ABSTRACT
The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.
Subject(s)
Cytoskeleton/metabolism , Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mechanotransduction, Cellular/physiology , Ovarian Follicle/metabolism , Protein Serine-Threonine Kinases/metabolism , Spectrin/metabolism , Animals , Cell Line , Cytoskeleton/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Ovarian Follicle/cytology , Protein Serine-Threonine Kinases/genetics , Spectrin/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , YAP-Signaling ProteinsABSTRACT
In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli.
Subject(s)
Drosophila Proteins/genetics , Microfilament Proteins/genetics , Microtubule-Associated Proteins/genetics , Microvilli/metabolism , Myosin Type V/genetics , rab GTP-Binding Proteins/genetics , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Animals , Cadherins/metabolism , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Polarity/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Epithelium/growth & development , Epithelium/metabolism , Humans , Microtubules/genetics , Microvilli/genetics , Myosin Type V/metabolism , Protein Transport/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.