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BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with mast cell-mediated inflammation and heightened oxidant stress. Kynurenine (KYN), an endogenous tryptophan metabolite, can promote allergen-induced mast cell activation through the aryl hydrocarbon receptor (AhR). OBJECTIVES: We sought to determine the role of the KYN/AhR axis and oxidant stress in mast cell activation and the development of CRSwNP. METHODS: We measured the expression of indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase, KYN, and oxidized calmodulin-dependent protein kinase II (ox-CaMKII) in nasal polyps and controls. KYN-potentiated ovalbumin (OVA)-induced ROS generation, cell activation, and ox-CaMKII expression were investigated in wild-type and AhR-deficient (AhR-/-) mast cells. The role of ox-CaMKII in mast cell activation was further investigated. RESULTS: Nasal polyps in CRSwNP showed an increased expression of indoleamine 2,3-dioxygenase 1, tryptophan2,3-dioxygenase, and KYN compared with controls. AhR was predominantly expressed in mast cells in nasal polyps. Activated mast cells and local IgE levels were substantially increased in eosinophilic polyps compared with noneosinophilic polyps and controls. Furthermore, KYN potentiated OVA-induced ROS generation, intracellular Ca2+ levels, cell activation, and expression of ox-CaMKII in wild-type, but not in AhR-/- mast cells. Compared with noneosinophilic polyps and controls, eosinophilic polyps showed increased expression of ox-CaMKII in mast cells. Mast cells from ROS-resistant CaMKII MMVVδ mice or pretreated with CaMKII inhibitor showed protection against KYN-promoted OVA-induced mast cell activation. CONCLUSIONS: These studies support a potentially critical but previously unidentified function of the KYN/AhR axis in regulating IgE-mediated mast cell activation through ROS and ox-CaMKII in CRSwNP.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Pólipos Nasais/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Receptores de Glutamato/imunologia , Rinite/imunologia , Sinusite/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/imunologia , Doença Crônica , Eosinófilos/imunologia , Eosinófilos/patologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Knockout , Pólipos Nasais/genética , Pólipos Nasais/patologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Glutamato/genética , Rinite/genética , Rinite/patologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Sinusite/genética , Sinusite/patologiaRESUMO
BACKGROUND: Mannose receptor (MRC1/CD206) has been suggested to mediate allergic sensitization and asthma to multiple glycoallergens, including cockroach allergens. OBJECTIVE: We sought to determine the existence of a protective mechanism through which MRC1 limits allergic inflammation through its intronic miR-511-3p. METHODS: We examined MRC1-mediated cockroach allergen uptake by lung macrophages and lung inflammation using C57BL/6 wild-type (WT) and Mrc1-/- mice. The role of miR-511-3p in macrophage polarization and cockroach allergen-induced lung inflammation in mice transfected with adeno-associated virus (AAV)-miR-511-3p (AAV-cytomegalovirus-miR-511-3p-enhanced green fluorescent protein) was analyzed. Gene profiling of macrophages with or without miR-511-3p overexpression was also performed. RESULTS: Mrc1-/- lung macrophages showed a significant reduction in cockroach allergen uptake compared with WT mice, and Mrc1-/- mice had an exacerbated lung inflammation with increased levels of cockroach allergen-specific IgE and TH2/TH17 cytokines in a cockroach allergen-induced mouse model compared with WT mice. Macrophages from Mrc1-/- mice showed significantly reduced levels of miR-511-3 and an M1 phenotype, whereas overexpression of miR-511-3p rendered macrophages to exhibit a M2 phenotype. Furthermore, mice transfected with AAV-miR-511-3p showed a significant reduction in cockroach allergen-induced inflammation. Profiling of macrophages with or without miR-511-3p overexpression identified 729 differentially expressed genes, wherein expression of prostaglandin D2 synthase (Ptgds) and its product PGD2 were significantly downregulated by miR-511-3p. Ptgds showed a robust binding to miR-511-3p, which might contribute to the protective effect of miR-511-3p. Plasma levels of miR-511-3p were significantly lower in human asthmatic patients compared with nonasthmatic subjects. CONCLUSION: These studies support a critical but previously unrecognized role of MRC1 and miR-511-3p in protection against allergen-induced lung inflammation.
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Hipersensibilidade/etiologia , Hipersensibilidade/metabolismo , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , MicroRNAs/genética , Receptores de Superfície Celular/metabolismo , Alérgenos/imunologia , Animais , Asma/etiologia , Asma/metabolismo , Asma/patologia , Baratas/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/genética , Hipersensibilidade/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Receptor de Manose , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Pneumonia/etiologia , Pneumonia/metabolismo , Pneumonia/patologia , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores ImunológicosRESUMO
Anastasis is a cell recovery mechanism that rescues dying cells from the brink of death. Reversal of apoptosis is the first example of anastasis. Here, we describe a comprehensive dataset containing time-course mRNA expression profiles for reversal of ethanol-induced apoptosis in mouse primary liver cells in νitro. This transcriptome dataset includes the conditions of the untreated cells, cells undergoing apoptosis triggered by incubating with cell death inducer of 4.5% ethanol for 5 hours, and apoptosis reversal of ethanol-induced cells at the early (3rd hour), middle (6th hour), and late (24th, 48th hour) stages after being washed with and incubated in fresh cell culture medium. By comparing this dataset with the transcriptomic profiles of other anastasis models generated with different combinations of cell types and cell death inducers, investigators can identify the key regulators governing reversal of apoptosis and other reversible cell death processes. Therefore, reusing or reanalysing this dataset will facilitate the future studies on the physiological, pathological, and therapeutic implications of anastasis.
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Apoptose , Etanol , Fígado , Transcriptoma , Animais , Reversão da Morte Celular , Etanol/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , CamundongosRESUMO
Globally, approximately 11% of all infants are born preterm, prior to 37 weeks' gestation. In these high-risk neonates, encephalopathy of prematurity (EoP) is a major cause of both morbidity and mortality, especially for neonates who are born very preterm (<32 weeks gestation). EoP encompasses numerous types of preterm birth-related brain abnormalities and injuries, and can culminate in a diverse array of neurodevelopmental impairments. Of note, posthemorrhagic hydrocephalus of prematurity (PHHP) can be conceptualized as a severe manifestation of EoP. PHHP impacts the immature neonatal brain at a crucial timepoint during neurodevelopment, and can result in permanent, detrimental consequences to not only cerebrospinal fluid (CSF) dynamics, but also to white and gray matter development. In this review, the relevant literature related to the diverse mechanisms of cell death in the setting of PHHP will be thoroughly discussed. Loss of the epithelial cells of the choroid plexus, ependymal cells and their motile cilia, and cellular structures within the glymphatic system are of particular interest. Greater insights into the injuries, initiating targets, and downstream signaling pathways involved in excess cell death shed light on promising areas for therapeutic intervention. This will bolster current efforts to prevent, mitigate, and reverse the consequential brain remodeling that occurs as a result of hydrocephalus and other components of EoP.
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Morte Celular , Hidrocefalia/patologia , Doenças do Prematuro/patologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Cílios/metabolismo , Epêndima/citologia , Epêndima/metabolismo , Humanos , Hidrocefalia/líquido cefalorraquidiano , Hidrocefalia/genética , Doenças do Prematuro/líquido cefalorraquidiano , Doenças do Prematuro/genética , Nascimento Prematuro , Transdução de SinaisRESUMO
Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.
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Mitocôndrias/fisiologia , Vimentina/fisiologia , Animais , Western Blotting , Linhagem Celular , Humanos , Imuno-Histoquímica , Imunoprecipitação , Microscopia de Fluorescência , RNA Interferente PequenoRESUMO
The classical view of cell death has long assumed that, once initiated, the dying process is irreversible. However, recent studies reveal that recovery of dying cells can actually occur, even after initiation of a cell suicide process called apoptosis. This discovery raised fundamental key questions about which forms of the cell death process could be reversible and how reversal is mediated. Here, we uncover an unanticipated reversibility of ferroptotic cell death process. Unlike apoptosis reversal, removal of ferroptosis inducers, such as erastin and glutamate, is insufficient to allow ferroptotic dying cells to escape the cell death process. However, by removing the cell death inducer and providing the reduced form of glutathione or the radical-trapping antioxidant ferrostatin-1, ferroptotic dying cells can be rescued and promoted to recover. Interestingly, although ferroptotic inhibitors such as aminooxyacetic acid, deferoxamine, dopamine and vitamin C can prevent initiation of ferroptosis, added alone they are unable to reverse the initiated ferroptosis, suggesting regulatory distinctions between preventing and reversing ferroptosis. Together, these results reveal the first evidence that ferroptosis is reversible and suggest strategies to enhance its reversibility, thereby providing a useful model for studying the physiological, pathological and therapeutic potentials of this cell recovery process.
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Anastasis is a natural cell recovery phenomenon that rescues cells from the brink of death. Programmed cell death such as apoptosis has been traditionally assumed to be an intrinsically irreversible cascade that commits cells to a rapid and massive demolition. Interestingly, recent studies have demonstrated recovery of dying cells even at the late stages generally considered immutable. Here, we examine the evidence for anastasis in cultured cells and in animals, review findings illuminating the potential mechanisms of action, discuss the challenges of studying anastasis and explore new strategies to uncover the function and regulation of anastasis, the identification of which has wide-ranging physiological, pathological and therapeutic implications.
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[This corrects the article DOI: 10.1098/rsos.180442.].
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Anastasis (Greek for "rising to life") is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.
Assuntos
Apoptose/fisiologia , Técnicas Biossensoriais/métodos , Drosophila melanogaster/patogenicidade , Animais , Feminino , Células HeLa , Humanos , MasculinoRESUMO
Accumulating evidence indicates the potential role of actin cytoskeleton in facilitating the mitochondrial recruitment of various pro-apoptotic proteins from the cytosol to initiate apoptosis. In the present paper, we report the observation of the increase in mitochondrial association of actin in early apoptosis. Using cell fractionation and Western blot analysis, we found that mitochondrial accumulation of beta-actin occurred before the mitochondrial insertion of Bax and release of cytochrome c in apoptosis. The mitochondrial accumulation of beta-actin was observed with various apoptotic stimuli in various cell lines, suggesting that this is a general apoptotic phenomenon in mammalian systems. Using fluorescence microscopy, we have shown that an apoptotic induction triggered the reorganization of the F-actin (filamentous actin) network with an increase in the association with mitochondria, which was observed before mitochondrial fission and nuclear condensation. Perhaps actin could contribute to the initiation of apoptosis by enabling cytosolic pro-apoptotic proteins to be carried to mitochondria by the cytoskeleton-driven trafficking system.
Assuntos
Actinas/fisiologia , Apoptose/fisiologia , Mitocôndrias/fisiologia , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Células COS/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Chlorocebus aethiops , Meios de Cultura Livres de Soro/farmacologia , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Células HeLa/efeitos dos fármacos , Células HeLa/efeitos da radiação , Humanos , Peróxido de Hidrogênio/farmacologia , Rim/citologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Estaurosporina/farmacologia , Raios UltravioletaRESUMO
Anastasis (Greek for "rising to life") is a cell recovery phenomenon that rescues dying cells from the brink of cell death. We recently discovered anastasis to occur after the execution-stage of apoptosis in vitro and in vivo. Promoting anastasis could in principle preserve injured cells that are difficult to replace, such as cardiomyocytes and neurons. Conversely, arresting anastasis in dying cancer cells after cancer therapies could improve treatment efficacy. To develop new therapies that promote or inhibit anastasis, it is essential to identify the key regulators and mediators of anastasis - the therapeutic targets. Therefore, we performed time-course microarray analysis to explore the molecular mechanisms of anastasis during reversal of ethanol-induced apoptosis in mouse primary liver cells. We found striking changes in transcription of genes involved in multiple pathways, including early activation of pro-cell survival, anti-oxidation, cell cycle arrest, histone modification, DNA-damage and stress-inducible responses, and at delayed times, angiogenesis and cell migration. Validation with RT-PCR confirmed similar changes in the human liver cancer cell line, HepG2, during anastasis. Here, we present the time-course whole-genome gene expression dataset revealing gene expression profiles during the reversal of apoptosis. This dataset provides important insights into the physiological, pathological, and therapeutic implications of anastasis.
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Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.
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Técnicas Biossensoriais , Caspases , Drosophila melanogaster/enzimologia , Animais , Ativação EnzimáticaRESUMO
Anastasis (Greek for "rising to life") refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor for identifying and tracking anastasis in mammalian cells.
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Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Rastreamento de Células/métodos , Animais , Células COS , Caspases/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Citocromos c/metabolismo , Dano ao DNA , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Células NIH 3T3 , Fosfatidilserinas/farmacologiaRESUMO
The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.
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Apoptose , Técnicas Biossensoriais , Caspases/metabolismo , Animais , Sobrevivência Celular , Drosophila , Ativação Enzimática , Imagem Molecular/métodosRESUMO
Apoptosis serves as a protective mechanism by eliminating damaged cells through programmed cell death. After apoptotic cells pass critical checkpoints, including mitochondrial fragmentation, executioner caspase activation, and DNA damage, it is assumed that cell death inevitably follows. However, this assumption has not been tested directly. Here we report an unexpected reversal of late-stage apoptosis in primary liver and heart cells, macrophages, NIH 3T3 fibroblasts, cervical cancer HeLa cells, and brain cells. After exposure to an inducer of apoptosis, cells exhibited multiple morphological and biochemical hallmarks of late-stage apoptosis, including mitochondrial fragmentation, caspase-3 activation, and DNA damage. Surprisingly, the vast majority of dying cells arrested the apoptotic process and recovered when the inducer was washed away. Of importance, some cells acquired permanent genetic changes and underwent oncogenic transformation at a higher frequency than controls. Global gene expression analysis identified a molecular signature of the reversal process. We propose that reversal of apoptosis is an unanticipated mechanism to rescue cells from crisis and propose to name this mechanism "anastasis" (Greek for "rising to life"). Whereas carcinogenesis represents a harmful side effect, potential benefits of anastasis could include preservation of cells that are difficult to replace and stress-induced genetic diversity.
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Apoptose/genética , Transformação Celular Neoplásica/genética , Dano ao DNA , Animais , Animais Recém-Nascidos , Anti-Infecciosos Locais/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Depsipeptídeos/farmacologia , Etanol/farmacologia , Perfilação da Expressão Gênica , Células HeLa , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Understanding how molecular dynamics leads to cellular behaviours that ultimately sculpt organs and tissues is a major challenge not only in basic developmental biology but also in tissue engineering and regenerative medicine. Here we use live imaging to show that the basal surfaces of Drosophila follicle cells undergo a series of directional, oscillating contractions driven by periodic myosin accumulation on a polarized actin network. Inhibition of the actomyosin contractions or their coupling to extracellular matrix (ECM) blocked elongation of the whole tissue, whereas enhancement of the contractions exaggerated it. Myosin accumulated in a periodic manner before each contraction and was regulated by the small GTPase Rho, its downstream kinase, ROCK, and cytosolic calcium. Disrupting the link between the actin cytoskeleton and the ECM decreased the amplitude and period of the contractions, whereas enhancing cell-ECM adhesion increased them. In contrast, disrupting cell-cell adhesions resulted in loss of the actin network. Our findings reveal a mechanism controlling organ shape and an experimental model for the study of the effects of oscillatory actomyosin activity within a coherent cell sheet.