Tension modulation of actomyosin ring assembly and RhoGTPases activity: Perspectives from the Xenopus oocyte wound healing model.

Cells are remarkably resilient structures; they are able to recover from injuries to their plasma membrane (PM) and cytoskeleton that would normally constitute existential threats. This capacity is exemplified by Xenopus laevis oocytes which can recover from very large PM defects through exocytotic and endocytic events and can repair damaged cortical cytoskeleton structures through the formation of a contractile actomyosin ring (AMR). Formation of the AMR involves the localized Ca2+ -dependent activation of RhoA and Cdc42, and the pre-patterning of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, this model fails to account for observations that suggest a link between cytoskeletal dynamics, intracellular tension, and AMR formation. It also does not explain why the formation of an AMR is not involved in the cytoskeletal repair program of adherent cells. We show here evidence for the support of tension as an essential regulatory signal for the formation of AMR. Indeed, oocytes in which global tension has been experimentally reduced were unable to form a functional AMR following injury, showing severely diminished RhoA activity at the wound site. These new insights place the cytoskeleton at the center of events involving changes in cell shape such as cytokinesis which also involves the formation and closure of an AMR.

Correcting an instance of synthetic lethality with a pro-survival sequence.

A human cDNA encoding the LIM domain containing 194 amino acid cysteine and glycine rich protein 3 (CSRP3) was identified as a BAX suppressor in yeast and a pro-survival sequence that abrogated copper mediated regulated cell death (RCD). Yeast lacks a CSRP3 orthologue but it has four LIM sequences, namely RGA1, RGA2, LRG1 and PXL1. These are known regulators of stress responses yet their roles in RCD remain unknown. Given that LIMs interact with other LIMs, we ruled out the possibility that overexpressed yeast LIMs alone could prevent RCD and that CSRP3 functions by acting as a dominant regulator of yeast LIMs. Of interest was the discovery that even though yeast cells lacking the LIM encoding PXL1 had no overt growth defect, it was nevertheless supersensitive to the effects of sublethal levels of copper. Heterologous expression of human CSPR3 as well as the pro-survival 14-3-3 sequence corrected this copper supersensitivity. These results show that the pxl1∆-copper synthetic lethality is likely due to the induction of RCD. This differs from the prevailing model in which synthetic lethality occurs because of specific defects generated by the combined loss of two overlapping but non-essential functions.

Actin dynamics and myosin contractility during plasma membrane repair and restoration: Does one ring really heal them all?

In order to survive daily insults, cells have evolved various mechanisms that detect, stabilize and repair damages done to their plasma membrane and cytoskeletal structures. Damage to the PM endangers wounded cells by exposing them to uncontrolled exchanges with the extracellular milieu. The processes and molecular machinery enabling PM repair are therefore at the center of the bulk of the investigations into single-cell repair program. Wounds are repaired by dynamically remodeling the composition and shape of the injured area through exocytosis-mediated release of intracellular membrane components to the wounded area, endocytosis-mediated removal of the injured area, or the shedding of the injury. The wound healing program of Xenopus oocytes and early Drosophila embryos is by contrast, mostly characterized by the rapid formation of a large membrane patch over the wound that eventually fuse with the plasma membrane which restores plasma membrane continuity and lead to the shedding of patch material into the extracellular space. Formation and contraction of actomyosin ring restores normal plasma membrane composition and organizes cytoskeletal repairs. The extend of the contributions of the cytoskeleton to the wound healing program of somatic cells have comparatively received little attention. This review offers a survey of the current knowledge on how actin dynamics, myosin-based contraction and other cytoskeletal structures affects PM and cortical cytoskeleton repair of somatic cells.

Dynamics of actin polymerisation during the mammalian single-cell wound healing response.

OBJECTIVE: The contribution of actomyosin contractile rings in the wound healing program of somatic cells as never been directly assessed. This contrast with the events characterising the wound healing response of in wounded Xenopus oocytes, in which formation and contraction of an actomyosin ring provides a platform for cytoskeletal repair and drives the restoration of proper plasma membrane composition at the site of injury. As such, we aimed to characterize, using high-resolution live-cell confocal microscopy, the cytoskeletal repair dynamics of HeLa cells. RESULTS: We confirm here that the F-actin enrichment that characterizes the late repair program of laser-wounded cells is mostly uniform and is not associated with co-enrichment of myosin-II or the formation of concentric zones of RhoA and Cdc42 activity.

Intracellular second messengers mediate stress inducible hormesis and Programmed Cell Death: A review.

Alterations in the levels of numerous second messengers are ubiquitous responses to all stresses that lead to apoptotic or hormetic responses. The sheer number and vast diversity of different second messenger systems activated in response to stresses belies a complexity that is often overlooked. This negligence is in large part due to the excessive focus on classical stress responsive second messenger mediators of stress especially Reactive Oxygen Species (ROS) but also others like calcium and ceramide. Here we review the many different intracellular second messengers that are involved in stress responses. We further integrate this information to emphasize that initial stress mediated responses consist of increased levels of a multitude of intracellular second messengers that serve to elicit the appropriate cell survival and/or cell death responses. We suggest that a greater focus on second messenger systems may shed more light on the processes that serve in the initiation of stress mediated PCD.

Stress is an agonist for the induction of programmed cell death: A review.

The prevailing models of stress induced Programmed Cell Death (PCD) posit that excess extracellular chemicals interact with or enter cells and disrupts cellular homeostasis. This activates signalling cascades involving the mitochondria, an increase in the steady state levels of Reactive Oxygen Species (ROS) as well as the activation of Bax and caspases. Further, the increased ROS also causes cellular damage that triggers or enhances PCD responses. The models have been modified in a number of ways, for example to include the existence of caspase and Bax independent forms of PCD. More recently, the ubiquity of ROS has also been challenged in part based on the failure of anti-oxidants to protect from diseases with increased intensity of oxidative stress. Here we focus on a number of other, often overlooked, observations regarding stress mediated responses that may further increase our mechanistic understanding of PCD. These include the concept of the "milieu intérieur" which suggests that cells actively protect themselves (adaptive homeostasis) in part by limiting entry to most extracellular chemicals. Of similar importance, stress also increases the levels of other stress inducible second messengers including ceramide, iron and calcium. This review focuses on the concept that stress is an agonist that conveys information that is transduced into the cell to activate the appropriate genetically encoded cell death and survival responses.

Regulation and Assembly of Actomyosin Contractile Rings in Cytokinesis and Cell Repair.

Cytokinesis and single-cell wound repair both involve contractile assemblies of filamentous actin (F-actin) and myosin II organized into characteristic ring-like arrays. The assembly of these actomyosin contractile rings (CRs) is specified spatially and temporally by small Rho GTPases, which trigger local actin polymerization and myosin II contractility via a variety of downstream effectors. We now have a much clearer view of the Rho GTPase signaling cascade that leads to the formation of CRs, but some factors involved in CR positioning, assembly, and function remain poorly understood. Recent studies show that this regulation is multifactorial and goes beyond the long-established Ca2+ -dependent processes. There is substantial evidence that the Ca2+ -independent changes in cell shape, tension, and plasma membrane composition that characterize cytokinesis and single-cell wound repair also regulate CR formation. Elucidating the regulation and mechanistic properties of CRs is important to our understanding of basic cell biology and holds potential for therapeutic applications in human disease. In this review, we present a primer on the factors influencing and regulating CR positioning, assembly, and contraction as they occur in a variety of cytokinetic and single-cell wound repair models. Anat Rec, 301:2051-2066, 2018. © 2018 Wiley Periodicals, Inc.

The Virtual Environment for Rapid Prototyping of the Intelligent Environment.

Advances in domains such as sensor networks and electronic and ambient intelligence have allowed us to create intelligent environments (IEs). However, research in IE is being held back by the fact that researchers face major difficulties, such as a lack of resources for their experiments. Indeed, they cannot easily build IEs to evaluate their approaches. This is mainly because of economic and logistical issues. In this paper, we propose a simulator to build virtual IEs. Simulators are a good alternative to physical IEs because they are inexpensive, and experiments can be conducted easily. Our simulator is open source and it provides users with a set of virtual sensors that simulates the behavior of real sensors. This simulator gives the user the capacity to build their own environment, providing a model to edit inhabitants' behavior and an interactive mode. In this mode, the user can directly act upon IE objects. This simulator gathers data generated by the interactions in order to produce datasets. These datasets can be used by scientists to evaluate several approaches in IEs.

Heterologous expression of anti-apoptotic human 14-3-3ß/α enhances iron-mediated programmed cell death in yeast.

The induction of Programmed Cell Death (PCD) requires the activation of complex responses involving the interplay of a variety of different cellular proteins, pathways, and processes. Uncovering the mechanisms regulating PCD requires an understanding of the different processes that both positively and negatively regulate cell death. Here we have examined the response of normal as well as PCD resistant yeast cells to different PCD inducing stresses. As expected cells expressing the pro-survival human 14-3-3ß/α sequence show increased resistance to numerous stresses including copper and rapamycin. In contrast, other stresses including iron were more lethal in PCD resistant 14-3-3ß/α expressing cells. The increased sensitivity to PCD was not iron and 14-3-3ß/α specific since it was also observed with other stresses (hydroxyurea and zinc) and other pro-survival sequences (human TC-1 and H-ferritin). Although microscopical examination revealed little differences in morphology with iron or copper stresses, cells undergoing PCD in response to high levels of prolonged copper treatment were reduced in size. This supports the interaction some forms of PCD have with the mechanisms regulating cell growth. Analysis of iron-mediated effects in yeast mutant strains lacking key regulators suggests that a functional vacuole is required to mediate the synergistic effects of iron and 14-3-3ß/α on yeast PCD. Finally, mild sub-lethal levels of copper were found to attenuate the observed inhibitory effects of iron. Taken together, we propose a model in which a subset of stresses like iron induces a complex process that requires the cross-talk of two different PCD inducing pathways.

Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae.

Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress.

Plasma membrane and cytoskeleton dynamics during single-cell wound healing.

Wounding leads not only to plasma membrane disruption, but also to compromised cytoskeleton structures. This results not only in unwarranted exchanges between the cytosol and extracellular milieu, but also in loss of tensegrity, which may further endanger the cell. Tensegrity can be described as the interplay between the tensile forces generated by the apparent membrane tension, actomyosin contraction, and the cytoskeletal structures resisting those changes (e.g., microtubules). It is responsible for the structural integrity of the cell and for its ability to sense mechanical signals. Recent reviews dealing with single-cell healing mostly focused on the molecular machineries controlling the traffic and fusion of specific vesicles, or their role in different pathologies. In this review, we aim to take a broader view of the different modes of single cell repair, while focussing on the different ways the changes in plasmalemma surface area and composition, plasmalemma tension, and cytoskeletal dynamics may influence and affect single-cell repair.

Human Thyroid Cancer-1 (TC-1) is a vertebrate specific oncogenic protein that protects against copper and pro-apoptotic genes in yeast.

The human Thyroid Cancer-1 (hTC-1) protein, also known as C8orf4 was initially identified as a gene that was up-regulated in human thyroid cancer. Here we show that hTC-1 is a peptide that prevents the effects of over-expressing Bax in yeast. Analysis of the 106 residues of hTC-1 in available protein databases revealed direct orthologues in jawed-vertebrates, including mammals, frogs, fish and sharks. No TC-1 orthologue was detected in lower organisms, including yeast. Here we show that TC-1 is a general pro-survival peptide since it prevents the growth- and cell death-inducing effects of copper in yeast. Human TC-1 also prevented the deleterious effects that occur due to the over-expression of a number of key pro-apoptotic peptides, including YCA1, YBH3, NUC1, and AIF1. Even though the protective effects were more pronounced with the over-expression of YBH3 and YCA1, hTC-1 could still protect yeast mutants lacking YBH3 and YCA1 from the effects of copper sulfate. This suggests that the protective effects of TC-1 are not limited to specific pathways or processes. Taken together, our results indicate that hTC-1 is a pro-survival protein that retains its function when heterologously expressed in yeast. Thus yeast is a useful model to characterize the potential roles in cell death and survival of cancer related genes.

C21-steroids inactivation and glucocorticoid synthesis in the developing lung.

Glucocorticoids (GCs) are important regulators of lung development. The genes normally involved in GC synthesis in adrenals are co-expressed with 20α-hydroxysteroid dehydrogenase (20α-HSD) in the developing lung. In this study, C21-steroid metabolism was investigated in fetal and postnatal mouse lungs. Incubation of [(3)H]-progesterone with lung explant cultures of different perinatal developmental time points revealed two different (antenatal vs. postnatal) complex metabolization patterns. Progesterone inactivation was predominant. 20αOH-derivatives were more abundant after birth and some metabolites were 5α-reduced. Using [(3)H]-progesterone as substrate, corticosterone synthesis was only observed in a fraction of lung explants from gestation day (GD) 15.5. Neither aldosterone synthase nor P450c17 activity was observed. With epithelial-enriched primary cell cultures, deoxycorticosterone synthesis from [(3)H]-progesterone was observed. With lung explants incubated with [(3)H]-corticosterone as substrate, [(3)H]-4-pregnen-21-ol-3,11,20-trione (11-dehydrocorticosterone), the product of 11ß-HSD2, accumulated in higher proportion on GD 15.5 than at later developmental time points. The temporal correlation observed between levels of progesterone inactivation by 20α-HSD (higher after birth) and the sensitivity of lung development to GCs suggests a role for 20α-HSD in the modulation of GR occupancy through the control of 21-hydroxylase substrate and product levels. In conclusion, the developing lung is characterized by effective inactivation of c21-steroids by 20α-HSD. The formation of active GCs from the "adrenal"-like pathway was observed with some lung explants and primary epithelial cell cultures. Coexistence of this GC synthesis pathway with 20α-HSD activity strongly suggests local regulation of GC action and is compatible with intracrine/paracrine actions of GC.

Ontogeny of adrenal-like glucocorticoid synthesis pathway and of 20α-hydroxysteroid dehydrogenase in the mouse lung.

BACKGROUND: Glucocorticoids exert recognized positive effects on lung development. The genes involved in the classical pathway of glucocorticoid synthesis normally occurring in adrenals were found to be expressed on gestation day (GD) 15.5 in the developing mouse lung. Recently, expression of two of these genes was also detected on GD 17.5 suggesting a more complex temporal regulation than previously expected. Here, we deepen the knowledge on expression of "adrenal" glucocorticoid synthesis genes in the mouse lung during the perinatal period and we also study expression of the gene encoding for the steroid inactivating enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). RESULTS: We performed an ontogenic study of P450scc, 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase 1 (3ß-HSD1), 21-hydroxylase, 11ß-hydroxylase, 11ß-HSD1, and 11ß-HSD2 expression up to post natal day (PN) 15. The substrate (progesterone) and the product (deoxycorticosterone) of 21-hydroxylase are substrates of 20α-HSD, thus 20α-HSD (Akr1c18) gene expression was investigated. In lung samples collected between GD 15.5 and PN 15, 11ß-hydroxylase was only detected on GD 15.5. In contrast, all the other tested genes were expressed throughout the analyzed period with different temporal expression patterns. P450scc, 21-hydroxylase, 20α-HSD and 11ß-HSD2 mRNA levels increased after birth with different patterns including an increase from PN 3 with a possible sex difference for 21-hydroxylase mRNA. Also, the 21-hydroxylase protein was observed by Western blot in perinatal lungs with higher levels after birth. CONCLUSION: Progesterone is present at high levels during gestation and the product of 21-hydroxylase, deoxycorticosterone, can bind the glucocorticoid receptor with an affinity close to that of corticosterone. Detection of 21-hydroxylase at the protein level during antenatal lung development is the first evidence that the adrenal-like glucocorticoid synthesis pathway detected during lung development has the machinery to produce glucocorticoids in the fetal lung. Glucocorticoids from lung 21-hydroxylase appear to modulate lung ontogenesis through paracrine/intracrine actions.

Glucocorticoid metabolism in the developing lung: adrenal-like synthesis pathway.

Glucocorticoids (GCs) are essential to normal lung development. They participate in the regulation of important developmental events including morphological changes, and lung maturation leading to the surge of surfactant synthesis by type II epithelial cells. Antenatal GC is administered to mothers at risk of premature delivery to reduce the risk of respiratory distress syndrome (RDS). Sex differences were reported in RDS, in the efficiency of antenatal GC treatment independently of surfactant levels, and in surfactant lipid synthesis. Type II epithelial cell maturation is regulated by epithelial-fibroblast cell-cell communication and involves paracrine factors secreted by fibroblasts under the stimulatory effect of GC. This positive action of GC can be inhibited by androgens through the androgen receptor (AR) present in fibroblasts. In fact, lung development is regulated not only by GC and androgens but also by GC and androgen metabolisms within the developing lung. We recently reviewed the metabolism of androgens in the fetal lung [45]. Here, we review multiple aspects of GC metabolism in the developing lung including inactivation and re-activation by 11ß-HSDs, synthesis from the adrenal-like synthesis pathway expressed within the lung and the putative role of CRH and ACTH originating from lung in the regulation of this pathway. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

Sex-specific perinatal expression of glutathione peroxidases during mouse lung development.

Reports indicate that antioxidant enzymes like the glutathione peroxidases (GPx) can be regulated by sex steroids. The GPx, a major class of antioxidants involved in H(2)O(2) and lipid hydroperoxides neutralization, showed an age- and sex-specific expression in many adult organs including the lung. High levels of androgens in the male lung are known to delay the surge of surfactant synthesis during gestation in several species. However, the impact of male androgens on antioxidant GPx early in life remains to be determined. The objective was to study the lung sex-specific expression of GPx during BALB/c mouse perinatal development. The mRNA expression of four seleno-dependent Gpx (Gpx1 to 4) in the lung of both sexes was characterized by real-time PCR from gestational day 15 to postnatal day 30, covering the entire canalicular, saccular and alveolar stages. Immunohistochemistry of GPx-1, -3 and -4, and seleno-dependent GPx enzymatic assays were also performed in the lung. We found a transient lower Gpx1 mRNA level in male than in female lungs during the first 5 days after birth, corresponding to the saccular phase. This dimorphic expression was concomitant to a sex difference in GPx enzymatic activity corrected for blood. It is, to our knowledge, the first report of a sex dimorphism for murine lung enzymatic antioxidant defenses during the perinatal period.

Levels of dihydrotestosterone, testosterone, androstenedione, and estradiol in canalicular, saccular, and alveolar mouse lungs.

Androgens and estrogens are known regulators of fetal and postnatal lung development, but their levels in the developing lung have never been determined. We present here, for the first time, a gas chromatography-mass spectrometry (GC/MS) quantification of dihydrotestosterone, testosterone, androstenedione, and estradiol in canalicular, saccular, and alveolar stage lungs of both sexes. Testosterone, androstenedione, and estradiol were observed in all the analyzed lung samples from gestation day (GD) 16.5 to postnatal day (PN) 30, totalizing 383 individual mice. Levels of these three steroids decreased between birth and PN 5. In contrast, dihydrotestosterone was detected only in male samples on GD 19.5, PN 0, and PN 30. A significant sex difference was observed for testosterone and androstenedione but not for estradiol. Steroid levels were also determined in skinned hind legs for comparison. Three-way analysis of variance revealed that tissue (lung or leg) had a significant effect on testosterone levels for both sexes, but not on androstenedione and estradiol levels. Low but significant testosterone and androstenedione levels were observed in all the females and in prepubertal male samples. These levels must be sufficient to induce androgen receptor activation, as suggested by our recent report showing the presence of androgen receptor in the nucleus of several lung cells in corresponding developmental ages and sexes.

Androgen receptor and 17beta-HSD type 2 regulation in neonatal mouse lung development.

A QPCR analysis of androgen receptor and several androgen metabolizing genes was performed during the saccular and alveolar stages of mouse lung development. Androgen receptor expression showed a statistically significant increase during the alveolar stage while levels of 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD 2) expression significantly decreased at the end of the saccular stage and remained low throughout the alveolar period. 17beta-HSD 1, 17beta-HSD 5, 5alpha-reductase type 1, and mouse 3alpha-HSD did not present such a regulation. The androgen receptor protein was primarily detected in the nucleus of airway epithelial cells and of a subset of respiratory epithelial cells. 17beta-HSD 2 mRNA co-localized with androgen receptor protein during saccularization, but was absent from airway epithelium during alveolarization. Taken together, our results demonstrate temporal and spatial regulation of androgen receptor and 17beta-HSD 2 during the sacculo-alveolar transition period of mouse lung development suggesting control of androgen action.

Apolipoprotein A-I, A-II, C-II, and H expression in the developing lung and sex difference in surfactant lipids.

A sex difference in surfactant lipids is associated with a higher incidence of respiratory distress syndrome for males in cases of preterm birth. In animal models, the sex difference in surfactant lipids was shown to be androgen receptor-dependent. This report examines expression of apolipoprotein (apo)A-I, apoA-II, apoC-II, apoE, apoH, and lipoprotein lipase (LPL) by quantitative real-time PCR in pools of male and female fetal lung tissues from various mouse litters from gestation day (GD) 15.5 to 18.5, and in various adult tissues. Although the expression profiles of ApoA-I, ApoA-II, ApoC-II, and ApoH are complex, these genes are co-regulated and they all present a sex difference (P=0.0896, 0.0896, 0.0195, and 0.0607 respectively) with higher expression for females for several litters. Pulmonary expression of apoA-I, apoA-II, and apoH were specific to the developing lung. ApoE and LPL mRNAs showed a significant increase from GD 17.5 to 18.5. An increase in apoA-I-, apoA-II-, apoC-II-, and apoH-mRNA accumulation was observed from GD 16.5 to 17.5 in correlation with the emergence of mature type II pneumonocytes. These four apolipoprotein genes are co-regulated with type 2 and 5 17beta-hydroxysteroid dehydrogenases, which are respectively involved in inactivation and synthesis of androgens. Finally, apoC-II was detected by immunohistochemistry in epithelial cells of the distal epithelium. Positive signals looking like secretory granules were located near the basal membrane. Our results are compatible with a role for apolipoproteins in lipid metabolism and transport in the developing lung in association with the sex difference in surfactant lipid synthesis.