Genetic interaction between integrins and moleskin,a gene encoding a Drosophila homolog of importin-7

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. If alphaPS subunits are inappropriately expressed during early pupariation, the epithelia separate, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: wing blisters are not generated efficiently by misexpression of loss-of-function alphaPS2 subunits with mutations that inhibit ligand binding, and gain-of-function, hyperactivated mutant alphaPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen for dominant suppressors of wing blisters generated null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has recently been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK. Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import. These data are discussed with respect to the possible regulation of integrin function by cytoplasmic ERK.     

Nuclear localization of the ERK MAP kinase mediated by Drosophila alphaPS2betaPS integrin and importin-7

The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants, either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation (on Westerns) is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7-deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein.     

The Ketel gene encodes a Drosophila homologue of importin-beta

The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.     

Long persistence of importin-beta explains extended survival of cells and zygotes that lack the encoding gene

Importin-beta is an essential component of nuclear protein import, spindle formation and nuclear envelope assembly. Formerly, the function of the Drosophila Ketel gene, which encodes importin-beta and is essential for the survival to adulthood, seemed to be required only in the mitotically active cells. We report here that importin-beta function is required in every cell and that this protein possesses an exceptionally long life span. Mosaic analysis, using gynanders, indicated that zygotic function of the Ketel gene is essential in a large group of cells in the embryos. Expression of a UAS-Ketel transgene by different tissue specific Gal4 drivers on ketel(null)/- hemizygous background revealed the requirement of Ketel gene function in the ectoderm. Elimination of the Ketel gene function using a UAS-Ketel-RNAi transgene driven by different Gal4 drivers confirmed the indispensability of the Ketel gene in the ectoderm. Using GFP-tagged importin-beta (encoded by a ketel(GFP) allele) we revealed that the maternally provided GFP-importin-beta molecules persist up to larval life. The zygotic Ketel gene is expressed in every cell during early gastrulation. Although the gene is then turned off in the non-dividing cells, the produced importin-beta molecules persist long and carry out nuclear protein import throughout the subsequent stages of development. In the continuously dividing diploid cells, the Ketel gene is constitutively expressed to fulfill all three functions of importin-beta.     

The Ketel(D) dominant-negative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation

The Ketel(D) dominant female-sterile mutations and their ketel(r) revertant alleles identify the Ketel gene, which encodes the importin-beta (karyopherin-beta) homologue of Drosophila melanogaster. Embryogenesis does not commence in the Ketel(D) eggs deposited by the Ketel(D)/+ females due to failure of cleavage nuclei formation. When injected into wild-type cleavage embryos, cytoplasm of the Ketel(D) eggs does not inhibit nuclear protein import but prevents cleavage nuclei formation following mitosis. The Ketel(+) transgenes slightly reduce effects of the Ketel(D) mutations. The paternally derived Ketel(D) alleles act as recessive zygotic lethal mutations: the Ketel(D)/- hemizygotes, like the ketel(r)/ketel(r) and the ketel(r)/- zygotes, perish during second larval instar. The Ketel maternal dowry supports their short life. The Ketel(D)-related defects originate most likely following association of the Ketel(D)-encoded mutant molecules with a maternally provided partner. As in the Ketel(D) eggs, embryogenesis does not commence in eggs of germline chimeras with ketel(r)/- germline cells and normal soma, underlining the dominant-negative nature of the Ketel(D) mutations. The ketel(r) homozygous clones are fully viable in the follicle epithelium in wings and tergites. The Ketel gene is not expressed in most larval tissues, as revealed by the expression pattern of a Ketel promoter-lacZ reporter gene.     

Msk is required for nuclear import of TGF-{beta}/BMP-activated Smads

Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-beta. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-beta and BMP into the nucleus.     

“Importin” signaling roles for import proteins: The function of Drosophila importin-7 (DIM-7) in muscle-tendon signaling

The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.     

“Importin” signaling roles for import proteins

The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.     

Perturbing Nuclear Transport in Drosophila Eye Imaginal Discs Causes Specific Cell Adhesion and Axon Guidance Defects

To study nucleocytoplasmic transport during multicellular development, we developed a sensitive nuclear protein import assay in living blastoderm embryos. We show that dominant negative truncations of the human nuclear transport receptor karyopherinbeta/Importinbeta (DNImpbeta) disrupt mRNA export and protein import in Drosophila. To test the sensitivity of different developmental processes to nuclear trafficking perturbations, we expressed DNImpbeta behind the morphogenetic furrow of the eye disc, at a time when photoreceptors are patterned and project their axons to the brain. DNImpbeta expression does not disrupt the correct specification of different photoreceptors, but causes a defect in cell adhesion that leads to some photoreceptors descending below the layer of ommatidia. The photoreceptors initially project their axons correctly to the posterior, but later their axons are unable to enter the optic stalk en route to the brain and continue to project an extensive network of misguided axons. The axon guidance and cell adhesion defects are both due to a disruption in the function of Ketel, the Drosophila ortholog of Importinbeta. We conclude that cell adhesion and axon guidance in the eye have specific requirements for nucleocytoplasmic transport, despite involving processes that occur primarily at the cell surface.     

Importin-alpha3 is required at multiple stages of Drosophila development and has a role in the completion of oogenesis

The Drosophila importin-alpha3 gene was isolated through its interaction with the large subunit of the DNA polymerase alpha in a two-hybrid screen. The predicted protein sequence of Importin-alpha3 is 65-66% identical to those of the human and mouse importin-alpha3 and alpha4 and 42.7% identical to that of Importin-alpha2 (Oho31/Pendulin), the previously reported Drosophila homologue. Both Importin-alpha3 and Importin-alpha2 interact with similar subsets of proteins in vitro, one of which is Ketel, the importin-beta homologue of Drosophila. importin-alpha3 is an essential gene, whose encoded protein is expressed throughout development. During early embryogenesis, Importin-alpha3 accumulates at the nuclear membrane of cleavage nuclei, whereas after blastoderm formation it is characteristically found within the interphase nuclei. Nuclear localisation is seen in several tissues throughout subsequent development. During oogenesis its concentration within the nurse cell nuclei increases during stages 7-10, concomitant with a decline in levels in the oocyte nucleus. Mutation of importin-alpha3 results in lethality throughout pupal development. Surviving females are sterile and show arrest of oogenesis at stages 7-10. Thus, Importin-alpha3-mediated nuclear transport is essential for completion of oogenesis and becomes limiting during pupal development. Since they have different expression patterns and subcellular localisation profiles, we suggest that the two importin-alpha homologues are not redundant in the context of normal Drosophila development.     

Phosphorylation of dystrophin Dp71d by Ca2+/calmodulin-dependent protein kinase II modulates the Dp71d nuclear localization in PC12 cells

We have shown that the splicing isoform of Dp71 (Dp71d) localizes to the nucleus of PC12 cells, an established cell line derived from a rat pheochromocytoma; however, the mechanisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of proteins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphorylated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phosphorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphorylation was activated by 2-O-tetradecanoyl phorbol 13-acetate (TPA), but this effect was blocked by EGTA. Supporting the role of intracellular calcium on Dp71d phosphorylation, we observed that the stimulation of calcium influx by cell depolarization increased Dp71d phosphorylation, and that the calcium-calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca2+/diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression vectors as well as the use of KN-62, a CaMKII-specific inhibitor, demonstrated that CaMKII is also involved in Dp71d phosphorylation. Stimulation of Dp71d phosphorylation by cell depolarization and/or the overexpression of CaMKII favored the Dp71d nuclear accumulation. Overall, our results indicate that CAMKII-mediated Dp71d phosphorylation modulates its nuclear localization.     

Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid

The non-beta-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10T1/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase C alpha (PKC alpha), and PKC beta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.