Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6-RACK1-ERK1/2-FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked

Collective Cell Migration Drives Morphogenesis of the Kidney Nephron

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis

JNK2 Promotes Endothelial Cell Alignment under Flow

Endothelial cells in straight, unbranched segments of arteries elongate and align in the direction of flow, a feature which is highly correlated with reduced atherosclerosis in these regions. The mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) is activated by flow and is linked to inflammatory gene expression and apoptosis. We previously showed that JNK activation by flow is mediated by integrins and is observed in cells plated on fibronectin but not on collagen or basement membrane proteins. We now show thatJNK2 activation in response to laminar shear stress is biphasic, with an early peak and a later peak. Activated JNK localizes to focal adhesions at the ends of actin stress fibers, correlates with integrin activation and requires integrin binding to the extracellular matrix. Reducing JNK2 activation by siRNA inhibits alignment in response to shear stress. Cells on collagen, where JNK activity is low, align slowly. These data show that an inflammatory pathway facilitates adaptation to laminar flow, thereby revealing an unexpected connection between adaptation and inflammatory pathways

The role of the β-1,6-endoglucanase gene vegB in physiology and virulence of Verticillium dahliae

The β-1,6-endoglucanase gene (vegB) of Verticillium dahliae was isolated using a genome walking technique. Nucleotide and deduced amino acid sequences of the gene showed high identity with the PAN1 sequence deposited at the Verticillium genome database (Broad Institute), but significant differences in intron numbers and sites of insertion. Detailed in silico analysis, accompanied by sequencing of both genomic and cDNA, as well as RT-PCR experiments, provided the correct size of the gene and the exact number, length and positions of introns. The putative protein of this gene was compared with corresponding β-1,6-endoglucanases from other fungi, and sequences were used to construct a phylogenetic tree. A clear differentiation between enzymes derived from plant pathogenic and mycoparasitic fungi was observed, fully supported by bootstrap data. An internal fragment (1.2kb) of vegB was used to disrupt the wild-type gene of a V. dahliae tomato race 2 strain, and the mutant strain, vegB-, was tested for pathogenicity on tomato plants. Results showed a small but constant reduction in disease symptoms only on eggplants for the vegB- strain in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose was similar to the wild type

Endothelial Shc Regulates Arteriogenesis Through Dual Control of Arterial Specification and Inflammation via the Notch and Nuclear Factor- -Light-Chain-Enhancer of Activated B-Cell Pathways

Arteriogenesis, the shear stress-driven remodeling of collateral arteries, is critical in restoring blood flow to ischemic tissue following a vascular occlusion. Our previous work has shown that the adaptor protein Shc mediates endothelial responses to shear stress in vitro

Platelet Endothelial Cell Adhesion Molecule-1 Mediates Endothelial-Cardiomyocyte Communication and Regulates Cardiac Function

BackgroundDilated cardiomyopathy is characterized by impaired contractility of cardiomyocytes, ventricular chamber dilatation, and systolic dysfunction. Although mutations in genes expressed in the cardiomyocyte are the best described causes of reduced contractility, the importance of endothelial‐cardiomyocyte communication for proper cardiac function is increasingly appreciated. In the present study, we investigate the role of the endothelial adhesion molecule platelet endothelial cell adhesion molecule (PECAM‐1) in the regulation of cardiac function.Methods and ResultsUsing cell culture and animal models, we show that PECAM‐1 expressed in endothelial cells (ECs) regulates cardiomyocyte contractility and cardiac function via the neuregulin‐ErbB signaling pathway. Conscious echocardiography revealed left ventricular (LV) chamber dilation and systolic dysfunction in PECAM‐1−/− mice in the absence of histological abnormalities or defects in cardiac capillary density. Despite deficits in global cardiac function, cardiomyocytes isolated from PECAM‐1−/− hearts displayed normal baseline and isoproterenol‐stimulated contractility. Mechanistically, absence of PECAM‐1 resulted in elevated NO/ROS signaling and NRG‐1 release from ECs, which resulted in augmented phosphorylation of its receptor ErbB2. Treatment of cardiomyocytes with conditioned media from PECAM‐1−/− ECs resulted in enhanced ErbB2 activation, which was normalized by pre‐treatment with an NRG‐1 blocking antibody. To determine whether normalization of increased NRG‐1 levels could correct cardiac function, PECAM‐1−/− mice were treated with the NRG‐1 blocking antibody. Echocardiography showed that treatment significantly improved cardiac function of PECAM‐1−/− mice, as revealed by increased ejection fraction and fractional shortening.ConclusionsWe identify a novel role for PECAM‐1 in regulating cardiac function via a paracrine NRG1‐ErbB pathway. These data highlight the importance of tightly regulated cellular communication for proper cardiac function

Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion

Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces