RESUMEN
Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. We show that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing, and that autophagosomes that appear near the wound edge acquire plasma membrane markers. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo-membranes and the lateral plasma membrane, while apical and basal membranes and epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent destruction of the larval epidermis by autophagy, in a process that depends on phagophore initiation and expansion but does not require autophagosomes fusion with lysosomes. Autophagy induction can also affect other sub-cellular membranes, as shown by its suppression of experimentally induced laminopathy-like nuclear defects. Our findings reveal a function for TORC1-mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing.
Asunto(s)
Autofagosomas , Autofagia , Animales , Autofagosomas/metabolismo , Membrana Celular , Drosophila , Células Gigantes/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
An effective healing response is critical to promote and ensure healthy aging. Major discoveries in both fields-repair and aging-have led to a better understanding of the mechanisms regulating the healing response and of the complexity of the aging process. It will now be important to translate and connect those findings to improve our insights into the decline of regeneration in the elderly. Furthermore, we need to understand how this process can be stalled to maintain and promote tissue resilience. Furthermore, it remains to be explored how the findings in model organisms are conserved in human wounds and how these findings might be translated into the clinic.
Asunto(s)
Inmunosenescencia , Queratinocitos/patología , Piel/lesiones , Cicatrización de Heridas/inmunología , Animales , Movimiento Celular/inmunología , Proliferación Celular , Senescencia Celular/inmunología , Modelos Animales de Enfermedad , Humanos , Queratinocitos/inmunología , Piel/citología , Piel/inmunología , Piel/patologíaRESUMEN
The Drosophila larva has been used to investigate many processes in cell biology, including morphogenesis, physiology and responses to drugs and new therapeutic compounds. Despite its enormous potential as a model system, longer-term live imaging has been technically challenging because of a lack of efficient methods for immobilizing larvae for extended periods. We describe here a simple procedure for anesthetization and uninterrupted long-term in vivo imaging of the epidermis and other larval organs, including gut, imaginal discs, neurons, fat body, tracheae, muscles and hemocytes, for up to 8 h. We also include a procedure for probing cell properties by laser ablation. We provide a survey of the effects of different anesthetics, demonstrating that short exposure to diethyl ether is the most effective for long-term immobilization of larvae. This protocol does not require specific expertise beyond basic Drosophila genetics and husbandry, and confocal microscopy. It enables high-resolution studies of many systemic and subcellular processes in larvae.
Asunto(s)
Drosophila/anatomía & histología , Microscopía Confocal/métodos , Grabación en Video/métodos , Animales , Éter , Inmovilización , Larva/anatomía & histología , Factores de TiempoRESUMEN
In epithelia, cells adhere to each other in a dynamic fashion, allowing the cells to change their shape and move along each other during morphogenesis. The regulation of adhesion occurs at the belt-shaped adherens junction, the zonula adherens (ZA). Formation of the ZA depends on components of the Par-atypical PKC (Par-aPKC) complex of polarity regulators. We have identified the Lin11, Isl-1, Mec-3 (LIM) protein Smallish (Smash), the orthologue of vertebrate LMO7, as a binding partner of Bazooka/Par-3 (Baz), a core component of the Par-aPKC complex. Smash also binds to Canoe/Afadin and the tyrosine kinase Src42A and localizes to the ZA in a planar polarized fashion. Animals lacking Smash show loss of planar cell polarity (PCP) in the embryonic epidermis and reduced cell bond tension, leading to severe defects during embryonic morphogenesis of epithelial tissues and organs. Overexpression of Smash causes apical constriction of epithelial cells. We propose that Smash is a key regulator of morphogenesis coordinating PCP and actomyosin contractility at the ZA.
Asunto(s)
Uniones Adherentes/metabolismo , Proteínas de Drosophila/metabolismo , Epidermis/embriología , Células Epiteliales/metabolismo , Morfogénesis/fisiología , Uniones Adherentes/genética , Animales , Proteínas de Drosophila/genética , Drosophila melanogasterRESUMEN
The TOR and Insulin/IGF signalling (IIS) network controls growth, metabolism and ageing. Although reducing TOR or insulin signalling can be beneficial for ageing, it can be detrimental for wound healing, but the reasons for this difference are unknown. Here we show that IIS is activated in the cells surrounding an epidermal wound in Drosophila melanogaster larvae, resulting in PI3K activation and redistribution of the transcription factor FOXO. Insulin and TOR signalling are independently necessary for normal wound healing, with FOXO and S6K as their respective effectors. IIS is specifically required in cells surrounding the wound, and the effect is independent of glycogen metabolism. Insulin signalling is needed for the efficient assembly of an actomyosin cable around the wound, and constitutively active myosin II regulatory light chain suppresses the effects of reduced IIS. These findings may have implications for the role of insulin signalling and FOXO activation in diabetic wound healing.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Transcripción Forkhead/metabolismo , Insulina/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Cicatrización de Heridas/fisiología , Actomiosina/biosíntesis , Animales , Activación Enzimática , Regulación de la Expresión Génica/genética , Glucógeno/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Miosina Tipo II/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Mutations in the hedgehog pathway drive the formation of tumors in many different organs, including the development of basal cell carcinoma in the skin. However, little is known about the role of epidermal Indian hedgehog (Ihh) in skin physiology. Using mouse genetics, we identified overlapping and distinct functions of Ihh in different models of epidermal tumorigenesis. Epidermal deletion of Ihh resulted in increased formation of benign squamous papilloma. Strikingly, Ihh-deficient mice showed an increase in malignant squamous cell carcinoma and developed lung and lymph node metastases. In a sebaceous gland tumor model, Ihh deficiency inhibited tumor cell differentiation. More mechanistically, IHH stimulated cell proliferation by activating the transcription factor GLI2 in human keratinocytes and human tumors. Thus, our results uncover important functions for Ihh signaling in controlling proliferation, differentiation, malignant progression, and metastasis of epithelial cancer, establishing Ihh as a gatekeeper for controlling the grade of tumor malignancy.