RESUMO
Cell-cell contact formation of polarized epithelial cells is a multi-step process that involves the co-ordinated activities of Rho family small GTPases. Consistent with the central role of Rho GTPases, a number of Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) have been identified at cell-cell junctions at various stages of junction maturation. As opposed to RhoGEFs and RhoGAPs, the role of Rho GDP dissociation inhibitors (GDIs) during cell-cell contact formation is poorly understood. Here, we have analyzed the role of RhoGDI1/ARHGDIA, a member of the RhoGDI family, during cell-cell contact formation of polarized epithelial cells. Depletion of RhoGDI1 delays the development of linear cell-cell junctions and the formation of barrier-forming tight junctions. In addition, RhoGDI1 depletion impairs the ability of cells to stop migration in response to cell collision and increases the migration velocity of collectively migrating cells. We also find that the cell adhesion receptor JAM-A promotes the recruitment of RhoGDI1 to cell-cell contacts. Our findings implicate RhoGDI1 in various processes involving the dynamic reorganization of cell-cell junctions.
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Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca2+ levels. Through this they act as Ca2+-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca2+-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.
Assuntos
Anexinas , Paladar , Animais , Anexinas/química , Membrana Celular/metabolismo , Transdução de Sinais , Transporte Biológico , Cálcio/metabolismo , Mamíferos/metabolismoRESUMO
Weibel Palade bodies (WPB) are lysosome-related secretory organelles of endothelial cells. Commonly known for their main cargo, the platelet and leukocyte receptors von-Willebrand factor (VWF) and P-selectin, WPB play a crucial role in hemostasis and inflammation. Here, the authors identify the glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) as a WPB cargo protein and show that GDPD5 is transported to WPB following uptake from the plasma membrane via an unique endocytic transport route. GDPD5 cleaves GPI-anchored, plasma membrane-resident proteins within their GPI-motif, thereby regulating their local activity. The authors identify a novel target of GDPD5 , the complement regulator CD59, and show that it is released from the endothelial surface by GDPD5 following WPB exocytosis. This results in increased deposition of complement components and can enhance local inflammatory and thrombogenic responses. Thus, stimulus-induced WPB exocytosis can modify the endothelial cell surface by GDPD5-mediated selective release of a subset of GPI-anchored proteins.
Assuntos
Exocitose , Diester Fosfórico Hidrolases , Corpos de Weibel-Palade , Corpos de Weibel-Palade/metabolismo , Exocitose/fisiologia , Humanos , Diester Fosfórico Hidrolases/metabolismo , Células Endoteliais/metabolismoRESUMO
Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.
Assuntos
Selectina-P , Canais de Dois Poros , Camundongos , Humanos , Animais , Selectina-P/metabolismo , Células Endoteliais/metabolismo , Corpos de Weibel-Palade/metabolismo , Adesão Celular , Leucócitos/metabolismo , Endotélio Vascular/metabolismoRESUMO
Scribble (Scrib) is a multidomain polarity protein and member of the leucine-rich repeat and PDZ domain (LAP) protein family. A loss of Scrib expression is associated with disturbed apical-basal polarity and tumor formation. The tumor-suppressive activity of Scrib correlates with its membrane localization. Despite the identification of numerous Scrib-interacting proteins, the mechanisms regulating its membrane recruitment are not fully understood. Here, we identify the cell adhesion receptor TMIGD1 as a membrane anchor of Scrib. TMIGD1 directly interacts with Scrib through a PDZ domain-mediated interaction and recruits Scrib to the lateral membrane domain in epithelial cells. We characterize the association of TMIGD1 with each Scrib PDZ domain and describe the crystal structure of the TMIGD1 C-terminal peptide complexed with PDZ domain 1 of Scrib. Our findings describe a mechanism of Scrib membrane localization and contribute to the understanding of the tumor-suppressive activity of Scrib.
Assuntos
Células Epiteliais , Complexo Glicoproteico GPIb-IX de Plaquetas , Membranas , Adesão CelularRESUMO
Cholesterol is a sterol lipid found in all higher eukaryotic organisms. It is required to consolidate the basic structural integrity and dynamic principles of cellular membranes and participates in many essential cellular processes that range from signal transduction to membrane traffic and metabolism. Moreover, a growing number of clinically highly relevant diseases such as immunological disorders or cancer has been linked to changes or misfunctions in cholesterol homeostasis. Therefore, the development of molecular tools that help to further unravel the role of cholesterol in essential cellular processes is of high relevance. Herein, we report the synthesis and proof-of-concept of a novel bifunctional imidazolium-based cholesterol analog (X-CHIM) that we envision to serve as a broadly applicable tool for the simultaneous investigation of cellular cholesterol distributions as well as cholesterol-protein interactions.
Assuntos
Colesterol , Esteróis , Colesterol/química , Esteróis/química , Membrana Celular/metabolismo , Homeostase , Transdução de SinaisRESUMO
The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, it is demonstrated that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) VAMP2, which is crucial for efficient membrane repair. Thus, the newly identified Ca2+ -evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.
Assuntos
Células Endoteliais , Proteínas SNARE , Humanos , Células Endoteliais/metabolismo , Membrana Celular/metabolismo , Proteínas SNARE/metabolismo , Endossomos/metabolismo , Exocitose/fisiologiaRESUMO
After damage, cells repair their plasma membrane in an active process that is driven by Ca2+ entering through the wound. This triggers a range of Ca2+-regulated events such as the translocation of different Ca2+-binding proteins to the wound site which likely function in the repair process. The translocated proteins include Ca2+/phospholipid binding proteins of the annexin (ANX) family and S100A11, an EF hand-type Ca2+-binding protein which can interact with ANX. The molecular mechanism by which S100A11 mediates PM wound repair remains poorly understood although it likely involves interactions with ANX. Here, using S100A11 knockout endothelial cells and expression of S100A11 mutants, we show that endothelial S100A11 is essential for efficient plasma membrane wound repair and engages in Ca2+-dependent interactions with ANXA1 and ANXA2 through its C-terminal extension (residues 93-105). ANXA2 but not ANXA1 translocation to the wound is substantially inhibited in the absence of S100A11; however, the repair defect in S100A11 knockout cells is rescued by ectopic expression of an ANX interaction-defective S100A11 mutant, suggesting an ANX-independent role of S100A11 in membrane wound repair. In search for other interaction partners that could mediate this action of S100A11 we identify extended synaptotagmin 1 (E-Syt1), a protein tether that regulates endoplasmic reticulum-plasma membrane contact sites. E-Syt1 binds to S100A11 in the presence of Ca2+ and depletion of E-Syt1 interferes with wound site recruitment of S100A11 and proper membrane resealing. Thus, the role of S100A11 in membrane wound repair does not exclusively dependent on ANX interactions and a Ca2+-regulated S100A11-E-Syt1 complex acts as a yet unrecognized component of the membrane resealing machinery.
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Entosis is a cell competition process during which tumor cells engulf other tumor cells. It is initiated by metabolic stress or by loss of matrix adhesion, and it provides the winning cell with resources derived from the internalized cell. Using micropatterns as substrates for single cell migration, we find that the depletion of the cell adhesion receptor JAM-A strongly increases the rate of entosis in matrix-adherent cells. The activity of JAM-A in suppressing entosis depends on phosphorylation at Tyr280, which is a binding site for C-terminal Src kinase, and which we have previously found to regulate tumor cell motility and contact inhibition of locomotion (CIL). Loss of JAM-A triggers entosis in matrix-adherent cells but not matrix-deprived cells. Our findings strongly suggest that the increased motility and the perturbed CIL response after the depletion of JAM-A promote entotic cell engulfment, and they link a dysregulation of CIL to entosis in breast cancer cells.
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Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9-tetramers preventing TLR4-binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the quaternary structure of these S100-proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100-dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100-tetramer/CD69).
Assuntos
Monócitos , Receptor 4 Toll-Like , Humanos , Receptor 4 Toll-Like/metabolismo , Cálcio/metabolismo , Calgranulina B/metabolismo , Calgranulina A/química , Calgranulina A/metabolismo , Inflamação/metabolismoRESUMO
Intestinal epithelial cells absorb nutrients through the brush border, composed of dense arrays of highly ordered microvilli at their apical membranes. A protocadherin-based intermicrovillar adhesion complex localized at microvilli tips mediates microvilli packing and organization. Here, we identified a second adhesion complex localized at the proximal base region of microvilli. This complex contained the immunoglobulin superfamily member TMIGD1, which directly interacted with the microvillar scaffolding proteins EBP50 and E3KARP. Complex formation with EBP50 required the activation of EBP50 by the actin-binding protein ezrin and was enhanced by the dephosphorylation of Ser162 in the PDZ2 domain of EBP50 by the phosphatase PP1α. Binding of the EBP50-ezrin complex to TMIGD1 enhanced the dynamic turnover of EBP50 at microvilli. Enterocyte-specific inactivation of Tmigd1 in mice resulted in microvillar blebbing, loss of intermicrovillar adhesion, and perturbed brush border formation. Thus, we identified a second adhesion complex in microvilli and propose a mechanism that promotes microvillar formation and dynamics.
Assuntos
Células Epiteliais , Intestinos , Glicoproteínas de Membrana/metabolismo , Animais , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/metabolismoRESUMO
Cationic amphiphiles have been reported to show broad antimicrobial activity. The potential for antimicrobial resistance to these molecules is low owing to their general cell membrane permeabilizing mode of action. However, their applications are often limited by toxicity resulting from their low selectivity for microbial cell membranes. Herein, we report a library of cationic, steroid-based imidazolium amphiphiles that show tunable antifungal activity in a variety of fungal pathogens of the genus Candida. We show that adoption of an ergosterol-derived backbone increases antifungal activity while modestly affecting hemolytic activity, thereby increasing overall selectivity by more than 8-fold in comparison to cholesterol-derived imidazolium salts. We hypothesize that this effect is caused by a privileged integration of the ergosterol-derived salts into fungal membranes leading to increased membrane disorder. We propose that these findings offer a useful platform for the development of improved amphiphilic fungicides.
Assuntos
Antifúngicos , Sais , Antifúngicos/farmacologia , Candida , Cátions/farmacologia , Ergosterol , Esteroides/farmacologiaRESUMO
Membrane polarity, defined as the asymmetric distribution of lipids and proteins in the plasma membrane, is a critical prerequisite for the development of multicellular tissues, such as epithelia and endothelia. Membrane polarity is regulated by polarized trafficking of membrane components to specific membrane domains and requires the presence of intramembrane diffusion barriers that prevent the intermixing of asymmetrically distributed membrane components. This intramembrane diffusion barrier is localized at the tight junctions (TJs) in these cells. Both the formation of cell-cell junctions and the polarized traffic of membrane proteins and lipids are regulated by Rho and Rab family small GTPases. In this review article, we will summarize the recent developments in the regulation of apico-basal membrane polarity by polarized membrane traffic and the formation of the intramembrane diffusion barrier in epithelial cells with a particular focus on the role of Rho and Rab family small GTPases.
RESUMO
Weibel-Palade bodies (WPB) are unique secretory granules of endothelial cells that store the procoagulant von-Willebrand factor (VWF) in a highly compacted form. Upon exocytosis the densely packed VWF unfurls into long strands that expose binding sites for circulating platelets and thereby initiate the formation of a platelet plug at sites of blood vessel injury. Dense packing of VWF requires the establishment of an acidic pH in the lumen of maturing WPB but the mechanism responsible for this acidification has not yet been fully established. We show here that subunits of the vacuolar-type H+-ATPase are present on mature WPB and that interference with the proton pump activity of the ATPase employing inhibitors of different chemical nature blocks a reduction in the relative internal pH of WPB. Furthermore, depletion of the V-ATPase subunit V0d1 from primary endothelial cells prevents WPB pH reduction and the establishment of an elongated morphology of WPB that is dictated by the densely packed VWF tubules. Thus, the vacuolar-type H+-ATPase present on WPB is required for proper acidification and maturation of the organelle.
Assuntos
ATPases Vacuolares Próton-Translocadoras , Corpos de Weibel-Palade , Células Cultivadas , Células Endoteliais/metabolismo , Exocitose , Concentração de Íons de Hidrogênio , ATPases Vacuolares Próton-Translocadoras/metabolismo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Endothelial cells form the inner layer of blood vessels, making them the first barrier between the blood and interstitial tissues; thus endothelial cells play a crucial role in inflammation. In the inflammatory response, one important element is the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). While other pro-inflammatory agents like thrombin and histamine induce acute but transient changes in endothelial cells, which have been well studied biologically as well as mechanically, TNF-α is primarily known for its sustained effects on permeability and leukocyte recruitment. These functions are associated with transcriptional changes that take place on the timescale of hours and days. Here, we investigated the early mechanical action of TNF-α and show that even just 4 min after TNF-α was added onto human umbilical vein endothelial cell monolayers, there was a striking rise in mechanical substrate traction force and internal monolayer tension. These traction forces act primarily at the boundary of the monolayer, as was to be expected. This increased internal monolayer tension may, in addition to TNF-α's other well-studied biochemical responses, provide a mechanical signal for the cells to prepare to recruit leukocytes.
Assuntos
Endotélio Vascular , Fator de Necrose Tumoral alfa , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Trombina/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Rationale: Capillary leakage frequently occurs during sepsis and after major surgery and is associated with microvascular dysfunction and adverse outcome. Procalcitonin is a well-established biomarker in inflammation without known impact on vascular integrity. Objectives: We determined how procalcitonin induces endothelial hyperpermeability and how targeting procalcitonin protects vascular barrier integrity. Methods: In a prospective observational clinical study, procalcitonin levels were assessed in 50 patients who underwent cardiac surgery and correlated to postoperative fluid and vasopressor requirements along with sublingual microvascular functionality. Effects of the procalcitonin signaling pathway on endothelial barrier and adherens junctional integrity were characterized in vitro and verified in mice. Inhibition of procalcitonin activation by dipeptidyl-peptidase 4 (DPP4) was evaluated in murine polymicrobial sepsis and clinically verified in cardiac surgery patients chronically taking the DPP4 inhibitor sitagliptin. Measurements and Main Results: Elevated postoperative procalcitonin levels identified patients with 2-fold increased fluid requirements (P < 0.01), 1.8-fold higher vasopressor demand (P < 0.05), and compromised microcirculation (reduction to 63.5 ± 2.8% of perfused vessels, P < 0.05). Procalcitonin induced 1.4-fold endothelial and 2.3-fold pulmonary capillary permeability (both Ps < 0.001) by destabilizing VE-cadherin. Procalcitonin effects were dependent on activation by DPP4, and targeting the procalcitonin receptor or DPP4 during sepsis-induced hyperprocalcitonemia reduced capillary leakage by 54 ± 10.1% and 60.4 ± 6.9% (both Ps < 0.01), respectively. Sitagliptin before cardiac surgery was associated with augmented microcirculation (74.1 ± 1.7% vs. 68.6 ± 1.9% perfused vessels in non-sitagliptin-medicated patients, P < 0.05) and with 2.3-fold decreased fluid (P < 0.05) and 1.8-fold reduced vasopressor demand postoperatively (P < 0.05). Conclusions: Targeting procalcitonin's action on the endothelium is a feasible means to preserve vascular integrity during systemic inflammation associated with hyperprocalcitonemia.
Assuntos
Dipeptidil Peptidase 4 , Sepse , Animais , Permeabilidade Capilar , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidase 4/farmacologia , Dipeptidil Peptidase 4/uso terapêutico , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Camundongos , Pró-Calcitonina , Sepse/tratamento farmacológico , Sepse/metabolismoRESUMO
Weibel-Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB-plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2.
Assuntos
Corpos de Weibel-Palade , Fator de von Willebrand , Células Cultivadas , Exocitose/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.
Assuntos
Inibição de Contato , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Inibição de Contato/genética , Receptores de Vitronectina , TetraspaninasRESUMO
Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cell-cell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here, we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen-I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen-I-binding α3ß1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3ß1 integrin and regulate α3ß1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3ß1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3ß1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3ß1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells.