Extrusion is a mechanism used to eliminate unfit, excess, or dying cells from epithelial tissues. The initial events guiding which cells will be selectively extruded from the epithelium are not well ...understood. Here, we induced damage in a subset of epithelial cells in the developing zebrafish and used time-lapse imaging to examine cell and cytoskeletal dynamics leading to extrusion. We show that cell extrusion is preceded by actomyosin contractions that are pulsatile. Our data show that pulsatile contractions are induced by a junctional to medial re-localization of myosin. Analysis of cell area during contractions revealed that cells pulsing with the longest duration and highest amplitude undergo progressive area loss and extrude. Although pulses were driven by local increases in tension, damage to many cells promoted an overall decrease in the tensile state of the epithelium. We demonstrate that caspase activation leads to sphingosine-1-phosphate enrichment that controls both tissue tension and pulses to dictate areas of extrusion. These data suggest that the kinetics of pulsatile contractions define a key behavioral difference between extruding and non-extruding cells and are predictive of extrusion. Altogether, our study provides mechanistic insight into how localized changes in physical forces are coordinated to remove defective cells for homeostatic maintenance of living epithelial tissues.
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•Cell extrusion is preceded by actomyosin pulsatile contractions•Kinetics of pulsatile contractions are predictive of cell extrusion•Pulsatile contractions are generated by an apicomedial myosin network•Caspase activation and S1P enrichment induce pulsatile contractions
Atieh et al. investigate the mechanics underlying epithelial cell extrusion. They show that dynamic pulsatile contractions emerge following cellular damage and promote extrusion of apoptotic cells. Caspase activation leads to sphingosine-1-phosphate enrichment that controls both tissue tension and pulses to dictate areas of extrusion.
Cell-extracellular matrix (ECM) interactions represent fundamental exchanges during tumor progression, yet how particular signal-transduction factors prompt the conversion of tumor cells into ...migratory populations capable of systemic spread during metastasis remains elusive. We demonstrate that the noncanonical Wnt receptor, Ror2, regulates tumor cell-driven matrix remodeling and invasion in breast cancer. Ror2 loss-of-function (LOF) triggers the disruption of E-cadherin within tumor cells, accompanied by an increase in tumor cell invasion and collagen realignment in three-dimensional cultures. RNA sequencing of Ror2-deficient organoids further uncovered alterations in actin cytoskeleton, cell adhesion, and collagen cross-linking gene expression programs. Spatially, we pinpoint the up-regulation and redistribution of α
and β
integrins together with the production of fibronectin in areas of invasion downstream of Ror2 loss. Wnt/β-catenin-dependent and Wnt/Ror2 alternative Wnt signaling appear to regulate distinct functions for tumor cells regarding their ability to modify cell-ECM exchanges during invasion. Furthermore, blocking either integrin or focal adhesion kinase (FAK), a downstream mediator of integrin-mediated signal transduction, abrogates the enhanced migration observed upon Ror2 loss. These results reveal a critical function for the alternative Wnt receptor, Ror2, as a determinant of tumor cell-driven ECM exchanges during cancer invasion and metastasis.
The early use of fresh frozen plasma as a resuscitative agent after hemorrhagic shock has been associated with improved survival, but the mechanism of protection is unknown. Hemorrhagic shock causes ...endothelial cell dysfunction and we hypothesized that fresh frozen plasma would restore endothelial integrity and reduce syndecan-1 shedding after hemorrhagic shock. A prospective, observational study in severely injured patients in hemorrhagic shock demonstrated significantly elevated levels of syndecan-1 (554±93 ng/ml) after injury, which decreased with resuscitation (187±36 ng/ml) but was elevated compared to normal donors (27±1 ng/ml). Three pro-inflammatory cytokines, interferon-γ, fractalkine, and interleukin-1β, negatively correlated while one anti-inflammatory cytokine, IL-10, positively correlated with shed syndecan-1. These cytokines all play an important role in maintaining endothelial integrity. An in vitro model of endothelial injury then specifically examined endothelial permeability after treatment with fresh frozen plasma orlactated Ringers. Shock or endothelial injury disrupted junctional integrity and increased permeability, which was improved with fresh frozen plasma, but not lactated Ringers. Changes in endothelial cell permeability correlated with syndecan-1 shedding. These data suggest that plasma based resuscitation preserved endothelial syndecan-1 and maintained endothelial integrity, and may help to explain the protective effects of fresh frozen plasma after hemorrhagic shock.
TNF-α (tumor necrosis factor-α) is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL) in ...several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1) mechanical properties, employing atomic force microscopy; 2) cytoskeletal organization, through fluorescence microscopy; and 3) membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h), for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells); the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with controls. Collectively, these results demonstrate that sufficiently high levels of circulating TNF-α have similar effects on different endothelial districts, and provide additional information for unraveling the possible correlations between circulating pro-inflammatory cytokines and systemic vascular dysfunction.
After exposure to an agonist, platelets are activated and become aggregated. They also shed membrane microparticles that participate in the pathogenesis of thrombosis, hyper-coagulation and ...inflammation. However, microvesiculation can potentially disrupt the integrity of platelet aggregation by shedding the membrane receptors and phosphatidylserine critical for forming and stabilising a platelet clot. We tested the hypothesis that adhesion and microvesiculation are functions of different subsets of platelets at the time of haemostasis by real-time monitoring of agonist-induced morphological changes and microvesiculation of human platelets.We identified two types of platelets that are adherent to fibrinogen: a high density bubble shape (HDBS) and low-density spread shape (LDSS). Adenosine diphosphate (ADP) predominantly induced HDBS platelets to vesiculate, whereas LDSS platelets were highly resistant to such vesiculation. Thrombin-receptor activating peptide (TRAP) stabilised platelets against microvesiculation by promoting a rapid HDBS-to-LDSS morphological transition. These activities of ADP and TRAP were reversed for platelets in suspension, independent of an engagement integrin αIIbβ3. As the result of membrane contact, LDSS platelets inhibited the microvesiculation of HDBS platelets in response to ADP. Aspirin and clopidogrel inhibited ADP-induced microvesiculation through different mechanisms. These results suggest that platelet aggregation and microvesiculation occur in different subsets of platelets and are differently regulated by agonists, platelet-platelets and platelet-fibrinogen interactions.
Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same ...cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-Ras
favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-Ras
PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-Ras
membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.
Abstract
Circulating platelets are anucleated and multi-functional cells that participate in hemostasis and arterial thrombosis. Multiple ligands and mechanical forces activate platelets, leading to ...cytoskeletal rearrangement and dramatic shape-changes. Such dramatic changes in platelets membrane structures are commonly detected by optical and electron microscopy after platelets are fixed. We have recently developed a method to study the membrane morphology of live platelets using Hopping Probe Ion Conductance Microscopy (HPICM). We have successfully used this technology to study the process of platelet microvesiculation upon exposure to selective agonists. Here, we further discussed technical details of using HPICM to study platelet biology and compared results from HPICM to those from conventional atomic force microscopy and scanning electron microscopy. This method offers several advantages over current technologies. First, it monitors morphological changes of platelets in response to agonists in real time. Second, platelets can be repeatedly scanned over time without damages brought by heat and prolong light exposure. Third, there is no direct contact with platelet surface so that there will no or minimal mechanical damages brought by a cantilever of a conventional atomic force microscopy. Finally, it offers the potential to study platelet membrane ion channels, which have been technically challenging up-to-date. Our data show that HPICM has high-resolution in delineating changes of platelet morphology in response to stimulations and could help to unravel the complex role of platelet in thrombus formation.
Ion Currents Induced by ATP and Angiotensin Ⅱ in Cultured Follicular Cells of Xenopus laevis Montiel-Herrera, Marcelino, Universidad Nacional Autonoma de Mexico, Campus Juriquilla, Juriquilla, Queretaro, Mexico; Zaske, Ana Maria, Universidad Nacional Autonoma de Mexico, Campus Juriquilla, Juriquilla, Queretaro, Mexico; Garcia-Colunga, Jesus, Universidad Nacional Autonoma de Mexico, Campus Juriquilla, Juriquilla, Queretaro, Mexico ...
Molecules and cells,
11/2011, Letnik:
32, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their ...enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at -60 mV revealed that ATP (20-250 μM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ∼60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ∼5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at -60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known F∧in and S∧in-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using whole-cell recordings, 1 μM Ang Ⅱ yielded smooth inward currents of 157 ± 45 pA in ∼16% of the FC held at -60 mV. The net Ang Ⅱ response, mediated by the activation of the AT₁ receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang Ⅱ receptors in the physiology of X. laevis oocytes.
Collagen linearization is a hallmark of aggressive tumors and a key pathogenic event that promotes cancer cell invasion and metastasis. Cell‐generated mechanical tension has been proposed to ...contribute to collagen linearization in tumors, but it is unknown whether other mechanisms play prominent roles in this process. Here, we show that the secretome of cancer cells is by itself able to induce collagen linearization independently of cell‐generated mechanical forces. Among the tumor cell‐secreted factors, we find a key role in this process for the matricellular protein WISP1 (CCN4). Specifically, WISP1 directly binds to type I collagen to promote its linearization in vitro (in the absence of cells) and in vivo in tumors. Consequently, WISP1‐induced type I collagen linearization facilitates tumor cell invasion and promotes spontaneous breast cancer metastasis, without significantly affecting gene expression. Furthermore, higher WISP1 expression in tumors from cancer patients correlates with faster progression to metastatic disease and poor prognosis. Altogether, these findings reveal a conceptually novel mechanism whereby pro‐metastatic collagen linearization critically depends on a cancer cell‐secreted factor.
Synopsis
Extracellular matrix collagen linearization is associated with aggressive tumors and promotes metastasis. The tumor cell‐secreted factor WISP1 induces collagen linearization independently of cell‐generated mechanical forces.
Breast carcinoma cells secrete WISP1 to induce collagen linearization.
WISP1 specifically remodels type I collagen.
TGF‐β1 promotes collagen linearization by inducing WISP1 expression.
WISP1‐induced collagen linearization facilitates tumor cell invasion and promotes spontaneous breast cancer metastasis.
Higher expression of WISP1 in tumors from cancer patients correlates with faster progression to metastatic disease.
The secretome of cancer cells induces type I collagen linearization independently of cell‐generated mechanical tension.
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