RESUMO
Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.
Assuntos
Junções Aderentes , Proteínas de Drosophila , Desenvolvimento Embrionário , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Junções Aderentes/metabolismo , Microtúbulos/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/citologia , alfa Catenina/metabolismo , Actomiosina/metabolismo , Morfogênese , Drosophila/embriologia , Forma Celular , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Membrana Celular , Centrossomo , Proteínas de Drosophila , Drosophila melanogaster , Animais , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Membrana Celular/metabolismo , Centrossomo/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Dineínas/metabolismo , Exocitose , Microtúbulos/metabolismoRESUMO
Actin networks are central to shaping and moving cells during animal development. Various spatial cues activate conserved signal transduction pathways to polarize actin network assembly at sub-cellular locations and to elicit specific physical changes. Actomyosin networks contract and Arp2/3 networks expand, and to affect whole cells and tissues they do so within higher-order systems. At the scale of tissues, actomyosin networks of epithelial cells can be coupled via adherens junctions to form supracellular networks. Arp2/3 networks typically integrate with distinct actin assemblies, forming expansive composites which act in conjunction with contractile actomyosin networks for whole-cell effects. This review explores these concepts using examples from Drosophila development. First, we discuss the polarized assembly of supracellular actomyosin cables which constrict and reshape epithelial tissues during embryonic wound healing, germ band extension, and mesoderm invagination, but which also form physical borders between tissue compartments at parasegment boundaries and during dorsal closure. Second, we review how locally induced Arp2/3 networks act in opposition to actomyosin structures during myoblast cell-cell fusion and cortical compartmentalization of the syncytial embryo, and how Arp2/3 and actomyosin networks also cooperate for the single cell migration of hemocytes and the collective migration of border cells. Overall, these examples show how the polarized deployment and higher-order interactions of actin networks organize developmental cell biology.
Assuntos
Actinas , Proteínas de Drosophila , Animais , Actinas/metabolismo , Drosophila , Actomiosina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Contração Muscular , Drosophila melanogasterRESUMO
Expansive Arp2/3 actin networks and contractile actomyosin networks can be spatially and temporally segregated within the cell, but the networks also interact closely at various sites, including adherens junctions. However, molecular mechanisms coordinating these interactions remain unclear. We found that the SCAR/WAVE complex, an Arp2/3 activator, is enriched at adherens junctions of the leading edge actomyosin cable during Drosophila dorsal closure. Myosin activators were both necessary and sufficient for SCAR/WAVE accumulation at leading edge junctions. The same myosin activators were previously shown to recruit the cytohesin Arf-GEF Steppke to these sites, and mammalian studies have linked Arf small G protein signaling to SCAR/WAVE activation. During dorsal closure, we find that Steppke is required for SCAR/WAVE enrichment at the actomyosin-linked junctions. Arp2/3 also localizes to adherens junctions of the leading edge cable. We propose that junctional actomyosin activity acts through Steppke to recruit SCAR/WAVE and Arp2/3 for regulation of the leading edge supracellular actomyosin cable during dorsal closure.
Assuntos
Actomiosina , Proteínas de Drosophila , Actinas/metabolismo , Actomiosina/metabolismo , Junções Aderentes/metabolismo , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mamíferos/metabolismo , Miosinas/metabolismoRESUMO
Drosophila anterior-posterior axis specification occurs in the oocyte, but the initial symmetry break has been unclear. A new study reveals that a posterior domain of cortical myosin is induced with unique post-translational modification and dynamics and that this domain recruits downstream posterior determinants.
Assuntos
Padronização Corporal , Proteínas de Drosophila , Animais , Drosophila , Miosinas , OócitosRESUMO
Cytohesin Arf-GEFs promote actin polymerization and protrusions of cultured cells, whereas the Drosophila cytohesin, Steppke, antagonizes actomyosin networks in several developmental contexts. To reconcile these findings, we analyzed epidermal leading edge actin networks during Drosophila embryo dorsal closure. Here, Steppke is required for F-actin of the actomyosin cable and for actin-based protrusions. steppke mutant defects in the leading edge actin networks are associated with improper sealing of the dorsal midline, but are distinguishable from effects of myosin mis-regulation. Steppke localizes to leading edge cell-cell junctions with accumulations of the F-actin regulator Enabled emanating from either side. Enabled requires Steppke for full leading edge recruitment, and genetic interaction shows the proteins cooperate for dorsal closure. Inversely, Steppke over-expression induces ectopic, actin-rich, lamellar cell protrusions, an effect dependent on the Arf-GEF activity and PH domain of Steppke, but independent of Steppke recruitment to myosin-rich AJs via its coiled-coil domain. Thus, Steppke promotes actin polymerization and cell protrusions, effects that occur in conjunction with Steppke's previously reported regulation of myosin contractility during dorsal closure.
Assuntos
Actinas/metabolismo , Extensões da Superfície Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Actomiosina/metabolismo , Animais , Desenvolvimento Embrionário/fisiologia , Junções Intercelulares/metabolismo , Miosinas/metabolismo , Domínios Proteicos/fisiologiaRESUMO
Tissue dynamics require regulated interactions between adherens junctions and cytoskeletal networks. For example, myosin-rich adherens junctions recruit the cytohesin Arf-GEF Steppke, which down-regulates junctional tension and facilitates tissue stretching. We dissected this recruitment mechanism with structure-function and other analyses of Steppke and Stepping stone, an implicated adaptor protein. During Drosophila dorsal closure, Steppke's coiled-coil domain was necessary and sufficient for junctional recruitment. Purified coiled-coil domains of Steppke and Stepping stone heterodimerized through a hydrophobic surface of the Steppke domain. This mapped surface was required for Steppke's junctional localization and tissue regulation. Stepping stone colocalized with Steppke at junctions, and was required for junctional Steppke localization and proper tissue stretching. A second conserved region of Stepping stone was necessary and largely sufficient for junctional localization. Remarkably, this region could substitute for the Steppke coiled-coil domain for junction localization and regulation, suggesting the main role of the Steppke coiled-coil domain is linkage to the junctional targeting region of Stepping stone. Thus, coiled-coil heterodimerization with Stepping stone normally recruits Step to junctions. Intriguingly, Stepping stone's junctional localization also seems partly dependent on Steppke.
Assuntos
Actomiosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Citoesqueleto/metabolismo , Epitélio/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Domínios Proteicos/fisiologia , Relação Estrutura-AtividadeRESUMO
Cell structure depends on the cortex, a thin network of actin polymers and additional proteins underlying the plasma membrane. The cell polarity kinase Par-1 is required for cells to form following syncytial Drosophila embryo development. This requirement stems from Par-1 promoting cortical actin caps that grow into dome-like metaphase compartments for dividing syncytial nuclei. We find the actin caps to be a composite material of Diaphanous (Dia)-based actin bundles interspersed with independently formed, Arp2/3-based actin puncta. Par-1 and Dia colocalize along extended regions of the bundles, and both are required for the bundles and for each other's bundle-like localization, consistent with an actin-dependent self-reinforcement mechanism. Par-1 helps establish or maintain these bundles in a cortical domain with relatively low levels of the canonical formin activator Rho1-GTP. Arp2/3 is required for displacing the bundles away from each other and toward the cap circumference, suggesting interactions between these cytoskeletal components could contribute to the growth of the cap into a metaphase compartment.
Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fase de Clivagem do Zigoto/metabolismo , Citoesqueleto/metabolismo , Forminas/metabolismo , Microtúbulos/metabolismo , Interferência de RNA , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. We demonstrate that Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, our findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity.
Assuntos
Junções Aderentes/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Miosinas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Células Cultivadas , Drosophila , Feminino , Células HEK293 , Células HeLa , HumanosRESUMO
Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Animais , Desenvolvimento Ósseo , Desenvolvimento Embrionário , Humanos , Neurogênese , Biossíntese de Proteínas , Transporte ProteicoRESUMO
We present a vertex-based model for Drosophila dorsal closure that predicts the mechanics of cell oscillation and contraction from the dynamics of the PAR proteins. Based on experimental observations of how aPKC, Par-6, and Bazooka translocate from the circumference of the apical surface to the medial domain, and how they interact with each other and ultimately regulate the apicomedial actomyosin, we formulate a system of differential equations that captures the key features of dorsal closure, including distinctive behaviors in its early, slow, and fast phases. The oscillation in cell area in the early phase of dorsal closure results from an intracellular negative feedback loop that involves myosin, an actomyosin regulator, aPKC, and Bazooka. In the slow phase, gradual sequestration of apicomedial aPKC by Bazooka clusters causes incomplete disassembly of the actomyosin network over each cycle of oscillation, thus producing a so-called ratchet. The fast phase of rapid cell and tissue contraction arises when medial myosin, no longer antagonized by aPKC, builds up in time and produces sustained contraction. Thus, a minimal set of rules governing the dynamics of the PAR proteins, extracted from experimental observations, can account for all major mechanical outcomes of dorsal closure, including the transitions between its three distinct phases.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Quinase 3 da Glicogênio Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Quinase C/metabolismo , Actomiosina/metabolismo , Animais , Polaridade Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Embrião não Mamífero/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Quinase C/genética , Transporte ProteicoRESUMO
The early Drosophila embryo is a large syncytial cell that compartmentalizes mitotic spindles with furrows. Before furrow ingression, an Arp2/3 actin cap forms above each nucleus and is encircled by actomyosin. We investigated how these networks transform a flat cortex into a honeycomb-like compartmental array. The growing caps circularize and ingress upon meeting their actomyosin borders, which become the furrow base. Genetic perturbations indicate that the caps physically displace their borders and, reciprocally, that the borders resist and circularize their caps. These interactions create an actomyosin cortex arrayed with circular caps. The Rac-GEF Sponge, Rac-GTP, Arp3, and actin coat the caps as a growing material that can drive cortical bending for initial furrow ingression. Additionally, laser ablations indicate that actomyosin contraction squeezes the cytoplasm, producing counterforces that swell the caps. Thus, Arp2/3 caps form clearances of the actomyosin cortex and control buckling and swelling of these clearances for metaphase compartmentalization.
Assuntos
Actinas/metabolismo , Actomiosina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Células Gigantes/fisiologia , Fuso Acromático/fisiologia , Animais , Membrana Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Células Gigantes/citologia , Microtúbulos/metabolismoRESUMO
Changes to cell shape and interaction drive animal tissue morphogenesis. Reporting now in Developmental Cell, Del Signore et al. (2018) describe active lengthening of cell-cell contacts by Arp2/3-based actin networks. Through cycles of contact lengthening and shortening, the developing Drosophila eye passes through various multicellular configurations to achieve its complex architecture.
Assuntos
Actinas , Drosophila , Complexo 2-3 de Proteínas Relacionadas à Actina , Animais , Forma Celular , MorfogêneseRESUMO
Drosophila research has revealed how planar polarized actomyosin networks affect various types of tissue morphogenesis. The networks are positioned by both tissue-wide patterning factors (including Even-skipped, Runt, Engrailed, Invected, Hedgehog, Notch, Wingless, Epidermal Growth Factor, Jun N-terminal kinase, Sex combs reduced and Fork head) and local receptor complexes (including Echinoid, Crumbs and Toll receptors). Networks with differing super-structure and contractile output have been discovered. Their contractility can affect individual cells or can be coordinated across groups of cells, and such contractility can drive or resist physical change. For what seem to be simple tissue changes, multiple types of actomyosin networks can contribute, acting together as contractile elements or braces within the developing structure. This review discusses the positioning and effects of planar polarized actomyosin networks for a number of developmental events in Drosophila, including germband extension, dorsal closure, head involution, tracheal pit formation, salivary gland development, imaginal disc boundary formation, and tissue rotation of the male genitalia and the egg chamber.
Assuntos
Actomiosina/fisiologia , Polaridade Celular/fisiologia , Proteínas de Drosophila/metabolismo , Epitélio/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Movimento Celular/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The scaffold protein Par-3 ( Drosophila Bazooka) is a central organizer of cell polarity across animals. This review focuses on how the clustering of Par-3 contributes to cell polarity. It begins with the Par-3 homo-oligomerization mechanism and its regulation by Par-1 phosphorylation. The role of polarized cytoskeletal networks in distributing Par-3 clusters to one end of the cell is then discussed, as is the subsequent maintenance of polarized Par-3 clusters through hindered mobility and inhibition from the opposite pole. Finally, specific roles of Par-3 clusters are reviewed, including the bundling of microtubules, the cortical docking of centrosomes, the growth and positioning of cadherin-catenin clusters, and the inhibition of the Par-6-aPKC kinase cassette. Examples are drawn from Drosophila, Caenorhabditis elegans, mammalian cell culture, and biochemical studies.
RESUMO
In response to a pulling force, a material can elongate, hold fast, or fracture. During animal development, multi-cellular contraction of one region often stretches neighboring tissue. Such local contraction occurs by induced actomyosin activity, but molecular mechanisms are unknown for regulating the physical properties of connected tissue for elongation under stress. We show that cytohesins, and their Arf small G protein guanine nucleotide exchange activity, are required for tissues to elongate under stress during both Drosophila dorsal closure (DC) and zebrafish epiboly. In Drosophila, protein localization, laser ablation, and genetic interaction studies indicate that the cytohesin Steppke reduces tissue tension by inhibiting actomyosin activity at adherens junctions. Without Steppke, embryogenesis fails, with epidermal distortions and tears resulting from myosin misregulation. Remarkably, actomyosin network assembly is necessary and sufficient for local Steppke accumulation, where live imaging shows Steppke recruitment within minutes. This rapid negative feedback loop provides a molecular mechanism for attenuating the main tension generator of animal tissues. Such attenuation relaxes tissues and allows orderly elongation under stress.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Reguladores de Proteínas de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Biosynthetic traffic from the Golgi drives plasma membrane growth. For Drosophila embryo cleavage, this growth is rapid but regulated for cycles of furrow ingression and regression. The highly conserved small G protein Arf1 organizes Golgi trafficking. Arf1 is activated by guanine nucleotide exchange factors, but essential roles for Arf1 GTPase-activating proteins (GAPs) are less clear. We report that the conserved Arf GAP Asap is required for cleavage furrow ingression in the early embryo. Because Asap can affect multiple subcellular processes, we used genetic approaches to dissect its primary effect. Our data argue against cytoskeletal or endocytic involvement and reveal a common role for Asap and Arf1 in Golgi organization. Although Asap lacked Golgi enrichment, it was necessary and sufficient for Arf1 accumulation at the Golgi, and a conserved Arf1-Asap binding site was required for Golgi organization and output. Of note, Asap relocalized to the nuclear region at metaphase, a shift that coincided with subtle Golgi reorganization preceding cleavage furrow regression. We conclude that Asap is essential for Arf1 to function at the Golgi for cleavage furrow biosynthesis. Asap may recycle Arf1 to the Golgi from post-Golgi membranes, providing optimal Golgi output for specific stages of the cell cycle.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Fase de Clivagem do Zigoto/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Transporte ProteicoRESUMO
Cadherin-based adherens junctions are critical for connecting cells in tissues. Regulated cadherin trafficking also makes these complexes amazingly dynamic, with permissive and instructive consequences on multicellular development. Here, we review how cadherin trafficking affects various forms of tissue morphogenesis from Drosophila and Caenorhabditis elegans to zebrafish, Xenopus and mouse. We describe how core trafficking machinery (such as clathrin, dynamin, Rab small G proteins and the exocyst complex) integrates with other molecular systems (transcriptional factors, signaling pathways, microtubules, actin networks, apico-basal polarity proteins and planar cell polarity proteins) to control cadherin endocytosis, exocytosis and recycling. This control can occur at all cell-cell contacts or specific junctions for distinct effects on tissue morphogenesis during animal development.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Desenvolvimento Embrionário/fisiologia , Morfogênese/fisiologia , Organogênese/fisiologia , Animais , Polaridade Celular/fisiologia , Endocitose/fisiologia , Exocitose/fisiologia , Humanos , Transporte ProteicoRESUMO
The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Estrutura Terciária de ProteínaRESUMO
To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells [1-3]. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy [4], but AJs are dynamic during epithelial development and remodeling [5, 6], and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation [7]. Here, we show that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer [5, 8-10], and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, our loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization [8, 11, 12]. We find that Par-1 promotes the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy [13-16]. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference.