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The TP53 gene is renowned as a tumor suppressor, playing a pivotal role in overseeing the cell cycle, apoptosis, and maintaining genomic stability. Dysregulation of p53 often contributes to the initiation and progression of various cancers, including lung cancer (LC) subtypes. The review explores the intricate relationship between p53 and its role in the development and progression of LC. p53, a crucial tumor suppressor protein, exists in various isoforms, and understanding their distinct functions in LC is essential for advancing our knowledge of this deadly disease. This review aims to provide a comprehensive literature overview of p53, its relevance to LC, and potential clinical applications.
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The microbiota in humans and animals play crucial roles in defense against pathogens and offer a promising natural source for immunomodulatory products. However, the development of physiologically relevant model systems and protocols for testing such products remains challenging. In this study, we present an experimental condition where various natural products derived from the registered lactic acid bacteria Ligilactobacillus salivarius CECT 9609, known for their immunomodulatory activity, were tested. These products included live and inactivated bacteria, as well as fermentation products at different concentrations and culture times. Using our established model system, we observed no morphological changes in the airway epithelium upon exposure to Pasteurella multocida, a common respiratory pathogen. However, early molecular changes associated with the innate immune response were detected through transcript analysis. By employing diverse methodologies ranging from microscopy to next-generation sequencing (NGS), we characterized the interaction of these natural products with the airway epithelium and their potential beneficial effects in the presence of P. multocida infection. In particular, our discovery highlights that among all Ligilactobacillus salivarius CECT 9609 products tested, only inactivated cells preserve the conformation and morphology of respiratory epithelial cells, while also reversing or altering the natural immune responses triggered by Pasteurella multocida. These findings lay the groundwork for further exploration into the protective role of these bacteria and their derivatives.
Assuntos
Produtos Biológicos , Ligilactobacillus salivarius , Infecções por Pasteurella , Pasteurella multocida , Humanos , Animais , Imunidade Inata , Células Epiteliais , Produtos Biológicos/farmacologia , Infecções por Pasteurella/microbiologia , Infecções por Pasteurella/veterináriaRESUMO
The airway epithelium is a critical component of the respiratory system, serving as a barrier against inhaled pathogens and toxins. It is composed of various cell types, each with specific functions essential to proper airway function. Chronic respiratory diseases can disrupt the cellular composition of the airway epithelium, leading to a decrease in multiciliated cells (MCCs) and an increase in secretory cells (SCs). Basal cells (BCs) have been identified as the primary stem cells in the airway epithelium, capable of self-renewal and differentiation into MCCs and SCs. This review emphasizes the role of transcription factors in the differentiation process from BCs to MCCs and SCs. Recent advancements in single-cell RNA sequencing (scRNAseq) techniques have provided insights into the cellular composition of the airway epithelium, revealing specialized and rare cell types, including neuroendocrine cells, tuft cells, and ionocytes. Understanding the cellular composition and differentiation processes within the airway epithelium is crucial for developing targeted therapies for respiratory diseases. Additionally, the maintenance of BC populations and the involvement of Notch signaling in BC self-renewal and differentiation are discussed. Further research in these areas could provide valuable insights into the mechanisms underlying airway epithelial homeostasis and disease pathogenesis.
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Células Epiteliais , Doenças Respiratórias , Humanos , Células Epiteliais/metabolismo , Mucosa Respiratória/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica , Doenças Respiratórias/metabolismoRESUMO
Tight-junction (TJ) proteins are essential for establishing the barrier function between neighbor epithelial cells, but also for recognition of pathogens or cell migration. Establishing the expression pattern and localization of different TJ proteins will help to understand the development and physiology of the airway. Here we identify that the junctional adhesion molecule 3 (Jam3) expression is restricted to multiciliated cells (MCCs) in the airway epithelium. In vitro, Jam3 expression varies along airway basal stem cell (BSC) differentiation and upon DAPT treatment or IL6 exposure. However, Jam3 is not required for BSC differentiation to specific cell types. In addition, we found that MCC lacking Jam3 display normal cilia morphology and cilia beating frequency with a delay in BB assembly/positioning in MCCs during differentiation. Remarkably, Jam3 in MCC is mostly localized to subapical organelles, which are negative for the apical recycling endosome marker Rab11 and positive for EEA1. Our data show that Jam3 expression is connected to mature MCC in the airway epithelium and suggest a Jam3 role unrelated to its known barrier function.
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Proper physiological function of mammalian airways requires the differentiation of basal stem cells into secretory or multiciliated cells, among others. In addition, the self-renewal ability of these basal stem cells is crucial for developing a quick response to toxic agents in order to re-establish the epithelial barrier function of the airways. Although these epithelial missions are vital, little is known about those mechanism controlling airway epithelial regeneration in health and disease. p53 has been recently proposed as the guardian of homeostasis, promoting differentiation programs, and antagonizing a de-differentiation program. Here, we exploit mouse and human tracheal epithelial cell culture models to study the role of MDM2-p53 signaling in self-renewal and differentiation in the airway epithelium. We show that p53 protein regulation by MDM2 is crucial for basal stem cell differentiation and to keep proper cell proliferation. Therefore, we suggest that MDM2/p53 interaction modulation is a potential target to control regeneration of the mammalian airway epithelia without massively affecting the epithelium integrity and differentiation potential.
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Diferenciação Celular/fisiologia , Epitélio/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Mucosa Respiratória/metabolismo , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Feminino , Homeostase/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Traqueia/metabolismoRESUMO
Planar cell polarity (PCP) is evolutionary conserved and play a critical role in proper tissue development and function. During central nervous system development, PCP proteins exhibit specific patterns of distribution and are indispensable for axonal growth, dendritogenesis, neuronal migration, and neuronal differentiation. The retina constitutes an excellent model in which to study molecular mechanisms involved in neural development. The analysis of the spatiotemporal expression of PCP proteins in this model constitutes an useful histological approach in order to identify possible roles of these proteins in retinogenesis. Immunohistochemical techniques revealed that Frz6, Celsr1, Vangl1, Pk1, Pk3, and Fat1 were present in emerging axons from recently differentiated ganglion cells in the chicken retina. Except for Vangl1, they were also asymmetrically distributed in differentiated amacrine cells. Pk1 and Pk3 were restricted in the outer nuclear layer to the outer segment of photoreceptors. Vangl1 was also located in the cell somata of Müller glia. Given these findings together, the distribution of PCP proteins in the developing chicken retina suggest essential roles in axonal guidance during early retinogenesis and a possible involvement in the establishment of cell asymmetry and maintenance of retinal cell phenotypes.
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Axônios/metabolismo , Polaridade Celular/fisiologia , Neuroglia/metabolismo , Retina/embriologia , Células Ganglionares da Retina/metabolismo , Animais , Diferenciação Celular , Embrião de Galinha , Modelos Animais , Retina/metabolismo , Células Ganglionares da Retina/citologiaRESUMO
IL6 is an essential cytokine in metabolism regulation and for intercommunication among different organs and tissues. IL6 produced by different tissues has different functions and therefore it is very important to understand the mechanism of its expression in adipose tissue. In this work we demonstrated that IL6 expression in mouse preadipocytes, like in human, is partially dependent on Wnt5a and JNK. Using mouse preadipocytes lacking each one of the p38 SAPK family members, we have shown that IL6 expression is also p38γ and p38δ dependent. In fact, the lack of some of these two kinases increases IL6 expression without altering that of Wnt5a. Moreover, we show that the absence of p38δ promotes greater ERK1/2 phosphorylation in a MEK1/2 independent manner, and that this increased ERK1/2 phosphorylation state is contributing to the higher IL6 expression in p38δ-/- preadipocytes. These results suggest a new crosstalk between two MAPK signaling pathway, p38δ and ERK1/2, where p38δ modulates the phosphorylation state of ERK1/2.
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BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMAL1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-of-function of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination.
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Fatores de Transcrição ARNTL/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição ARNTL/fisiologia , Animais , Diferenciação Celular/fisiologia , Ritmo Circadiano/genética , Metabolismo Energético/fisiologia , Expressão Gênica/genética , Glicólise , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Mitocôndrias/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/citologiaRESUMO
Organelle positioning as many other morphological parameters in a cell is not random. Centriole positioning as centrosomes or ciliary basal bodies is not an exception to this rule in cell biology. Indeed, centriole positioning is a tightly regulated process that occurs during development, and it is critical for many organs to function properly, not just during development but also in the adulthood. In this book chapter, we overview our knowledge on centriole positioning in different and highly specialized animal cells like photoreceptor or ependymal cells. We will also discuss recent advances in the discovery of molecular pathways involved in this process, mostly related to the cytoskeleton and the cell polarity pathways. And finally, we present quantitative methods that have been used to assess centriole positioning in different cell types although mostly in epithelial cells.
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Compartimento Celular , Centríolos/metabolismo , Animais , Polaridade Celular , Citoesqueleto/metabolismoRESUMO
Proper ciliary basal body positioning within a cell is key for cilia functioning. Centriole and basal body positioning depends on signaling pathways such as the planar cell polarity pathway (PCP) governed by Frizzled (Fz-PCP). There have been described two PCP pathways controlled by different protein complexes, the Frizzled-PCP and the Fat-PCP pathway. Centriole planar polarization in non-dividing cells is a dynamic process that depends on the Fz-PCP pathway to properly occur during development from flies to humans. However, the function of the Ft-PCP pathway in centrioles polarization is elusive. Here, we present a descriptive initial analysis of centrioles polarization in Fat-PCP loss of function (LOF) conditions. We found that Fat (Ft) and Dachsous (Ds) LOF showed a marked centrioles polarization defect similar to what we have previously reported in Fz-PCP alterations. Altogether, our data suggest that centriole planar polarization in Drosophila wings depends on both Ft-PCP and Fz-PCP pathways. Further analyses in single and double mutant conditions will be required to address the functional connection between PCP and centriole polarization in flies.
RESUMO
In vertebrates, planar polarization of ciliary basal bodies has been associated with actin polymerization that occurs downstream of the Frizzled-planar cell polarity (Fz-PCP) pathway. In Drosophila wing epithelial cells, which do not have cilia, centrioles also polarize in a Fz-PCP-dependent manner, although the relationship with actin polymerization remains unknown. By combining existing and new quantitative methods, we unexpectedly found that known PCP effectors linked to actin polymerization phenotypes affect neither final centriole polarization nor apical centriole distribution. But actin polymerization is required upstream of Fz-PCP to maintain the centrioles in restricted areas in the apical-most planes of those epithelial cells before and after the actin-based hair is formed. Furthermore, in the absence of proper core Fz-PCP signalling, actin polymerization is insufficient to drive this off-centred centriole migration. Altogether, the results reveal that there are at least two pathways controlling centriole positioning in Drosophila pupal wings - an upstream actin-dependent mechanism involved in centriole distribution that is PCP independent, and an unknown mechanism that links core Fz-PCP and centriole polarization.
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Polaridade Celular , Centríolos/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Asas de Animais/citologia , Asas de Animais/metabolismo , Actinas/metabolismo , Animais , Polaridade Celular/efeitos dos fármacos , Centríolos/efeitos dos fármacos , Citocalasina D/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Mutação com Ganho de Função/genética , Mutação com Perda de Função/genética , Fenótipo , PolimerizaçãoRESUMO
Planar cell polarity (PCP)-signaling and associated tissue polarization are evolutionarily conserved. A well documented feature of PCP-signaling in vertebrates is its link to centriole/cilia positioning, although the relationship of PCP and ciliogenesis is still debated. A recent report in Drosophila established that Frizzled (Fz)-PCP core signaling has an instructive input to polarized centriole positioning in non-ciliated Drosophila wing epithelia as a PCP read-out. Here, we review the impact of this observation in the context of recent descriptions of the relationship(s) of core Fz-PCP signaling and cilia/centriole positioning in epithelial and non-epithelial cells. All existing data are consistent with a model where Fz-PCP signaling functions upstream of centriole/cilia positioning, independent of ciliogenesis. The combined data sets indicate that the Fz-Dsh PCP complex is instructive for centriole/ciliary positioning via an actin-based mechanism. Thereby, centriole/cilia/centrosome positioning can be considered an evolutionarily conserved readout and common downstream effect of PCP-signaling from flies to mammals.
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Polaridade Celular , Centríolos/fisiologia , Células Epiteliais/fisiologia , Receptores Frizzled/fisiologia , Transdução de Sinais , Animais , Células Epiteliais/metabolismoRESUMO
Most mutant alleles in the Fz-PCP pathway genes were discovered in classic Drosophila screens looking for recessive loss-of-function (LOF) mutations. Nonetheless, although Fz-PCP signaling is sensitive to increased doses of PCP gene products, not many screens have been performed in the wing under genetically engineered Fz overexpression conditions, mostly because the Fz phenotypes were strong and/or not easy to score and quantify. Here, we present a screen based on an unexpected mild Frizzled gain-of-function (GOF) phenotype. The leakiness of a chimeric Frizzled protein designed to be accumulated in the endoplasmic reticulum (ER) generated a reproducible Frizzled GOF phenotype in Drosophila wings. Using this genotype, we first screened a genome-wide collection of large deficiencies and found 16 strongly interacting genomic regions. Next, we narrowed down seven of those regions to finally test 116 candidate genes. We were, thus, able to identify eight new loci with a potential function in the PCP context. We further analyzed and confirmed krasavietz and its interactor short-stop as new genes acting during planar cell polarity establishment with a function related to actin and microtubule dynamics.
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Polaridade Celular/genética , Proteínas de Drosophila/genética , Drosophila/embriologia , Drosophila/genética , Genes Modificadores , Testes Genéticos , Organogênese/genética , Asas de Animais/embriologia , Alelos , Animais , Animais Geneticamente Modificados , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Estudos de Associação Genética , Patrimônio Genético , Genótipo , FenótipoRESUMO
Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. Here we report instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. We show that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals.
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Polaridade Celular , Centríolos/metabolismo , Proteínas de Drosophila/metabolismo , Receptores Frizzled/metabolismo , Transdução de Sinais , Animais , Citoesqueleto/metabolismo , Drosophila melanogaster , Pupa/citologia , Asas de Animais/citologiaRESUMO
A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo.
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Fator 1 de Ribosilação do ADP/genética , Complexo 1 de Proteínas Adaptadoras/genética , Polaridade Celular/genética , Proteínas de Drosophila/genética , Drosophila/embriologia , Asas de Animais/embriologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Desgrenhadas , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fosfoproteínas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid-base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS(812)LL, a dileucine motif in CLC-2's C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel's dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI(623)LL and QVVA(635)LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI(623)LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI(623)LL with a highly conserved pocket in their γ1-σ1A hemicomplex.
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Complexo 1 de Proteínas Adaptadoras/metabolismo , Canais de Cloreto/metabolismo , Complexo 1 de Proteínas Adaptadoras/química , Motivos de Aminoácidos , Animais , Canais de Cloro CLC-2 , Canais de Cloreto/química , Cães , Células Madin Darby de Rim Canino , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte ProteicoRESUMO
Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.
Assuntos
Vias Biossintéticas/fisiologia , Polaridade Celular/fisiologia , Células Epiteliais/citologia , Rodopsina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Primers do DNA/genética , Cães , Complexo de Golgi/metabolismo , Immunoblotting , Imuno-Histoquímica , Células Madin Darby de Rim Canino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Plasmídeos/genética , Transporte Proteico/fisiologia , Rodopsina/biossíntese , Vesículas Transportadoras/metabolismoRESUMO
Correct patterning and polarization of epithelial and mesenchymal cells are essential for morphogenesis and function of all organs and organisms. Epithelial cells are generally polarized in two axes: (a) the ubiquitous apical-basal axis and (b) polarity within the plane of the epithelium. The latter is generally referred to as planar cell polarity (PCP) and also is found in several contexts of mesenchymal cell patterning. In Drosophila, all adult structures display PCP features, and two conserved molecular systems (the Fat [Ft]/Dachsous [Ds] system and the Frizzled [Fz]/PCP pathway) that regulate this process have been identified. Although significant progress has been made in dissecting aspects of PCP signaling within cells, much remains to be discovered about the mechanisms of long-range and local PCP cell-cell interactions. Here, we discuss the current models based on Drosophila studies and incorporate recent insights into this long-standing cell and developmental biology problem.
RESUMO
Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). We demonstrate in Drosophila melanogaster wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/genética , Asas de Animais/metabolismo , Proteínas Wnt/genética , Proteína Wnt1/genética , Animais , Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais , Asas de Animais/citologia , Proteínas Wnt/metabolismo , Proteína Wnt1/metabolismoRESUMO
Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelial-specific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.