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1.
The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane.  相似文献   

2.
Cellularization of the Drosophila embryo is a specialized form of cytokinesis that couples membrane growth with the formation of a polarized epithelium. We have identified a gene essential for polarized growth of the plasma membrane during cellularization. In slam mutant embryos, the furrow canal is disorganized, and polarized insertion of transmembrane proteins is disrupted. slam shows a striking developmental induction during the slow phase of cellularization, and Slam protein localizes to the furrow canal and the basal junction. Slam colocalizes with the junctional proteins Arm/beta-catenin, the PDZ domain-containing protein Dlt, and Myosin and is also required for their proper membrane localization. Our results suggest that developmental induction of Slam organizes the polarized growth of membrane via the recruitment of membrane-targeting proteins at adherens junctions.  相似文献   

3.
《Fly》2013,7(3-4):191-198
ABSTRACT

In this extra view, we comment on our recent work concerning the mRNA localization of the gene slow as molasses (slam). slam is a gene essential for the polarized invagination of the plasma membrane and separation of basal and lateral cortical domains during cellularization as well as for germ cell migration in later embryogenesis. We have demonstrated an intimate relationship between slam RNA and its encoded protein. Slam RNA co-localizes and forms a complex with its encoded protein. Slam mRNA localization not only is required for reaching full levels of functional Slam protein but also depends on Slam protein. The translation of slam mRNA is subject to tight spatio-temporal regulation leading to a rapid accumulation of Slam protein and zygotic slam RNA at the furrow canal. In this extra view, we first discuss the mechanism controlling localization and translation of slam RNA. In addition, we document in detail the maternal and zygotic expression of slam RNA and protein and provide data for a function in membrane stabilization. Furthermore, we mapped the region of Slam protein mediating cortical localization in cultured cells.  相似文献   

4.
Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help us understand animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. Here, we report the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. We find that Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, our data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction.  相似文献   

5.
During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe that allows the visualisation of small RhoGTPase activity in Drosophila, we present evidence that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination.  相似文献   

6.
Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)(Pbl)-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF(Pbl)-dependent control themselves. Microscopy of Drosophila melanogaster S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, we uncovered a separate RhoGEF(Pbl)-dependent pathway that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF(Pbl)-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. We propose that such interactions occur dynamically and transiently to promote furrow stability.  相似文献   

7.
Cytohesin Arf-GEFs are conserved plasma membrane regulators. The sole Drosophila cytohesin, Steppke, restrains Rho1-dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the syncytial embryo. By mass spectrometry, we identified a single major Steppke-interacting protein from syncytial embryos, which we named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homologue of the mammalian cytohesin adaptor FRMD4A. Our experiments supported this relationship. Specifically, heterophilic coiled-coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C-terminal region was required for Sstn localization to furrows. Sstn mutant and RNAi embryos displayed abnormal, Rho1-dependent membrane cytoskeleton expansion from the base of pseudocleavage and cellularization furrows, closely mimicking Steppke loss-of-function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting that Steppke acts downstream of Sstn and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled-coil domain of Steppke was required for Sstn binding and in addition homodimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall we propose that Sstn acts as a cytohesin adaptor that promotes Steppke activity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo.  相似文献   

8.
Plasma membrane ingression during cytokinesis involves both actin remodeling and vesicle-mediated membrane addition. Vesicle-based membrane delivery from the recycling endosome (RE) has an essential but ill-defined involvement in cytokinesis. In the Drosophila melanogaster early embryo, Nuf (Nuclear fallout), a Rab11 effector which is essential for RE function, is required for F-actin and membrane integrity during furrow ingression. We find that in nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane. Wild-type embryos treated with Latrunculin A or Rho inhibitor display similar defects. Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects. We also find that RhoGEF2 does not properly localize at the furrow in nuf mutant embryos and that RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf. We propose a model in which RE-derived vesicles promote furrow integrity by regulating the rate of actin polymerization through the RhoGEF2-Rho1 pathway.  相似文献   

9.
The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI''s cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1''s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.  相似文献   

10.
Drosophila spectrin: the membrane skeleton during embryogenesis   总被引:12,自引:9,他引:3       下载免费PDF全文
《The Journal of cell biology》1989,108(5):1697-1709
The distribution of alpha-spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo.  相似文献   

11.
Syntrophins are a family of 59 kDa peripheral membrane‐associated adapter proteins, containing multiple protein‐protein and protein‐lipid interaction domains. The syntrophin family consists of five isoforms that exhibit specific tissue distribution, distinct sub‐cellular localization and unique expression patterns implying their diverse functional roles. These syntrophin isoforms form multiple functional protein complexes and ensure proper localization of signalling proteins and their binding partners to specific membrane domains and provide appropriate spatiotemporal regulation of signalling pathways. Syntrophins consist of two PH domains, a PDZ domain and a conserved SU domain. The PH1 domain is split by the PDZ domain. The PH2 and the SU domain are involved in the interaction between syntrophin and the dystrophin‐glycoprotein complex (DGC). Syntrophins recruit various signalling proteins to DGC and link extracellular matrix to internal signalling apparatus via DGC. The different domains of the syntrophin isoforms are responsible for modulation of cytoskeleton. Syntrophins associate with cytoskeletal proteins and lead to various cellular responses by modulating the cytoskeleton. Syntrophins are involved in many physiological processes which involve cytoskeletal reorganization like insulin secretion, blood pressure regulation, myogenesis, cell migration, formation and retraction of focal adhesions. Syntrophins have been implicated in various pathologies like Alzheimer’s disease, muscular dystrophy, cancer. Their role in cytoskeletal organization and modulation makes them perfect candidates for further studies in various cancers and other ailments that involve cytoskeletal modulation. The role of syntrophins in cytoskeletal organization and modulation has not yet been comprehensively reviewed till now. This review focuses on syntrophins and highlights their role in cytoskeletal organization, modulation and dynamics via its involvement in different cell signalling networks.  相似文献   

12.
13.
Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis.  相似文献   

14.
Several G proteins of the Rho family have been shown to be required for cytokinesis. The activity of these proteins is regulated by GTP exchange factors (GEFs), which stimulate GDP/GTP exchange, and by GTPase activating proteins (GAPs), which suppress activity by stimulating the intrinsic GTPase activity. The role of Rho family members during cytokinesis is likely to be determined by their spatial and temporal interactions with these factors. Here we focus on the role of the pebble (pbl) gene of Drosophila melanogaster, a RhoGEF that is required for cytokinesis. We summarise the evidence that the primary target of PBL is Rho1 and describe genetic approaches to elucidating the function of PBL and identifying other components of the PBL-activated Rho signalling pathway.  相似文献   

15.
MARK/PAR-1 protein kinases play important roles in cell polarization in animals. Kin1 and Kin2 are a pair of MARK/PAR-1 orthologs in the budding yeast Saccharomyces cerevisiae. They participate in the regulation of secretion and ER stress response. However, neither the subcellular localization of these two kinases nor whether they may have other cellular functions is clear. Here, we show that Kin2 localizes to the sites of polarized growth in addition to localization on the plasma membrane. The localization to polarity sites is mediated by two targeting domains—TD1 and TD2. TD1 locates in the N-terminal region that spans the protein kinase domain whereas TD2 locates in the C-terminal end that covers the KA1 domain. We also show that an excess of Kin2 activity impaired growth, septin organization, and chitin deposition in the cell wall. Both TD1 and TD2 contribute to this function. Moreover, we find that the C-terminal region of Kin2 interacts with Cdc11, a septin subunit, and Pea2, a component of the polarisome that is known to play a role in septin organization. These findings suggest that Kin2 may play a role in the regulation of the septin cytoskeleton and the cell wall. Finally, we show that the C-terminal region of Kin2 interacts with Rho3, a Rho GTPase, whereas the N-terminal region of Kin2 interacts with Bmh1, a 14-3-3 protein. We speculate that Kin2 may be regulated by Bmh1, Rho3, or Pea2 in vivo. Our study provides new insight in the localization, function, and regulation of Kin2.  相似文献   

16.
Rho GTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. Here we found that a member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G(12/13) family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ RhoGEF and abolishes its ability to mediate the accumulation of Rho-GTP by Galpha13. Moreover, when overexpressed, active PAK4 was able to dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases.  相似文献   

17.
In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein–protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin–Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (ΔL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Δ5) or fused to the L27 domain of LIN7 (L27-IRSp53Δ5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7–IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.  相似文献   

18.
The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA.  相似文献   

19.
BACKGROUND INFORMATION: Rho GTPases are involved in many biological processes and participate in cancer development. Their activation is catalysed by exchange factors [RhoGEFs (Rho GTPase guanine nucleotide-exchange factor)] of the Dbl family. RhoGEFs display proto-oncogenic features, thus appearing as candidate targets for anticancer drugs. Dominant-negative Rho GTPase mutants have been widely used to block RhoGEF signalling. However, these tools suffer from limitations, due to the high number of RhoGEFs and the complex mechanisms that control Rho GTPase activation. RESULTS: RhoG-T17N is a poor inhibitor of its exchange factor TRIO-GEFD1 (first exchange domain of the exchange factor TRIO) in vivo: although it binds to TRIO-GEFD1, RhoG-T17N does not block the downstream signalling. Using the yeast exchange assay, we show that in the presence of TRIO-GEFD1, RhoG-T17N can bind to its effectors, which illustrates how negative mutants may produce misleading interpretations and emphasizes the need for new types of RhoGEF inhibitors. In that prospect, we adapted the yeast exchange assay method to identify RhoGEF inhibitors. Using this novel approach, we screened a 3500-chemical-compound library and identified a potential inhibitor of TRIO-GEFD1. This molecule inhibited TRIO-GEFD1 in vitro. Among the chemical analogues of this compound, we identified two molecules with better inhibitory activity. The three TRIO-GEFD1 inhibitors had no effect on ARHGEF17 and ARNO [ARF (ADP-ribosylation factor) nucleotide-binding-site opener], two exchange factors for RhoA and Arf1 respectively. CONCLUSIONS: The development of RhoGEF inhibitors appears as a valuable tool for the study of Rho GTPase signalling pathways. The yeast exchange assay adaptation we present here is suitable to screen for chemical or peptide libraries and identify candidate inhibitors.  相似文献   

20.
The ERBB receptors have a crucial role in morphogenesis and oncogenesis. We have identified a new PDZ protein we named ERBIN (ERBB2 interacting protein) that acts as an adaptor for the receptor ERBB2/HER2 in epithelia. ERBIN contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ (PSD-95/DLG/ZO-1) domain at its carboxy terminus, and belongs to a new PDZ protein family. The PDZ domain directly and specifically interacts with ERBB2/HER2. ERBIN and ERBB2/HER2 colocalize to the lateral membrane of human intestinal epithelial cells. The ERBIN-binding site in ERBB2/HER2 has a critical role in restricting this receptor to the basolateral membrane of epithelial cells, as mutation of the ERBIN-binding site leads to the mislocalization of the receptor in these cells. We suggest that ERBIN acts in the localization and signalling of ERBB2/HER2 in epithelia.  相似文献   

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