/metabolism , Adaptor Proteins, Signal Transducing , Animals , Binding Sites , Biomechanical Phenomena , Cell Adhesion , Cell Line , Fibronectins/metabolism , Focal Adhesions/metabolism , Humans , Integrin beta1/chemistry , Integrin beta1/metabolism , Mice , Models, Biological , Signal Transduction , Ubiquitin-Protein Ligases/metabolismABSTRACT
Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor ß1 integrin and occurs in the absence of blood flow. Downregulation of ß1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a ß1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.
Subject(s)
Cell Movement/physiology , Hemangioma, Cavernous, Central Nervous System/metabolism , Integrin beta1/metabolism , Kruppel-Like Transcription Factors/metabolism , Neovascularization, Pathologic/metabolism , Proteins/metabolism , Zebrafish Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Calcium-Binding Proteins , Cell Adhesion/physiology , Cell Movement/genetics , Central Nervous System Neoplasms/metabolism , EGF Family of Proteins , Hemangioma, Cavernous, Central Nervous System/genetics , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Mechanotransduction, Cellular/physiology , Membrane Proteins/metabolism , Mice , Nerve Tissue Proteins/metabolism , RNA, Small Interfering/genetics , Signal Transduction/physiology , ZebrafishABSTRACT
The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of ß1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased ß1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1-deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of ß1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.
Subject(s)
Fibronectins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Integrin beta1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cell Adhesion , Cells, Cultured , Human Umbilical Vein Endothelial Cells/cytology , Humans , Intracellular Signaling Peptides and Proteins/deficiency , KRIT1 Protein , Mice , Mice, Inbred Strains , Mice, Knockout , Microtubule-Associated Proteins/deficiency , Models, Biological , Proto-Oncogene Proteins/deficiencyABSTRACT
Cell migration is an integrated process requiring the continuous coordinated assembly and disassembly of adhesion structures. How cells orchestrate adhesion turnover is only partially understood. We provide evidence for a novel mechanistic insight into focal adhesion (FA) dynamics by demonstrating that integrin cytoplasmic domain-associated protein 1 (ICAP-1) slows down FA assembly. Live cell imaging, which was performed in both Icap-1-deficient mouse embryonic fibroblasts and cells expressing active beta(1) integrin, shows that the integrin high affinity state favored by talin is antagonistically controlled by ICAP-1. This affinity switch results in modulation in the speed of FA assembly and, consequently, of cell spreading and migration. Unexpectedly, the ICAP-1-dependent decrease in integrin affinity allows cell sensing of matrix surface density, suggesting that integrin conformational changes are important in mechanotransduction. Our results clarify the function of ICAP-1 in cell adhesion and highlight the central role it plays in the cell's integrated response to the extracellular microenvironment.
