RESUMEN
The formation and organization of complex blood vessel networks rely on various biophysical forces, yet the mechanisms governing endothelial cell-cell interactions under different mechanical inputs are not well understood. Using the dorsal longitudinal anastomotic vessel (DLAV) in zebrafish as a model, we studied the roles of multiple biophysical inputs and cerebral cavernous malformation (CCM)-related genes in angiogenesis. Our research identifies heg1 and krit1 (ccm1) as crucial for the formation of endothelial cell-cell interfaces during anastomosis. In mutants of these genes, cell-cell interfaces are entangled with fragmented apical domains. A Heg1 live reporter demonstrated that Heg1 is dynamically involved in the oscillatory constrictions along cell-cell junctions, whilst a Myosin live reporter indicated that heg1 and krit1 mutants lack actomyosin contractility along these junctions. In wild-type embryos, the oscillatory contractile forces at junctions refine endothelial cell-cell interactions by straightening junctions and eliminating excessive cell-cell interfaces. Conversely, in the absence of junctional contractility, the cell-cell interfaces become entangled and prone to collapse in both mutants, preventing the formation of a continuous luminal space. By restoring junctional contractility via optogenetic activation of RhoA, contorted junctions are straightened and disentangled. Additionally, haemodynamic forces complement actomyosin contractile forces in resolving entangled cell-cell interfaces in both wild-type and mutant embryos. Overall, our study reveals that oscillatory contractile forces governed by Heg1 and Krit1 are essential for maintaining proper endothelial cell-cell interfaces and thus for the formation of a continuous luminal space, which is essential to generate a functional vasculature.
RESUMEN
Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis.
Asunto(s)
Antígenos CD/genética , Encéfalo/metabolismo , Cadherinas/genética , Neovascularización Fisiológica/genética , Receptores de Lisoesfingolípidos/genética , Vía de Señalización Wnt , Proteínas de Pez Cebra/genética , Pez Cebra/genética , beta Catenina/genética , Animales , Animales Modificados Genéticamente , Antígenos CD/metabolismo , Barrera Hematoencefálica/crecimiento & desarrollo , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/crecimiento & desarrollo , Cadherinas/metabolismo , Capilares/crecimiento & desarrollo , Capilares/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Circulación Cerebrovascular/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo , beta Catenina/metabolismo , Proteína Fluorescente RojaRESUMEN
Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement.
Asunto(s)
Actomiosina/genética , Regulación del Desarrollo de la Expresión Génica , Fosfatasa de Miosina de Cadena Ligera/genética , Neovascularización Fisiológica/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas/genética , Proteínas Represoras/genética , Actomiosina/metabolismo , Animales , Tipificación del Cuerpo/genética , Embrión de Mamíferos , Embrión no Mamífero , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Pulmón/irrigación sanguínea , Pulmón/citología , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Retina/citología , Retina/crecimiento & desarrollo , Retina/metabolismo , Transducción de Señal , Pez CebraRESUMEN
Angiogenesis and vascular remodeling are driven by extensive endothelial cell movements. Here, we present in vivo evidence that endothelial cell movements are associated with oscillating lamellipodia-like structures, which emerge from cell junctions in the direction of cell movements. High-resolution time-lapse imaging of these junction-based lamellipodia (JBL) shows dynamic and distinct deployment of junctional proteins, such as F-actin, VE-cadherin and ZO1, during JBL oscillations. Upon initiation, F-actin and VE-cadherin are broadly distributed within JBL, whereas ZO1 remains at cell junctions. Subsequently, a new junction is formed at the front of the JBL, which then merges with the proximal junction. Rac1 inhibition interferes with JBL oscillations and disrupts cell elongation-similar to a truncation in ve-cadherin preventing VE-cad/F-actin interaction. Taken together, our observations suggest an oscillating ratchet-like mechanism, which is used by endothelial cells to move over each other and thus provides the physical means for cell rearrangements.
Asunto(s)
Actinas/metabolismo , Antígenos CD/fisiología , Cadherinas/fisiología , Movimiento Celular/fisiología , Células Endoteliales/fisiología , Seudópodos/fisiología , Animales , Animales Modificados Genéticamente , Comunicación Celular/fisiología , Embrión no Mamífero , Uniones Intercelulares/fisiología , Proteínas de Pez Cebra/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
VCP/p97 (valosin containing protein) is a key regulator of cellular proteostasis. It orchestrates protein turnover and quality control in vivo, processes fundamental for proper cell function. In humans, mutations in VCP lead to severe myo- and neuro-degenerative disorders such as inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS) or and hereditary spastic paraplegia (HSP). We analyzed here the in vivo role of Vcp and its novel interactor Washc4/Swip (WASH complex subunit 4) in the vertebrate model zebrafish (Danio rerio). We found that targeted inactivation of either Vcp or Washc4, led to progressive impairment of cardiac and skeletal muscle function, structure and cytoarchitecture without interfering with the differentiation of both organ systems. Notably, loss of Vcp resulted in compromised protein degradation via the proteasome and the macroautophagy/autophagy machinery, whereas Washc4 deficiency did not affect the function of the ubiquitin-proteasome system (UPS) but caused ER stress and interfered with autophagy function in vivo. In summary, our findings provide novel insights into the in vivo functions of Vcp and its novel interactor Washc4 and their particular and distinct roles during proteostasis in striated muscle cells.