InteractiveFly: GeneBrief

ketel: Biological Overview | References

BIOLOGICAL OVERVIEW

Importin-β is an essential component of nuclear protein import, spindle formation and nuclear envelope assembly. Previous studies have concluded that the function of the Drosophila Ketel gene, which encodes importin-β and is essential for the survival to adulthood, is required only in the mitotically active cells. This study reports that importin-β function is required in every cell; this protein possesses an exceptionally long life span. Mosaic analysis, using gynanders (carrying a functional Ketel gene in their female but not in male cells), 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-β (encoded by a ketel^GFP allele), this study revealed that the maternally provided GFP-importin-β 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-β 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-β (Villányi, 2008a).

The Ketel gene encodes for the Drosophila homologue of importin-ß, a key player in nuclear protein import. Importin-ß, the founding member of the importin-ß superfamily, was originally described to participate in import of proteins that carry a classical nuclear localization signal (cNLS) into the nucleus. The C-terminal section of importin-ß associates with importin-α, an adapter molecule, that binds to the cNLS-containing import substrate. Importin-ß forms 19 HEAT- and armadillo-resembling repeats and wraps around the importin-ß-binding (IBB) domain of importin-α (Cingolani, 1999). The substrate-importin-α-importin-ß complex docks, in an energy-independent manner, on the cytoplasmic side of the nuclear pore complexes (NPCs). During translocation through the NPCs, importin-ß interacts with a number of nucleoporins with its NPC binding domains located toward the N terminus (Kutay, 1997; Wozniak, 1998). Import of the cNLS-containing nuclear protein is completed on the nuclear surface of the NPCs, where following interaction of the transport complex with Ran-GTP (see Drosophila Ran), the substrate-importin-α-importin-ß complex disassembles. (Ran is a Ras-related G protein without a membrane anchoring site). The import substrate stays in the nucleus, while importin-α and importin-ß are recycled to initiate a new import cycle: importin-ß returns to the cytoplasm in a complex with Ran-GTP. In the cytoplasm Ran-GTP dissociates from importin-ß and is converted to Ran-GDP by RanGTP-ase activating protein (RanGAP) and the Ran binding protein 1 (RanBP1); thus importin-ß can participate in a new transport cycle (Lippai, 2000 and references therein).

Importin-β is an essential component of nuclear protein import (reviewed in Görlich, 1999; Fried, 2003]). In Drosophila, importin-β is encoded by the Ketel gene (Lippai, 2000). Analysis of Ketel^D dominant negative female-sterile mutations reveal that importin-β is engaged not only in nuclear protein import but also in the reassembly of the nuclear envelope towards the end of mitosis (Tirian, 2000; Tirian, 2003). Furthermore, analysis of the Ketel^D associated mutant phenotypes suggested the involvement of importin-β in the formation of the spindle apparatus (Lippai, 2000; Villányi, 2008a and references therein).

A second mutagenesis of the Ketel^D alleles lead to the induction of the so-called ketel^revertant alleles, some of which are complete loss-of-function (ketel^null) mutations while others are short deficiencies that remove the Ketel and a few of the adjacent loci (Szabad, 1989; Erdelyi, 1997; Villányi, 2008a and references therein).

Regarding the Ketel gene expression pattern and the function of importin-β, three enigmas emerged in course of the former studies. (1) Analysis of a reporter gene in which the Ketel gene regulatory sequences ensured expression of the LacZ gene and the formation of β-galactosidase showed that in late 3rd instar larvae the Ketel gene is expressed only in the mitotically active diploid imaginal and not in the non-dividing polytenic larval cells (Tirian, 2000). Results of the Western blot analysis were in accord with that of the reporter gene expression analysis (Lippai, 2000). The exclusive expression of the Ketel gene only in the diploid cells is puzzling since there is only a single importin-β coding gene in the Drosophila genome and nuclear protein import must also be going on in the metabolically highly active polytenic larval cells. How do polytenic cells import protein into their nuclei if all the known nuclear protein import mechanisms include importin-β or a closely related protein (Görlich, 1999)? Do polytenic cells use a yet unknown mechanism for nuclear protein import? Nonetheless, function of the Ketel gene must be indispensable in at least some cell type(s) since zygotes without the Ketel gene die during second larval instar. (2) Clones of cells that originate from mitotic recombination and become homozygous for a ketel^null allele, are fully viable and capable of differentiating normally in at least four different diploid cell types as if the function of the Ketel gene was not required in the diploid cells (Tirian, 2000). Where is the focus, i.e. in which cell type is the function of the Ketel gene indispensable? (3) Zygotes without the Ketel gene (the ketel^null/− hemizygotes) live for 3 days, up to the end of 2nd larval instar. It has been shown that the maternal dowry, supplied by the ketel^null/+ heterozygous females, supports their relatively short life (Tirian, 2000). Assuming that the function of the Ketel gene is required in the diploid cells only, death of the ketel^null/− hemizygous larvae in 2nd larval instar is astonishing since larvae without diploid cells have been known to develop up to the end of larval life (Villányi, 2008a).

To resolve the above rather perplexing observations, this study set out to localize the cell type in which the function of the Ketel gene is essential, and the classical gynander-based focusing analysis was performed. The experiment clearly showed that the focus of the Ketel gene action is huge and/or extends over large areas of the embryo. Use was made of the Gal4; UAS system and a UAS-Ketel transgene was driven with different tissue-specific Gal4 drivers on a ketel^null/− background and the ketel^null/− zygotes were tested for viability to adulthood. The Gal4; UAS system revealed the requirement of the Ketel gene function in the ectoderm, which comprises a rather large and also wide spread region of the blastoderm. The function of the Ketel gene was eliminated by driving a UAS-Ketel-RNAi transgene with different Gal4 drivers and the fate of the zygotes examined. Results of the RNAi experiments largely confirm the former conclusions (Villányi, 2008a).

A ketel^GFP mutant allele that encodes a GFP-tagged importin-β protein is described. The use of ketel^GFP clearly showed that some of the maternally derived GFP-importin-β molecules persist up to pupariation, 5 days after their formation. It appears that GFP-importin-β, and most likely wild type importin-β, is one of the longest living molecules inside the Drosophila cells. The paternally-derived ketel^GFP allele is first expressed during early gastrulation, in every cell of the embryo. Last but not least, GFP-importin-β clearly showed that importin-β is present in all of the larval cells though its concentration is very low in the polytenic as compared to the diploid cells. This observation makes sense knowing that polytenic cells do not divide and, thus, need importin-β only to accomplish nuclear protein import. Intensive expression of the Ketel gene in the diploid cells is plausible since diploid cells need importin-β not only for nuclear protein import but also for the formation of the microtubules of the spindle apparatus and the assembly of the nuclear envelope at the end of mitosis (Villányi, 2008a).

Previous results concluded that Ketel is expressed only in the dividing diploid imaginal and not in the non-dividing polytenic cells in late 3rd instar larvae (Tirian, 2000; Lippai, 2000). The observation raised the possibility that the function of the Ketel gene was not required in every cell and suggested the existence of a thus far unknown nuclear protein import mechanism that operates without importin-β. It was also difficult to understand how the diploid cells, in which the Ketel gene is expressed, can proliferate and function normally without the Ketel gene (Tirian, 2000). In any case, the function of the Ketel gene must be indispensable in at least some cell type(s) since larvae without the Ketel gene perish during mid larval life (Villányi, 2008a).

To identify the cell types in which the function of the Ketel gene is essential, gynanders were generated that carried a functional Ketel gene in their female but not in their male cells. Such gynanders do not survive to the end of larval life implying that the function of the Ketel gene is most likely required in a large group of cells. This conclusion is supported by the observation that when the elav-Gal4, the esg-Gal4 or the vg-Gal4 drivers ensured expression of a UAS-Ketel transgene, zygotes that otherwise lack a functional Ketel gene survive to adulthood. The former drivers induce gene expression in the ectoderm, the largest germ layer which comprises about 72% of the blastoderm cells (3600/5000 cells). In accordance with the former findings, elimination of Ketel gene activity in the primordial cells of the epidermis (by expressing a UAS-Ketel-RNAi transgene with the elav-Gal4, the esg-Gal4 or the vg-Gal4 drivers) led to death of the zygotes. How do e.g. the esg-Gal4; UAS-Ketel; ketel^null/− zygotes, in which the Ketel gene is expressed only in the ectoderm and the neuroectoderm but not in the other germ layers, survive to adulthood? How do the entoderm- and the mesoderm-derived cells acquire importin-β? The most likely source of importin-β in those cells is the maternal importin-β dowry, which persists and functions in the entoderm- and in the mesoderm-derived cells throughout development. This proposition presumes a very long persistence of importin-β, a feature that is supported by the following:

• (1) the GFP-importin-β molecules can persist up to 5 days. The life span of the wild type importin-β molecules may be even longer than 5 days since the GFP-importin-β molecules are barely functional and are probably prone to faster degradation than wild type importin-β, due to the misfolding induced by the GFP tag. Yet, the GFP-importin-β molecules survive longer than expected, stabilized by their intimate association with the nuclear pore complex. It has been suggested that being parts of large protein complexes, components of the anaphase promoting complex are stabilized; they persist unusually long and function in low concentrations (Pal, 2007b; Pal, 2007a; Wehman, 2007). The maternally provided importin-β molecules may be able to support the few cell divisions the entoderm- and the mesoderm-derived cells accomplish between the blastoderm stage and their final differentiation. Thereafter, they need to assist only in nuclear protein import in the protective milieu of the NPCs (Villányi, 2008a).
• (2) When transplanted into host embryos, pole cells without the Ketel gene but with the maternally provided importin-β inside perform the usual 4-5 mitosis during larval life, become functional oogonial cells, go through the cystocyte divisions and become sources of normal looking eggs. [However, when the eggs are fertilized embryogenesis does not commence inside (Tirian, 2000)]. The maternally derived importin-β molecules can thus support female germ line development for at least 10 days (Villányi, 2008a).
• (3) Elimination of zygotic Ketel gene function in e.g. the esg-Gal4; UAS-Ketel-RNAi zygotes did not lead to death in mid larval life as in the ketel^null/− larvae but only during late pupal life. The prolonged life of the zygotes can be accounted for by the persistence of those importin-β molecules that form during the short period of time between synthesis and demolition of the Ketel mRNA molecules (Villányi, 2008a).

The finding that importin-β molecules persist long and carry out their functions in low concentrations provides an explanation for the normal behavior of clones of wing disc cells without the Ketel gene. Such cells can accomplish as many as seven rounds of cell divisions following the induction of mitotic recombination and becoming homozygous for a ketel^null allele (Tirian, 2000). Perdurance of importin-β, inherited from the ketel^null/+ mother cell, sustains the life of the descending ketel^null homozygous cells (Villányi, 2008a).

The ketel^GFP allele encoded GFP-importin-β clearly showed that importin-β is present in every cell type, though in very different concentrations. Apparently, the non-dividing larval cells also make use of importin-β in nuclear protein import and, thus, there is no unknown mechanism of nuclear protein import to be discovered. However, the diploid cells contain a lot more importin-β as compared to the non-dividing larval cells. The low importin-β concentration in the non-dividing cells is reasonable since here the protein is engaged in the nuclear protein import only. (Precursor cells of the larval epidermis and the Malpighian tubules, for example, divide only two-to-three times following the blastoderm stage and become polytenic. The imaginal disc cells remain diploid and keep on proliferating throughout larval and early pupal life. Diploid cells need a higher importin-β concentration to accomplish three functions: nuclear protein import, formation of the spindle microtubule bundles and assembly of the nuclear envelope at the end of mitosis (Zhang, 2000; Nachury, 2001; Wiese, 2001; Gruss, 2001; Timinszky, 2002; Zhang, 2002; Tirian, 2003; Villányi, 2008a and referneces therein).

Apparently, as revealed by the analysis of the ketel^GFP encoded GFP-importin-β, the zygotic Ketel gene is expressed in every cell during early gastrulation in the same stage of development as the reporter gene in which the Ketel gene regulatory sequences control the expression of LacZ and the formation of β-galactosidase (Tirian, 2000; Lippai, 2000). However, the β-galactosidase molecules decompose in all the larval cells by late embryogenesis and leave the cells unstained for the rest of the larval development demonstrating the non-expressed status of the Ketel gene. Although the Ketel gene is not expressed in the larval cells, presence of GFP-importin-β clearly shows that there are importin-β molecules in the larval cells and they fulfill their function over a long period of time (Villányi, 2008a).

The Ketel gene encodes a Drosophila homologue of Importin-β

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. The Ketel gene was cloned 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-ß, 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 (Lippai, 2000).

During a genetic dissection of maternal effects in Drosophila, 75 dominant female sterile (Fs) mutations were isolated. In 32 of the Fs mutations the Fs/+ females deposit normal-looking eggs, and although the eggs are fertilized embryogenesis does not commence or ceases after a few abnormal cleavage divisions. The 32 Fs mutations identify 21 genes, suggesting that products of several genes are required for commencement and the initial steps of embryogenesis. This conclusion is supported by the fact that very few, if any, of the zygotic genes are expressed during early embryogenesis and evidently the initial steps of embryogenesis are under maternal (Lippai, 2000).

The Ketel gene, which was identified by four Fs(2)Ketel (= Ketel^D) mutations, is one of the 21 genes mentioned above. Embryogenesis is terminated in Ketel^D eggs, which are deposited by the Ketel^D/+ females, soon after fertilization due to the failure of cleavage nuclei formation. When injected into wild-type cleavage embryos, the Ketel^D egg cytoplasm is toxic: it hinders formation of cleavage nuclei following mitosis most likely through the prevention of nuclear envelope (NE) assembly and/or function. The mutant phenotype suggests involvement of the Ketel gene in a NE-related function and motivated cloning of the gene (Lippai, 2000).

A combined genetic, molecular, and cell biological approach reveals novel features of importin-ß in Drosophila. The Ketel protein shows characteristic features of importin-ß: (1) it supports import of a cNLS-containing substrate into nuclei of digitonin-permeabilized HeLa cells and (2) it is largely cytoplasmic with pronounced accumulation in the NE. Surprisingly, the highly 'toxic' Ketel^D egg cytoplasm, which prevents NE assembly following mitosis, does not prevent nuclear protein import. Unexpectedly, as revealed by Western blot analysis, the Ketel gene is not expressed in most cells of the larvae and adults, raising questions about cellular functions of importin-ß and nuclear import of the cNLS-containing proteins (Lippai, 2000).

As other members of the importin-ß family, the Ketel protein is largely cytoplasmic with pronounced accumulation in the NE. As predicted by features of the Ketel^D and ketel^r mutant phenotypes (Tirian, 2000), the Ketel protein is produced and is dumped into the oocyte cytoplasm during oogenesis and cleavage embryos make use of the Ketel maternal dowry. Surprisingly, however, the Ketel gene does not appear to be expressed in the fully differentiated larval cells. Apparently the Ketel protein appears to be present largely in mitotically active cells (Lippai, 2000).

When injected into wild-type cleavage embryos, traces of the Ketel^D egg cytoplasm exert deleterious effects through the prevention of cleavage nuclei formation (Tirian, 2000). Toxic effects of the Ketel^D-encoded molecules are perhaps an outcome of arrested nuclear protein import. To elaborate this possibility, cytosol was prepared from ovaries of the Ketel^D/+ females and its effects on nuclear protein import was examined. Unexpectedly, the Ketel^D cytosol preparations did not prevent nuclear import of the cNLS-PE substrate. In fact, the cNLS-PE molecules were equally efficiently imported into the nuclei in the presence of the Ketel^D or wild-type ovary cytosol. Consistent with this observation, the Ketel^D egg cytoplasm does not prevent import of the cNLS-PE molecules into interphase nuclei of wild-type cleavage embryos (Tirian, 2000). Knowing that the Ketel^D alleles are strong dominant-negative mutations, the above results may be surprising. A number of possibilities may come to light to explain the former observation. It is very unlikely that all four of the EMS-induced Ketel^D alleles alter expression of the Ketel gene such that the cytosol preparations do not contain Ketel^D-encoded molecules. Although the Ketel^D-encoded molecules block function of the normal ones, perhaps the cNSL-PE substrate is imported into the nuclei via another nuclear protein import route powered by unidentified components of the ovary cytosol. The existence of parallel import pathways is well established. It is also possible that although the Ketel^D-encoded molecules do not participate in nuclear protein import, they do not interfere with import function of the normal Ketel molecules, and their toxic effects become apparent when the importin-ß molecules perform a function other than nuclear protein import (Lippai, 2000).

Indeed, the deleterious effects of the Ketel^D mutations become apparent at the end of cleavage mitosis when the NE reassemble and daughter nuclei form. Remarkably, the Ketel^D cytosol does not disrupt HeLa cell nuclei and, along with this observation, Drosophila wild-type interphase cleavage nuclei remain intact in presence of the Ketel^D egg cytoplasm. Because the digitonin-permeabilized HeLa cells do not divide, they are inadequate to detect defects associated with NE assembly. It appears as if the Ketel^D mutations identify a novel function of importin-ß required during reassembly of the NE at the end of mitosis. Perhaps importin-ß is not only engaged in nuclear protein import but is also a structural component of the NPCs, and the Ketel^D mutations identify the nucleoporin function of the gene (Lippai, 2000).

NE assembly is a stepwise process. At first, every chromosome associates with Ran-GDP. The chromatin-associated Ran-GDP promotes binding to chromatin of membrane vesicles and recruits RCC1, the guanine nucleotide exchange factor for Ran, and promotes the association of nucleoporins. RCC1 generates Ran-GTP from Ran-GDP, and Ran-GTP causes fusion of the vesicles and formation of double nuclear membrane. Formation of the NE with NPCs establishes a condition for resumed nuclear protein import and the formation of functional nuclei. The process takes place in vitro where NE forms from egg cytoplasm extract components over the demembranated sperm chromatin in a process that is similar to NE assembly around the sperm chromatin during male pronucleus formation following fertilization. NEs form over Sepharose beads loaded with Ran-GDP in Xenopus egg extract in the absence of DNA or chromatin. However, the role of importin-ß in NE/NPC assembly waits to be elucidated (Lippai, 2000).

Anillin-mediated targeting of Peanut to pseudocleavage furrows is regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. This study demonstrates that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevents pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that form. Ran is required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin α and β to anillin prevents the binding of Peanut to anillin. Furthermore, RanGTP reverses the inhibitory action of importin α and β. On expression of a mutant form of anillin that lacks an importin α and β binding site, inhibition of Ran no longer restricts the depth and duration of furrow ingression in those pseudocleavage furrows that form. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran (Silverman-Gavrila, 2008).

During cytokinesis, the ingressing plasma membrane physically divides the mother cell into two daughter cells. Membrane ingression during cell division is both temporally and spatially regulated, ensuring that membrane scission occurs (1) only after the chromosomes have fully segregated and (2) between the two chromosomal masses. The signals within the cell that determine cytokinetic furrow positioning are complex, reflecting the strict control needed to ensure that cytokinesis is successful. Signals from astral microtubules (D'Avino, 2005), the spindle midbody (D'Avino, 2005), the nucleus, and the membrane itself (Janetopoulos, 2006) direct the assembly of the contractile ring to the equatorial cortex of the plasma membrane. The contractile ring is an actomyosin-based structure that constricts and generates the force needed to drive membrane ingression. As the membrane ingresses, it is remodeled and stabilized (Silverman-Gavrila, 2008).

Other membrane ingression events share many of the same features and involve many of the same proteins as cytokinetic furrows. In the syncytial Drosophila embryo before cellularization, up to 6000 closely packed nuclei exist in a common cytosol close to the cortex. To ensure faithful chromosome segregation during the rapid nuclear divisions, nuclei are isolated from one another to prevent neighboring spindles from contacting and fusing. To achieve this, plasma membrane ingressions form transiently between nuclei during the rapid nuclear cycles before cellularization. These membrane ingressions, termed pseudocleavage or metaphase furrows, are organized by the actin cytoskeleton and bear a close resemblance to cytokinetic cleavage furrows. First actin caps form at the plasma membrane above each nucleus. Then during interphase, as the centrosomes migrate to either side of the nucleus, the actin caps expand correspondingly. In prophase the cap reorganizes to drive membrane ingression into the embryo such that nuclei and newly forming spindles are separated from one another. Toward the end of metaphase, the furrows begin to retract and dissipate by anaphase. This process is repeated from the tenth through the thirteenth nuclear cycles. During the fourteenth nuclear cycle, the syncytial embryo cellularizes to form 6000 columnar epithelial cells. In this instance the cleavage furrows extend down into the embryo, before growing transversally and fusing to form a single layer of nucleated cells (Silverman-Gavrila, 2008).

Most components required for furrow ingression are conserved between cytokinetic furrows (during conventional mitosis) and pseudocleavage furrows (Mazumdar, 2002). However, there are some differences. Notably pseudocleavage furrows are membrane ingressions that do not meet and therefore do not lead to membrane fusion. Instead they extend into the embryo, perpendicular to the cortex, and then retract back toward the embryo cortex after the chromosomes have begun to segregate. In addition, there is a difference in the stage of the cell cycle when the furrow components assemble. Although the cytokinetic furrow begins to assemble during anaphase and is required to divide a cell in two, the syncytial embryo pseudocleavage furrows begin to assemble in prophase and serve to prevent neighboring spindles from contacting one another (Silverman-Gavrila, 2008).

A key protein involved in cytokinetic furrow function is anillin, which has multiple domains allowing it to bind and bundle actin filaments, target septins to the plasma membrane, and interact with components of the microtubule-bound centralspindilin complex (Gregory, 2008). Consequently anillin is thought to act as a scaffold for the correct assembly of the contractile ring (Piekny, 2007). It is not fully understood how the role of anillin in cytokinesis is regulated. However, its role in remodeling the actomyosin contractile ring in somatic cells is in part regulated by its differential spatial positioning in the cell during the cell cycle. In interphase anillin localizes to the nucleus where it cannot interact with actin and myosin at the plasma membrane. However, in mitosis upon nuclear envelope breakdown, anillin is released from the nucleus and is targeted to the cortex of the plasma membrane and later to the equatorial cortex of the plasma membrane in a RhoGTP-dependent manner. The spatial regulation of anillin during the cell cycle contributes to the restriction of its function to mitosis. However, in Drosophila syncytial embryos anillin is cytosolic, localizing to pseudocleavage furrows throughout the nuclear cycle, suggesting that it may be regulated by other mechanisms (Silverman-Gavrila, 2008).

One function of anillin is to target septins to the contractile ring. Septins are a family of GTP-binding proteins that can assemble into filaments. Septins have been attributed multiple roles: as membrane diffusion barriers, as stabilizers of the furrow, in membrane trafficking, and as a scaffold. In Drosophila there are five septins: Peanut, Sep1, Sep2, Sep4, and Sep5. Peanut, Sep1 and Sep2 have been isolated as a stoichiometric complex that in vitro can polymerize into filaments. In contrast, Xenopus laevis Sept2 can self assemble into filaments, suggesting that septins may function independently (Silverman-Gavrila, 2008 and references therein).

The GTPase Ran is a key positive regulator of mitosis (Ciciarello, 2007). RanGTP regulates a number of mitotic factors that are sequestered in the nucleus by nuclear transport receptors during interphase. In mitosis RanGTP antagonizes the binding of nuclear transport receptors to these proteins and thereby promotes their activity. RanGTP is at its highest concentration around the chromosomes, where RCC1 the nucleotide exchange factor for Ran is localized. Consequently, RanGTP has been proposed to act as a spatial cue by only activating these mitotic proteins close to the chromosomes (Caudron, 2005; Kalab, 2006). In so doing RanGTP is thought to specify where certain mitotic processes occur in the cell. For example, it could specify that spindle assembly only occurs around chromosomes. The full extent to which this mechanism regulates the mitotic cell is not known and continues to expand (Silverman-Gavrila, 2008).

This study demonstrates a new role for Ran in regulating pseudocleavage furrow ingression, a membrane invagination process in early Drosophila embryos. The Ran pathway regulates the interaction between anillin and the septin Peanut, thereby regulating furrow stability (Silverman-Gavrila, 2008).

A cytological screen was carried out to identify mitotic processes regulated by the Ran pathway (Silverman-Gavrila, 2006). Inhibitors of the Ran pathway were injected into GFP-α-tubulin-expressing embryos just before mitotic entry, and then microtubule organization was monitored by time-lapse microscopy (Silverman-Gavrila, 2006). One phenotype, the fusion of neighboring spindles, occurred more frequently upon the injection of inhibitors of the Ran pathway compared with control injections. In control injected embryos 0.2% of observed spindles fused to a neighboring spindle. In contrast, inhibition of the Ran pathway by injecting either the dominant negative allele of Ran, RanT24N, or importin α resulted in 8.4 and 7.8% of observed spindles fusing to neighboring spindles, respectively (Silverman-Gavrila, 2008).

Peanut is recruited to ingressing furrows by anillin, a multifunctional protein required for cytokinesis that interacts with myosin II, actin, and septins. Septins bind to the carboxy-terminus of anillin, which includes a pleckstrin homology (PH) domain. Drosophila anillin has three potential nuclear localization signals (NLS) that could bind to the nuclear transport receptors importin α and β. Two of the NLS motifs are located in or directly adjacent to the PH domain (Silverman-Gavrila, 2008).

To determine if the carboxy-terminus of anillin could bind to importin α and β, a fusion was constructed between GST and the carboxy-terminus of anillin (amino acids 815-1201, anillin-CT, and its ability to bind to recombinant importin α and β was analyzed. Both importin α and β bound to anillin-CT, and this binding was reversed in the presence of RanQ69L, a point mutant of Ran locked in the GTP-bound state. Of the two potential NLS motifs, the one located between amino acid residues 989 and 999, bares the closest resemblance to an archetypal bipartite NLS and is found in the same region of human anillin (amino acids 887-898). Mutation of lysines 997-999 to alanine (3A-anillin-CT) abrogate both importin α and β binding to this region of anillin, suggesting that amino acids 989-999 constitute a nuclear transport receptor-binding site (Silverman-Gavrila, 2008).

It was next asked if the anillin-CT could interact with Peanut. GST-anillin-CT was incubated with 0-3-h Drosophila embryo extract and then isolated using glutathione agarose beads. Anillin-CT copurified with Peanut and another septin, Sep2. However, the addition of exogenous importin α and importin β inhibited the binding of Peanut to anillin-CT in a concentration- and NLS-dependent manner. This inhibition was specific to Peanut, because Sep2 binding to anillin-CT was not inhibited by importins (Silverman-Gavrila, 2008).

To determine if the in vivo targeting of Peanut and Sep2 to the pseudocleavage furrows is differentially regulated, importin α was injected into syncytial embryos and GFP-Sep2 localization was determined by time-lapse microscopy. Consistent with in vitro results, GFP-Sep2 localization was not perturbed upon interfering with the Ran pathway. Furthermore, in fixed GFP-Sep2-expressing embryos in which the Ran pathway has been perturbed, Peanut fails to localize to nascent furrows, whereas GFP-Sep2 does localize to nascent furrows. These data suggest that Peanut and Sep2 are differentially regulated by Ran and that Sep2 can localize to pseudocleavage furrows independently of Peanut (Silverman-Gavrila, 2008).

This study has identified RanGTP as a regulator of the interaction between Peanut and anillin. This mechanism operates directly and independently of Ran's well-characterized role in regulating the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

Studies suggest that anillin is required for the recruitment of septins to the furrow. By perturbing the Ran pathway, this study has demonstrated that the recruitment of the septins Peanut and Sep2 is differentially regulated, consistent with previous observations that Sep1 recruitment to furrows is dependent on Peanut but Sep2 is not. Anillin lacking the importin binding site between residues 997 and 999 can bind to Peanut in the presence of importins, suggesting that importins directly block the anillin-Peanut interaction rather than disrupting the Peanut, Sep1, and Sep2 complex. These data suggest that although Peanut, Sep1, and Sep2 can exist in a single complex, they may be able to function independently of one another as has been demonstrated in vitro for a Xenopus septin (Silverman-Gavrila, 2008).

Perturbing the Ran pathway destabilizes pseudocleavage furrows. One mechanism for this is through the regulation of the anillin-Peanut interaction. In embryos that expressed an anillin mutant lacking the importin-binding site, Peanut recruitment to pseudocleavage furrows occurs even in the presence of exogenous importins, and furrows demonstrate wild-type dynamics. These data suggest that Peanut is required for pseudocleavage furrow stability. This role for anillin and Peanut is consistent with the observed role for these proteins in stabilizing the cellularization furrow later in Drosophila development. These findings may at first appear to contradict those studies, in which embryos lacking Peanut protein progressed through the syncytial nuclear divisions only showing the first defects during cellularization. However, these studies only analyzed syncytial furrows from the top, apical view and not from the lateral view to observe ingression dynamics. Therefore, these studies would not have detected changes in furrow ingression dynamics that were observed upon inhibition of Ran, which correlated with a failure to recruit Peanut to the furrow (Silverman-Gavrila, 2008).

The Ran pathway regulates pseudocleavage furrow ingression directly by regulating importin binding to anillin. It was previously shown that in Drosophila syncytial embryos the importin β, whose injection causes similar effects as importin α (Silverman-Gavrila, 2006), is released from the nucleus upon nuclear envelope breakdown and becomes diffuse throughout the cytosol during the rest of mitosis. During this period pseudocleavage furrows begin to retract. Therefore, as importin β is cytosolic during metaphase and anaphase it could act to prevent the interaction of Peanut and anillin. In turn this would lead to furrow instability and retraction (Silverman-Gavrila, 2008).

It cannot unequivocally be ruled out that some of the defects caused by perturbing the Ran pathway are due to a disruption of microtubule cytoskeleton. Indeed, one microtubule-dependent furrow phenotype, the formation of pseudocleavage furrows that encompassed a small area of cytosol around a nucleus, was observed. This phenotype has also seen in another study upon depolymerization of microtubules in embryos. However, microtubule depolymerization when instigated in interphase does not cause a failure in pseudocleavage furrow formation, a finding consistent with a previous study (Silverman-Gavrila, 2008).

Another mechanism through which Ran could affect pseudocleavage furrows is by disrupting nuclear trafficking. Indeed it was observed that nuclear trafficking can be reduced by up to 50% upon disruption of the Ran pathway. However, it seems unlikely that the changes in nuclear import kinetics in these experiments disrupted the function of anillin because anillin is a cytosolic protein in the syncytial embryo and localizes to the leading edge of the ingressing furrow during interphase. It is not understood how anillin is retained in the cytoplasm of syncytial embryos because it is imported into nuclei in other developmental stages. However, this phenomenon is not unique to anillin and is also exhibited by the kinesin Pavarotti, another protein involved in pseudocleavage furrow organization (Silverman-Gavrila, 2008).

These studies suggest that Ran regulates multiple factors involved in pseudocleavage furrow ingression, because embryos expressing the mutant anillin still exhibit a failure to form all the expected pseudocleavage furrows. Failure to fully suppress the phenotype could be due to the continued presence of endogenous anillin or reflect that other Ran pathway-sensitive factors are involved in pseudocleavage furrow formation. Regulation through the Ran pathway could define a spatial cue concentrated around chromosomes and extending to the cortex. Such a spatiotemporal regulatory mechanism could be involved in promoting cytokinetic furrows in other cells. A recent study in oocytes finds that Ran regulates myosin II (Deng, 2007), whose activity is required for cytokinetic cleavage furrows. In addition importin α is required for ring canal organization during oogenesis (Gorjanacz, 2002). Ring canals form as a result of incomplete cytokinesis, and many proteins involved in cytokinesis both localize to and are required for their formation, including anillin and septins (Silverman-Gavrila, 2008).

The data suggest that the anillin-Peanut interaction, which is inhibited by importins must occur in regions of the cell where there are low levels of importins or high levels of RanGTP. Recent studies have visualized a RanGTP-importin β gradient and found that it persists from the chromosomes to the centrosomes (Kalab, 2006), a distance similar to that between the metaphase plate and the cortex. Thus, RanGTP could play an important role in positioning the plane of cleavage by defining on the cell cortex where furrow proteins interact (Silverman-Gavrila, 2008).

Although there are clear differences between cytokinetic and pseudocleavage furrows, anillin and septins are involved in both. Therefore, this study suggests that Ran could also have a role in regulating cytokinetic furrows. Whether chromosomes play a significant role in cytokinesis remains controversial. However, studies where nuclei or chromosomes are asymmetrically positioned within a cell show that furrow ingression coincided with the region of the cell that contained the chromosomes, suggesting that signals from the nucleus and in particular the chromosomes had a role in specifying furrow ingression. Similarly, enucleated sea urchin eggs are able to duplicate their centrosomes and generate astral arrays of microtubules, but fail to form stable cleavage furrows. The current study proposes a molecular mechanism to explain, at least in part, these observations, suggesting that RanGTP generated around the chromosomes is a diffusible signal that facilitates multiple processes required for furrow formation. Whether RanGTP is required early in cytokinesis to 'prime' the cortex for a future ingression or acts directly later during the ingression process is unclear. Testing these hypotheses is not straightforward, since Ran is also required for organizing the mitotic microtubule cytoskeleton, which is required for cytokinesis. Taken together these findings suggest an additional mechanism involved in regulating cytokinesis that is dependent on signals from chromosomes in addition to those stemming from the different organizational states of the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

The DRE motif is a key component in the expression regulation of the importin-β encoding Ketel gene in Drosophila

Importin-β, encoded by the Ketel gene in Drosophila, is a key component of nuclear protein import, the formation of the spindle microtubules and the assembly of the nuclear envelope. The Drosophila embryos rely on the maternal importin-β dowry at the beginning of their life. Expression of the zygotic Ketel gene commences during gastrulation in every cell and while the expression is maintained in the mitotically active diploid cells it ceases in the non-dividing larval cells in which nuclear protein import is assured by the long persisting importin-β molecules. How is the expression of the Ketel gene regulated? In silico analysis revealed several conserved transcription factor binding sequences in the Ketel gene promoter. Reporter genes in which different segments of the promoter ensured transient expression of the luciferase gene in S2 cells identified the sequences required for normal Ketel gene expression level. Gel retardation and band shift assays revealed that the DREF and the CFDD transcription factors play key roles in the regulation of Ketel gene expression. Transgenic LacZ reporter genes revealed the sequences that ensure tissue-specific gene expression. Apparently, the regulation of Ketel gene expression depends largely on a DRE motif and action of the DREF, CFDD, CF2-II and BEAF transcription factors (Villanyi, 2008b).

The Ketel gene has been known to be expressed (1) in the egg primordia to provide importin-β for oogenesis and early embryogenesis, (2) in every cell of the gastrulating embryo and (3) in the diploid cells during larval life, but not in the polytenic larval cells (Lippai, 2000; Tirian, 2000; Villanyi, 2008a). The polytenic cells need relatively few importin-β molecules to accomplish nuclear protein import, and this duty is accomplished by the unusually long-lived importin-β molecules, some of which are maternally provided others are produced during early gastrulation (Villanyi, 2008a). Intensive expression of the Ketel gene in the diploid cells is comprehensible since these cells need importin-β not only for nuclear protein import but also for the formation of the spindle microtubules and the reassembly of the nuclear envelope at the end of mitosis. The aim of the present study was to understand the mechanisms that ensure the characteristic expression pattern of the Ketel gene. To achieve this goal, cis-acting control elements were examined that are engaged in (1) the proper loading of the egg cytoplasm with the Ketel gene products, (2) the regulation of the all-over type of importin-β production during gastrulation and (3) controlling tissue-specific expression of the Ketel gene during the later stages of development (Villanyi, 2008b).

Computer analysis revealed several evolutionarily conserved transcription factor binding sites in the Ketel promoter of which only the CF2-II, the CFDD, the DREF and perhaps the BEAF binding sites are of relevance. The CFDD, the DREF and the BEAF transcription factors have been known to be involved in the expression regulation of a number of genes engaged in cell cycle regulation. In fact, the CFDD binding sites are commonly present in the promoters of a number of DNA replication-related genes like PCNA and DREF. Since importin-β is required for spindle formation and nuclear envelope assembly, which are essential events in cell proliferation, it may not be surprising that the expression of the Ketel gene is regulated by the same transcription factors that control the expression of several genes engaged in cell cycle regulation (Villanyi, 2008b).

Transient expression of a luciferase reporter gene in S2 cells clearly showed that all the sequences which regulate Ketel gene expression reside within a 750 bp sequence towards the 5' region of the Ketel gene. The 'active' transcription factor binding sequences within the region were identified in gel-shift experiments, and the sequences that ensure tissue-specific expression of the Ketel gene were determined through the analysis of the expression patterns of LacZ reporter transgenes (Villanyi, 2008b).

It appears that the presence of an approximately 140 bp long sequence around the transcription start site is sufficient for a basic expression of the Ketel gene in the gonial cells. The simultaneous presence of two sequences is required for the expression of the Ketel gene in the nurse cells and for the loading of the egg cell cytoplasm with the Ketel gene products: a CFDD binding site in the first intron (around +247) and the DRE motif around −74. (Note that the importin-β-related maternal effect depends on the expression of the Ketel gene in the germ line components of the egg primordia; Tirian, 2000) Removal of either of these sequences leads to an absence of Ketel gene expression in the nurse cells. Similarly, the concurrent presence of the DRE motif at −74 and the CFDD site(s) around −250 is necessary for the expression of the Ketel gene in every cell of the gastrulating embryo. Removal of any of these sequences abolishes Ketel gene expression during early gastrulation. It appears that cooperative binding of transcription factors to the DRE motif and to either of the CFDD recognition sites establishes favourable conditions for tissue-specific expression of the Ketel gene. A CF2-II binding site around −483 is sufficient and necessary for the expression of the Ketel gene in the diploid cells of the imaginal discs, the neuroblasts and the follicle epithelium. CF2-II has been reported to be expressed in the follicle cells and seems to be the only candidate to control Ketel gene expression in the imaginal disc cells and in the neuroblasts (Villanyi, 2008b).

Interestingly, none of the six different types of LacZ reporter transgenes are expressed in any polytenic larval cell types. One possible explanation could be the different modes of action of DREF in the larval and in the diploid cells: DREF does not displace BEAF from the DRE motif in the larval cells and, thus, an insulator can form which blocks transcription of the Ketel gene. Three BEAF binding sites are necessary for the formation of an insulator, and the promoter of the Ketel gene contains three BEAF binding sites, one of which is part of the DRE motif. In the diploid cells, where DREF binds to the DRE motif and competes with BEAF, the insulator cannot form and, hence, there is no block to prevent expression of the Ketel gene. However, the above model is rather unlikely since when the DRE motif, and along with it one of the BEAF binding sites, is abolished the BEAF insulator cannot form. Yet, the Ketel gene is not expressed in the larval cells. The lack of Ketel gene expression in the larval cells can also be explained by the absence of CF2-II transcription factor in that cell type. Further studies are needed to ascertain whether this assumption is correct (Villanyi, 2008b).

In summary, the DRE motif is a key component in the regulation of Ketel gene expression: transcription factors that bind to the DRE motif interact with different CFDDs, which are bound to different CFDD binding sites, ensuring tissue-specific expression of the gene. The DRE motif and the CFDD sites are commonly present in the promoter of several genes engaged in DNA replication and cell cycle control, and interaction of DREF and CFDD could be a key component in the regulation of those genes as well (Villanyi, 2008v).

Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport

The phenylanine-glycine (FG)- rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. Thirty putative nucleoporins in cultured Drosophila S2 cells were inactivated by RNA interference, and the phenotypes on importin α/ß- mediated import and CRM1-dependent protein export were analyzed. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. There was a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. These data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events (Sabri, 2007).

Yeast and vertebrate nuclear pore complexes (NPCs) are structurally similar and consist of multiple copies of ~30 different nucleoporins. Approximately one third of all nucleoporins (Nups) carry phenylanine-glycine (FG) repeats of variable length. They are found at the nuclear basket, cytoplasmic fibers, and the central part of the NPC and can bind to both importins and exportins. X-ray crystallography has mapped the contact sites between FG repeats and importin ß, and mutations altering these amino acids in importin ß also reduce nuclear protein import (Bayliss, 2002). The extended conformation of the FG regions, their abundance in the NPC, and their differential affinity for transport receptors suggest that they are major determinants of transport through the channel. However, genetic and biochemical experiments in yeast show that half of the FG repeats can be removed without any defect in protein transport and cell viability (Sabri, 2007 and references therein).

The FG domain nucleoporins collectively provide a diffusion barrier to the pore. According to the virtual gating model, macromolecules are excluded from the pore by the fluctuations of unfolded peripheral FG domains. The local interaction between transport receptors and peripheral FG repeats traps the cargo, increases its residence time, and facilitates passage through the pore. In the selective phase partitioning model, intermolecular hydrophobic interactions between the FG repeats create a selective permeability barrier that prohibits free diffusion through the NPC. The interaction of nuclear transport receptors with distinct FG nucleoporins locally breaks the mesh and allows passage through the NPC. Are the mechanistic functions of all FG nucleoporins the same? Do individual metazoan FG nucleoporins contribute to protein transport differently than their yeast counterparts? These questions were addressed by functional analysis of the NPC using inducible GFP transport reporters in conjunction with RNAi in Drosophila melanogaster S2 cells (Sabri, 2007).

Inducible S2 cells were established expressing GFP, GFP fused to a classic NLS (cNLS [cNLS-GFP]), or GFP carrying a nuclear export signal (NES [GFP-NES]). Living cells expressing native GFP showed a homogenous distribution of the fluorescent signal. The cNLS-GFP reporter accumulated in nuclei, whereas the GFP-NES cargo was localized predominantly in the cytoplasm (Sabri, 2007).

Tests were performed to see whether the cNLS-GFP and GFP-NES reporters are cargoes of importin α/ßs and CRM1. The cell lines with double-stranded RNA (dsRNA) against the Drosophila homologues of importin α1, α2 (Pendulin), α3, ß (ketel), or kapß3. Only the addition of importin α3 and ß dsRNAs reduced the relative levels of nuclear cNLS-GFP. The distribution of the GFP and GFP-NES reporters was unaffected by the dsRNA treatments. Thus, the cNLS-GFP reporter is transported into the nucleus by importin α3/ß. In parallel, the reporter cell lines were tested with dsRNA for CRM1 (emb). The nuclear intensity of GFP-NES was increased in CRM1-depleted cells. This phenotype was comparable with the one generated by the treatment of GFP-NES cells with leptomycin, a CRM1-specific inhibitor. Therefore, the cytoplasmic accumulation of GFP-NES provides a functional assay for CRM1- mediated export (Sabri, 2007).

To assess the relative contributions of the NPC components on cNLS import and NES export, the Drosophila genome database was searched for nucleoporins. A set of 30 putative nucleoporins was identified along with a protein export cofactor, RanBP3. No Pom121 and Nup180 homologues were identified in the fly genome. The putative nucleoporin function of the selected Drosophila genes was also predicted by the Inparanoid algorithm, which classified them as orthologues of human genes encoding nucleoporins. For simplicity, the putative Drosophila nucleoporins are identified by the names of their human homologues (Sabri, 2007).

DsRNAs targeting each candidate nucleoporin were generated and gene inactivation efficiency was tested in the reporter cell lines by RT-PCR and by immunostainings and Western blots in cases in which specific antibodies were available. The dsRNA treatments considerably reduced the endogenous gene product after 4 d and allowed functional analysis of the genes in protein transport. The cellular distribution of each GFP reporter was assessed in parallel 4 d and 6 d after the addition of dsRNA to the cultures (Sabri, 2007).

Cells treated with dsRNAs for Nup358, Nup153, or Nup54 exhibited a clear reduction in cNLS-GFP nuclear concentration but showed no defects in GFP-NES and GFP localization, suggesting a selective role for Nup358, Nup153, and Nup54 in cNLS-protein import (Sabri, 2007).

Whether the RNAi inactivations caused defects in the localization or the amount of importin ß was examined by in situ stainings and Western blots. Untreated cells showed the characteristic rim-staining pattern of importin ß. Nup358 RNAi cells exhibited a weak cytoplasmic staining. The importin ß signal was also reduced in Nup54 dsRNA-treated cells, but its localization was not affected. In Nup153 RNAi cells, the levels of importin ß were not appreciably affected, but a substantial fraction of the protein was displaced from the rim into the cytoplasm. Thus, in all cases, the nuclear import deficit of the dsRNA-treated cells correlates with defects in the levels and/or localization of importin ß. Neither the distribution nor the intensity of CRM1 staining was appreciably changed in these cells, implying that Nup358, Nup153, and Nup54 are selectively required for importin ß-mediated import. Genetic analysis of Nup153 and Nup54 function in cNLS import is consistent with studies in yeast (Nup57), Xenopus laevis oocytes (Nup153 and Nup54), and HeLa cells (Nup153) using immunodepletion and overexpression experiments. However, the role of Drosophila Nup358 is surprising. Nup358 is the major component of the cytoplasmic filaments, and immunodepletion of its Xenopus homologue does not cause cNLS import defects in oocyte nuclei. Drosophila Nup358 is essential for importin ß expression or integrity, and the cNLS-GFP mislocalization in nup358i cells may be caused by the massive reduction of importin ß levels (Sabri, 2007).

A common feature of Nup358, Nup153, and Nup54 is the high content of FG repeats in their primary sequence. Does the FG-rich part of Nup153 contribute to nuclear import? To address this question, a V5-tagged full-length (V5-Nup153) and a truncated form of Nup153 lacking the FG domain (V5-Nup153DeltaFG) were overexpressed in Nup153 RNAi cells. Both chimeric proteins were expressed at similar levels and became localized at the nuclear envelope. The full-length form restored both the pore composition defects and the cNLS-GFP phenotype, indicating that Drosophila Nup153, like its vertebrate homologues, contributes to both pore integrity and importin ß transport (Walther, 2001). The Nup153DeltaFG fragment could rescue the defects in Nup214 and TPR localization in >98% of the expressing cells, suggesting that it contains all of the necessary sequences for Nup153 function in NPC integrity. Whether the Nup153DeltaFG fragment is also sufficient to restore the importin ß localization and cNLS import defects of Nup153 dsRNA-treated cells was examined. In 50% of the V5-positive cells, importin ß accumulation resembled its steady-state localization in untreated cells, suggesting that the Nup153 FG repeats are partly redundant for importin ß localization. Restoration of the import receptor at the NPC may be caused by the reinstatement of other importin ß-binding FG nucleoporins like Nup214 (Xylourgidis, 2006). However, only 10% of the transfected cells displayed an increased nuclear cNLS-GFP accumulation. The results argue that the role of Nup153 in protein import is independent of its function in NPC integrity. The FG region is required for importin ß-mediated transport, whereas the remainder of the protein ensures an intact NPC. A direct role of the Nup153-FG part in conveying importin α3/ß cargos through the pore is further supported by its localization along the entire channel and by its highly flexible conformation (Sabri, 2007).

None of the dsRNA treatments against nucleoporins caused detectable defects in GFP-NES distribution. However, the inactivation of RanBP3 increased the nuclear accumulation of the export reporter. The treatment had no effect on GFP and cNLS-GFP localization. Yrb2, the yeast homologue of RanBP3, is also essential for CRM1-dependent export. Vertebrate RanBP3 forms complexes with CRM1, RanGTP, and export substrates to stimulate NES nuclear protein export. RanBP3 and CRM1 were also found in complex with the chromatin-associated protein RanGEF. It was asked whether RanBP3 inactivation impacts CRM1 distribution by staining for CRM1. Untreated cells showed a predominantly nuclear accumulation of CRM1 with only a small fraction of the protein localized at the nuclear envelope. The nuclear CRM1 staining was severely reduced in RanBP3 RNAi cells. Instead, CRM1 became highly concentrated at the rim and, to some extent, in the cytoplasm of RanBP3 dsRNA-treated cells. The treatment had no effect on the accumulation of Nup214, Nup88, or any of the tested nucleoporins, suggesting a new function of RanBP3 in CRM1 localization. Reexpression of V5-tagged RanBP3 at low levels in ranBP3i cells restored both CRM1 depletion from the nucleus and the NES export defect. The results suggest that RanBP3 directly controls CRM1 localization and protein export (Sabri, 2007).

CRM1 forms complexes with Nup88 and Nup214, and, in Drosophila mutants lacking either of the nucleoporins, the NPC-bound CRM1 fraction accumulates in the nucleus. To determine whether Nup88 or Nup214 silencing causes similar phenotypes in S2 cells, cells treated with Nup88 or Nup214 dsRNA were stained for CRM1. The treatments reduced the CRM1 signal intensity at the nuclear envelope, suggesting that Nup88 and Nup214 anchor a CRM1 fraction at the NPC of S2 cells. However, unlike the defects of nup88 (mbo) and nup214 mutant larvae, the inactivation of Nup214 or Nup88 in S2 cells did not increase the cytoplasmic accumulation of the GFP-NES reporter. Thus, Nup214 or Nup88 depletion has no impact on CRM1 activity in S2 cultured cells. This difference between larval tissues and S2 cells can be attributed to the relatively high levels of CRM1 bound to the NPCs of distinct larval tissues. The redundancy of Nup214 for NES-GFP export in S2 cells is consistent with the lack of detectable defects in the nuclear export of NLS-GFP-NES in HeLa cells depleted for Nup214. Surprisingly, RNAi inactivation of Nup214 in the same cell line resulted in defects in nuclear export of the NFAT (nuclear factor of activated T cells) transcription factor and, to a lesser extent, in export of the Rev-GR-GFP reporter. The different phenotypes may suggest specific requirements of the different export cargoes used in the two studies (Sabri, 2007).

CRM1 can shuttle between the nucleus and the cytoplasmic face of the NPC in an energy-independent manner, and the inactivation of Nup214 and RanBP3 show opposing phenotypes in its localization. Therefore, CRM1 accumulation was investigated in cells treated simultaneously with both Nup214 and RanBP3 dsRNAs. In these cells, CRM1 was found inside the nucleus, suggesting that RanBP3 and Nup214 antagonize each other to determine the nuclear concentration of CRM1. The C-terminal FG-rich region of Nup214 binds to CRM1 directly, and it was asked whether it is also required for its antagonistic role in CRM1-mediated export. V5-tagged full-length or FG-deleted versions of Nup214 was expressed in cells lacking both RanBP3 and Nup214, where CRM1 accumulates inside the nucleus. The V5-Nup214 protein complemented the Nup88 deficit at the nuclear envelope and prohibited the nuclear accumulation of CRM1. The V5-Nup214DeltaFG protein was expressed at similar levels as the wild-type protein and rescued the Nup88 degradation defect caused by the Nup214 inactivation. Thus, the N-terminal part of Nup214 is sufficient for the interaction with Nup88 and NPC. However, the V5-Nup214DeltaFG fragment only slightly increased the NPC-bound fraction of CRM1. This small amount of CRM1 at the rim may be attracted by Nup88, which also binds to the export receptor. The results suggest that the antagonistic function of Nup214 on CRM1 localization is dependent on the Nup214 FG repeats (Sabri, 2007).

How do the opposing roles of Nup214 and RanBP3 on CRM1 accumulation influence its activity in NES export? Treatment of GFP-NES-expressing cells with dsRNAs against both Nup214 and RanBP3 resulted in the cytoplasmic distribution of the reporter, closely resembling its accumulation in untreated cells. Thus, unleashing the pore-bound fraction of CRM1 through Nup214 inactivation largely restores the GFP-NES export defect caused by the depletion of RanBP3. The results suggest that CRM1 NES export activity can be tuned by the opposing functions of Nup214 and RanBP3. Overexpression of the full-length Nup214 construct in Nup214 and RanBP3 RNAi cells resulted in a nuclear accumulation of GFP-NES closely resembling the phenotype caused by single RanBP3 inactivation. In contrast, the distribution of GFP-NES remained unaffected in V5-Nup214DeltaFG-expressing cells lacking both Nup214 and RanBP3. The results indicate that the ability of Nup214 to antagonize the function of RanBP3 in NES export requires the Nup214 FG repeats (Sabri, 2007).

CRM1 is reduced in the nucleus of RanBP3i cells, arguing that RanBP3 retains it inside the nucleus. To further examine the proposed new role of RanBP3, it was overexpressed in S2 cells and its effects on CRM1 and GFP-NES localization was examined. The expression of V5-tagged RanBP3 increased the nuclear intensity of CRM1), further arguing for a dynamic equilibrium between the Nup214- and RanBP3-bound forms of CRM1. In parallel experiments, the effect of RanBP3 overexpression in GFP-NES distribution was assessed. Although low levels of V5-RanBP3 did not change the predominantly cytoplasmic distribution of GFP-NES, high amounts of the exogenous protein increased the nuclear intensity of the reporter. This phenotype is consistent with in vitro experiments in which high levels of RanBP3 inhibit the assembly of CRM1 export complexes. In summary, a dual function of RanBP3 is proposed: one maintaining high nuclear levels of CRM1 and one aiding the assembly of CRM1-RanGTP cargo complexes (Sabri, 2007).

This in vivo analysis of nucleoporin function by RNAi did not detect protein transport phenotypes for the majority of the nucleoporins. This could be the result of functional redundancy, incomplete gene inactivation, or the relatively insensitive reporter assays. Nevertheless, the data provide some new insights into the function of NPCs. (1) Nup214 and RanBP3 antagonize each other to determine CRM1 localization and function. RanBP3 has a primary role in maintaining CRM1 inside the nucleus. This function of RanBP3 becomes redundant when Nup214 is codepleted. (2) Genetic evidence is provided arguing that individual FG domains are essential for distinct transport pathways in Drosophila. The importance of the Nup153 FG motif in mediating cNLS import was already suggested by overexpression experiments in permeabilized HeLa cells. Surprisingly, the FG repeats of Nup214 do not facilitate NES-GFP export but rather inhibit it. The FG regions from Nup153 or Nup214 are indispensable for the distinct transport roles of Nup153 and Nup214, yet they are not expected to affect the total mass of FG repeats and the barrier function of the NPC. The genetic analysis of nucleoporins in Drosophila argues that the Nup153 and Nup214 FG regions have specific functions in import and export, respectively, and suggest that peripheral nucleoporins have acquired additional roles during metazoan evolution. Understanding the mechanistic roles of animal nucleoporins in endogenous protein transport may provide new insights into the regulatory potential of the NPC (Sabri, 2007).

The importin-ß P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope

Three of the four independently induced Ketel^D dominant-negative female sterile mutations that identify the Drosophila importin-ß gene, originated from a C4114-->T transition and the concurrent replacement of Pro446 by Leu (P446L). CD spectroscopy of representative peptides with Pro or Leu in the crucial position revealed that upon the Pro-->Leu exchange the P446L mutant protein loses flexibility and attains most likely an open conformation. The P446L mutation abolishes RanGTP binding of the P446L mutant form of importin-ß protein and results in increased RanGDP binding ability. Notably, the P446L mutant importin-ß does not exert its dominant-negative effect on nuclear protein import and has no effect on mitotic spindle-related functions and chromosome segregation. However, it interferes with nuclear envelope formation during mitosis-to-interphase transition, revealing a novel function of importin-ß (Timinszky, 2002).

The fact that in three of the four independently isolated Ketel^D mutations the same C-->T transition and the concurrent replacement of Pro446 by Leu is the basis of dominant female sterility underlines the importance of Pro446 in importin-ß function. It is assumed that during a nuclear import cycle, whereas importin-ß interacts with the NLS containing protein (directly or through importin-ß), nucleoporins and RanGTP, the conformation of importin-ß changes significantly. In fact the region around HEAT repeat 10 was suggested, based on X-ray crystallography, to be a flexible point during switching between the IBB- and the Ran-bound forms. Pro446 resides in the linker region connecting HEAT repeats 10 and 11 and, as described in this study, plays a crucial role in enduring flexibility of importin-ß. CD spectra of model peptides representing the wild-type and the P446L mutant proteins reveal loss of flexibility upon Pro446-->Leu replacement. The lost flexibility is most likely the consequence of fusion of the small alpha-helix in the linker region with the alpha-helix of HEAT 10B. Computer 3D modeling of the P446L protein structure, based on results of CD spectroscopy, shows altered molecular structure: the P446L molecule takes on an open conformation such that its inner hydrophobic surface becomes exposed to water, explaining the reduced hydrophilic nature of the P446L protein (Timinszky, 2002).

The significant conformational change due to the exchange of Pro446 to Leu in the Ketel protein is further supported by the S317T suppressor mutation that restores Ketel gene function. In human importin-ß the corresponding Ser311 (in the linker region between HEAT repeats 7 and 8) and Pro441 are 32.5 Å apart and yet the Ser-->Thr exchange in the Drosophila homologue restores function of importin-ß. The 10 Å area surrounding serine is hydrophobic. The stronger hydrophobicity of threonine compared with serine does perhaps increase apolar interactions and bend the molecule back to its functional structure (Timinszky, 2002).

Experiments with digitonin-permeabilized HeLa cells show that, to a reduced extent, the P446L proteins do participate in formation of the nuclear import complexes and in their docking to the cytoplasmic surface of the NE; however, they do not support import of the complexes into the nuclei in the presence of Ran, energy source, RanGAP and RanBP1. In fact, the import complexes do not form upon the addition of the latter components. Apparently the main structural domains of the P446L protein are intact (binds importin-alpha, NPC and Ran) but the interaction with Ran is altered. Indeed, the binding of wild-type and P446L Ketel proteins to Ran are very different: the P446L cannot bind to RanGTP, to which the wild-type importin-ß binds strongly, but shows elevated affinity to RanGDP, to which the wild-type protein shows very little affinity. It is noteworthy that a single amino acid exchange outside the classical Ran-binding domain can change Ran binding ability dramatically. The change in Ran-binding ability is most likely the source of the Ketel^D-associated dominant female sterility. However, the Ketel^D-associated dominant-negative effect is not manifested via nuclear protein import but rather through the prevention of cleavage nuclei formation: revealing a novel importin-ß function (Timinszky, 2002).

Injection experiments into wild-type cleavage embryos revealed that the P446L mutant protein does not inhibit nuclear protein import: when co-injected with P446L, a fluorescent nuclear substrate readily enteres the nuclei. Furthermore, although the cleavage nuclei enter mitosis and the chromosomes segregate normally, intact NE never forms in the presence of P446L mutant importin-ß. Failure of NE assembly in the presence of P446L is revealed by the following observations: (1) the homogenous distribution of a fluorescent nuclear substrate, the high molecular weight dextrane and the GFP-tubulin; (2) the absence of the nuclear lamina lining. Thus the mutant P446L importin-ß reveals a novel importin-ß function required during the mitosis-to-interphase transition, a function distinct from the already known functions of importin-ß in nuclear protein import and in mitotic spindle assembly (Timinszky, 2002).

The P446L mutant importin-ß possesses altered Ran-binding properties: it does not bind RanGTP but shows elevated affinity to RanGDP. A series of experiments showed that altered RanGTP-RanGDP balance leads to a similar phenotype in yeast (i.e., arrest in mitosis-to-interphase transition). Results of enzyme assays described in the present paper show that the altered Ran-binding ability of P446L importin-ß does not interfere with the GTP hydrolysis and nucleotide exchange on Ran and thus it is unlikely that the Ketel^D-related defects are consequences of distorted Ran metabolism. Most probably importin-ß is a downstream effector of Ran during mitosis-to-interphase transition, as in nuclear protein import and mitotic spindle assembly (Timinszky, 2002).

Although several functions of Ran and importin-ß during the cell cycle have been described, the exact molecular mechanisms are still missing. This study describes a novel function of Drosophila importin-ß during mitosis-to-interphase transition where it is involved in the formation of intact nuclear envelope (NE). There seem to be three feasible explanations for the P446L-associated defects. First, since the P446L importin-ß shows higher affinity to RanGDP than wild-type importin-ß, a possible explanation may be the depletion of significant amounts of RanGDP that is required for NE reassembly at the end of mitosis. Removal of RanGDP by P446L may lead to the failure of cleavage nuclei formation. This explanation is thought not to be very likely for the following reasons. (1) Binding and nucleotide exchange assays revealed that the affinity of the P446L to RanGDP is low and hence depletion of a significant fraction of Ran from the cytoplasm is rather unlikely. (2) Interestingly, defects do not evolve in nuclear protein import or in spindle formation and chromosome segregation following injection of P446L despite the fact that both nuclear protein import and spindle formation have been shown to be Ran dependent. Ran's involvement in NE assembly has also been described but since none of the aforementioned Ran-related processes were disturbed, the P446L protein does not seem to disturb the Ran cycle. A second possible explanation of the P446L-related defects is perhaps the inability of the P446L protein to bind RanGTP and, consequently, the inability to release factors required for proper chromatin decondensation and/or NE assembly. In this case the role of importin-ß in the above processes would resemble its function in mitotic spindle formation, where it is thought to be required for the release of factors needed for spindle assembly [e.g. NuMA, TPX2. A third possibility is that the P446L-related defects are not associated with the change in Ran-binding ability. The P446L mutation may disturb the association of thus far unidentified factors (e.g. nucleoporins). In the case of the second and third possibilities the factor(s) required for the newly described importin-ß-related functions remain to be identified (Timinszky, 2002).

Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, Rolled. However, the molecular details of Rolled translocation are largely unknown. In this regard, D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin have been identified. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated Rolled. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated Rolled in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic rolled mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated Rolled. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin beta homolog Ketel also reduce the nuclear localization of activated Rolled. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated Rolled (Lorenzen, 2001).

The activation of ERK represents the focal point of a conserved signaling module used by a diverse array of extracellular stimuli. The potency and duration of ERK activation and its accompanying translocation to the nucleus can profoundly affect the fate of a cell. This is apparent in PC12 cells where the decision to proliferate or differentiate depends upon the number and duration of receptors stimulated. Throughout development, cells respond to spatial and temporal signals and must interpret gradients to produce qualitative differences in gene expression. In Drosophila, the terminal system or Torso RTK signaling pathway illustrates one example whereby quantitative differences in D-ERK activity generate distinct cell fates. Distinct quantitative levels of D-ERK activity inside a cell may be achieved within the RTK pathway by modulating D-ERK phosphorylation. Moreover, it is apparent that mechanisms exist to control the localization of ERK activity by either regulating its retention in the cytoplasm and/or its nucleocytoplasmic shuttling. In this regard, nuclear translocation of dpERK is not always a compulsory consequence of RTK signaling. In Drosophila as photoreceptors are recruited into the developing retina, dpERK is held in the cytoplasm for up to several hours following EGFR and Sevenless RTK signaling. This raises the possibility that import is differentially controlled relative to D-ERK phosphorylation and/or dimerization. Interest in the active transport of dpERK also partly stems from the observation that dimer formation is a common property of mammalian MAP kinase family members (Lorenzen, 2001).

Presumably, D-ERK shares this property, since the residues involved in dimer formation have been conserved. Although monomeric dpERK can enter the nucleus passively, it has been shown that import of dimeric ERK is an active process. This study has clarified mechanistic issues in the nuclear relocalization of dpERK through the identification of DIM-7, the Drosophila homolog of importin 7 and member of the importin superfamily of nuclear transport proteins. DIM-7 exhibits several properties that establish its participation in RTK signaling. These include a physical interaction with CSW and dpERK, and the finding that DIM-7 is tyrosine phosphorylated in stimulated cells. Furthermore, alleles of msk, the gene that encodes DIM-7, dominantly interact with hypomorphic and hypermorphic alleles of D-ERK (Lorenzen, 2001).

The primary structure of DIM-7 originally suggested that it might play a role in nuclear transport. In addition to exhibiting significant sequence identity with its Xenopus and human homologs, DIM-7 also possesses a conserved Ran-binding domain. This latter property is a hallmark of nuclear transport proteins that display a RanGDP versus RanGTP regulated interaction with their cargo proteins. In addition to physically binding to phosphorylated D-ERK, DIM-7 and dpERK have overlapping nuclear localization patterns in developing tissues subject to RTK regulation. This made dpERK an attractive candidate cargo for DIM-7. To address this possibility during embryogenesis and in the absence of functional DIM-7, tracheal placodes and tracheal pits were assayed for defects in the accumulation of dpERK. Significantly, embryos in which the genomic interval encoding the msk gene is deleted or mutated exhibit a fivefold reduction in the number of dpERK-positive nuclei. Importantly and demonstrating that DIM-7 is essential for nuclear uptake of dpERK, expression of wild-type DIM-7 in a msk mutant background restores dpERK nuclear accumulation. These findings reinforce the idea that not only is dpERK actively transported through the nuclear pore complex but also that DIM-7 functions as either the transport receptor and/or adapter for dpERK (Lorenzen, 2001).

Finally, this study has implicated KET in the nuclear import of dpERK. As vertebrate importin 7 and importin b form an abundant heterodimeric complex, it was asked whether the Drosophila homolog of importin β, KET participates in the dpERK transport mechanism. Supporting a model whereby DIM-7 and KET function together in the nuclear transport of dpERK, it was found that nuclear localization of dpERK is impaired in homozygous ket mutant embryos. Although there are other possibilities, two models are envisioned by which DIM-7 could function in the nuclear import cycle of dpERK. In one, DIM-7 and KET could function together in the same import cycle, where DIM-7 and KET serve as the import adapter and import receptor, respectively. Alternatively, DIM-7 and KET could function independently of each other in separate import cycles that could be, at least partially, redundant. However, it is unlikely that DIM-7 and KET serve totally redundant functions for two reasons. First, each individual locus when deleted (msk) or mutated (msk and ket) reduces substantially the number of dpERK-positive nuclei, and, second, both msk and ket mutations, alone, are lethal (Lorenzen, 2001).

In the literature there appears to be an increasing number of transport receptors with complex cargo specificities. If importin 7 is the functional vertebrate homolog of DIM-7, then this transport factor can import at least three different proteins, dpERK, histone H1 and rpL23a. An additional point concerning specificity of DIM-7 regards its nearest homolog, Nmd5p, in Saccharomyces cerevisiae. Nmd5p is essential for the nuclear import of HOG1, a p38 MAP kinase family member that is activated in response to osmotic stress. Interestingly, the movement of HOG1 into the nucleus does not require the importin b homolog, RSL1. This work raises the possibility that DIM-7 might also mediate the nuclear transport of one or more other Drosophila MAPkinase family members, D-p38a (Mpk2), D-p38b (Mpk34C) and D-JNK (JUN kinase). The combinatorial use of different transport factors may provide a means for specific recognition of the various MAP kinase family members. For example, DIM-7 alone may bind and import D-p38; however, recognition of dpERK may require the simultaneous pairing of DIM-7 and KET. Determining the mechanism(s) employed to establish recognition between a cargo and its transport receptor will require a precise molecular dissection of the interactions between several transport receptor/cargo pairs (Lorenzen, 2001).

CSW was first demonstrated to have a positive function during embryogenesis in the Torso RTK pathway. In this pathway CSW serves two functions. First, the adapter protein DRK does not bind Torso; instead, CSW functions as an adapter linking Torso to RAS. Second, CSW is able to dephosphorylate the Torso autophosphorylation site that binds RasGAP. The work presented in this paper suggests a third function for CSW as an adapter to facilitate the physical interaction of DIM-7 with its import cargo dpERK. When two, or three, of these CSW functions are used within one signaling module, interpreting the epistatic relationships of CSW with various signaling components could become problematic. For example, previous epistasis experiments have suggested that CSW carries out its function either upstream or downstream of RAS1 and/or D-RAF. Now it appears these differences could simply reflect the differential use of the various signaling capabilities of CSW within a given RTK pathway (Lorenzen, 2001).

Whether or not the association of DIM-7 with CSW constitutes part of a regulatory process at the level of the receptor that governs D-ERK redistribution has yet to be determined. However, it appears that nuclear import is a fundamental mechanism used by cells to modulate incoming signals throughout development. It is expected, then, that the development of reagents to modulate the nuclear entry of specific molecules may have profound effects for controling both disease and oncogenic states (Lorenzen, 2001).

Perturbing nuclear transport in Drosophila eye imaginal discs causes specific cell adhesion and axon guidance defects

To study nucleocytoplasmic transport during multicellular development, a sensitive nuclear protein import assay was developed in living blastoderm embryos. Dominant negative truncations of the human nuclear transport receptor karyopherinβ/Importinβ (DNImpβ) disrupt mRNA export and protein import in Drosophila. To test the sensitivity of different developmental processes to nuclear trafficking perturbations, DNImpβ was expressed behind the morphogenetic furrow of the eye disc, at a time when photoreceptors are patterned and project their axons to the brain. DNImpβ 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 Importinβ. It is concluded 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 (Kumar, 2001).

The results are consistent with the fact that, in permeabilised human cells and in Xenopus, a similar truncation of Impb inhibits most nuclear trafficking except tRNA export and localizes to the inner surface of the nuclear envelope. DNImpβ probably acts by binding Nup153, a key shuttling nucleoporin required for most nucleocytoplasmic trafficking including mRNA export and all known protein import and export, but not for tRNA export. This study has shown that DNImpβ causes a similar nuclear transport block in Drosophila embryos and that fluorescently labeled DNImpβ accumulates at the nuclear envelope around the site of injection. The nuclear trafficking inhibition caused by DNImpβ injection in Drosophila embryos is probably complete. However, DNImpβ expression in the eye disc causes a partial inhibition of nuclear transport, revealing processes that have particularly high requirements for nuclear transport machinery (Kumar, 2001).

It is difficult to be certain whether mRNA export, nuclear protein import, or protein export are the primary cause of the eye phenotypes observed. The interpretation is favored that DNImpβ causes a disruption of nuclear protein import because the results show that recessive alleles of Ketel enhance all aspects of the eye phenotype. The human ortholog of Ketel, Impβ, is known to play a role in protein import but not in RNA and protein export. However, it is also possible that Impβ inhibits the Ketel-dependent nuclear import of factors required for mRNA export. To address these issues will require the identification of the factors disrupted by Impβ expression in the eye (Kumar, 2001).

Cell adhesion in the eye, as in other epithelial sheets, involves components of adherens junctions, extracellular matrix, transmembrane proteins, and actin, all of which are thought to act at the periphery of the cell. Surprisingly, the results suggest that cell adhesion is more sensitive to perturbations in nuclear trafficking than are other processes such as nuclear import of some transcription factors. The disruption of adhesion observed may either be due to a direct role for nuclear trafficking in the supply of adhesion components, or a role in the signals that regulate cell adhesion (Kumar, 2001).

The results also suggest that communication between the cytoplasm and nucleus have a role in axon guidance decisions, a process which is still poorly understood. Key molecules that have been found to play direct roles in axon guidance include ligands and receptors that attract or repel the growth cone. Guidance decisions are made when the growth cone assesses the relative balance of attractive and repulsive forces and selects appropriate routes based on multiple cues. However, nuclear proteins with specific roles in axon guidance have also been identified. Furthermore, guidance cues are likely to induce changes in gene expression requiring the import of signals into the nucleus. For example, in the case of the axon guidance receptor Roundabout (Robo), its expression is dramatically increased when axons cross the midline of the CNS, thus preventing further crossing of the midline. Identifying why axon guidance in the eye is particularly sensitive to perturbations in nuclear transport will require the identification of the axon guidance factor whose trafficking is disrupted by Impβ in the eye (Kumar, 2001).

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-ß (karyopherin-ß) homologue of Drosophila. 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 (Tirian, 2000).

HURP is a Ran-importin beta-regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes

Formation of a bipolar mitotic spindle in somatic cells requires the cooperation of two assembly pathways, one based on kinetochore capture by centrosomal microtubules, the other on RanGTP-mediated microtubule organization in the vicinity of chromosomes. How RanGTP regulates kinetochore-microtubule (K-fiber) formation is not presently understood. This study identifies the mitotic spindle protein HURP as a novel target of RanGTP. HURP is a direct cargo of importin beta and in interphase cells, it shuttles between cytoplasm and nucleus. During mitosis, HURP localizes predominantly to kinetochore microtubules in the vicinity of chromosomes. Overexpression of importin beta or RanT24N (resulting in low RanGTP) negatively regulates its spindle localization, whereas overexpression of RanQ69L (mimicking high RanGTP) enhances HURP association with the spindle. Thus, RanGTP levels control HURP localization to the mitotic spindle in vivo, a conclusion supported by the analysis of tsBN2 cells (mutant in RCC1). Upon depletion of HURP, K-fiber stabilization is impaired and chromosome congression is delayed. Nevertheless, cells eventually align their chromosomes, progress into anaphase, and exit mitosis. HURP is able to bundle microtubules and, in vitro, this function is abolished upon complex formation with importin beta and regulated by Ran. These data indicate that HURP stabilizes K-fibers by virtue of its ability to bind and bundle microtubules. In conclusion this study identifies HURP as a novel component of the Ran-importin beta-regulated spindle assembly pathway, supporting the conclusion that K-fiber formation and stabilization involves both the centrosome-dependent microtubule search and capture mechanism and the RanGTP pathway (Sillje, 2006).

Drosophila importin α1 performs paralog-specific functions essential for gametogenesis

Importin αs mediate nuclear transport by linking nuclear localization signal (NLS)-containing proteins to importin beta1. Animal genomes encode three conserved groups of importin α's, α1's, α2's, and α3's, each of which are competent to bind classical NLS sequences. Using Drosophila melanogaster, the isolation and phenotypic characterization of the first animal importin α1 mutant (Drosophila karyopherin α1) is described. Animal α1's are more similar to ancestral plant and fungal α1-like genes than to animal α2 and α3 genes. Male and female importin α1 (Dα1) null flies developed normally to adulthood (with a minor wing defect) but were sterile with defects in gametogenesis. The Dα1 mutant phenotypes were rescued by Dα1 transgenes, but not by Dα2 or Dα3 transgenes. Genetic interactions between the ectopic expression of Dα1 and the karyopherins CAS and importin β1 suggest that high nuclear levels of Dα1 are deleterious. It is concluded that Dα1 performs paralog-specific activities that are essential for gametogenesis and that regulation of subcellular Dα1 localization may affect cell fate decisions. The initial expansion and specialization of the animal importin α-gene family may have been driven by the specialized needs of gametogenesis. These results provide a framework for studies of the more complex mammalian importin α-gene family (Ratan, 2008).

The genetic interactions between coectopic expression of Dα1 and Dcas and Ketel are consistent with the idea that the tergite defects and lethality are the result of increases in the levels of importin α in nuclei. Genetic manipulations that would be expected to decrease nuclear levels of Dα1 (overexpression of Dcas or loss-of-function Ketel mutants) mitigated the effects of overexpressing Dα1. Likewise, manipulations that would be expected to increase nuclear levels of Dα1 (overexpression of Ketel or loss-of-function Dcas mutants) enhanced Dα1 overexpression phenotypes. Interestingly, an increase in cNLS cargo levels also enhanced the Dα1 overexpression defects. Here, higher cNLS cargo levels could be expected to recruit more Dα1 into targeting complexes with importin β1 (Ketel), resulting in higher steady state nuclear levels of Dα1. Taken together, these results argue that higher than normal nuclear levels of Dα1 are deleterious, and that the nucleocytoplasmic trafficking of nuclear transport factors must be carefully balanced during development (Ratan, 2008).

Search PubMed for articles about Drosophila Ketel

Bayliss, R., T. Littlewood, L.A. Strawn, S.R. Wente, and M. Stewart. 2002. GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta. J. Biol. Chem. 277: 50597-50606. PubMed ID: 12372823

Caudron, M., Bunt, G., Bastiaens, P. and Karsenti, E. (2005). Spatial coordination of spindle assembly by chromosome-mediated signaling gradients. Science 309: 1373-1376. PubMed ID: 16123300

Ciciarello, M., Mangiacasale, R. and Lavia, P. (2007). Spatial control of mitosis by the GTPase Ran. Cell Mol. Life Sci. 64: 1891-1914. PubMed ID: 17483873

Cingolani, G., et al. (1999). Structure of importin-β bound to the IBB domain of importin-α. Nature 399: 221-229. PubMed ID: 10353244

D'Avino, P. P., Savoian, M. S. and Glover, D. M. (2005). Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy. J. Cell Sci. 118: 1549-1558. PubMed ID: 15811947

Deng, M., Suraneni, P., Schultz, R. M. and Li, R. (2007). The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes. Dev. Cell 12: 301-308. PubMed ID: 17276346

Erdelyi, M., Mathe, E. and Szabad, J. (1997). Genetic and developmental analysis of mutant Ketel alleles that identify the Drosophila importin-beta homologue. Acta Biol. Hung. 48: 323-338. PubMed ID: 9406612

Fried, H. and Kutay, U. (2003). Nucleocytoplasmic transport: taking an inventory. Cell. Mol. Life Sci. 60: 1659-1688. PubMed ID: 14504656

Gorjanacz, M., Adam, G., Torok, I., Mechler, B. M., Szlanka, T. and Kiss, I. (2002). Importin-alpha 2 is critically required for the assembly of ring canals during Drosophila oogenesis. Dev. Biol. 251: 271-282. PubMed ID: 12435357

Görlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm, Annu. Rev. Cell Dev. Biol. 15: 607-660. PubMed ID: 10611974

Gregory, S. L., et al. (2008). Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring. Curr. Biol. 18(1): 25-9. PubMed ID: 18158242

Gruss, O. J., et al. (2001). Ran induces spindle assembly by reversing the inhibitory effect of importin-α on TPX2 activity. Cell 104: 83-93. PubMed ID: 11163242

Janetopoulos, C. and Devreotes, P. (2006). Phosphoinositide signaling plays a key role in cytokinesis. J. Cell Biol. 174: 485-489. PubMed ID: 16908667

Kalab, P., Pralle, A., Isacoff, E. Y., Heald, R. and Weis, K. (2006). Analysis of a RanGTP-regulated gradient in mitotic somatic cells. Nature 440: 697-701. PubMed ID: 16572176

Kumar, J. P., Wilkie, G. S., Tekotte, H., Moses, K. and Davis, I. (2001). Perturbing nuclear transport in Drosophila eye imaginal discs causes specific cell adhesion and axon guidance defects. Dev. Biol. 240(2): 315-25. PubMed ID: 11784066

Kutay, U., et al. (1997). Dominant-negative mutants of importin-β block multiple pathways of import and export through the nuclear pore complex. EMBO J. 16: 1153-1163. PubMed ID: 9135132

Lippai, M., et al. (2000). The Ketel gene encodes a Drosophila homologue of importin-beta. Genetics 156: 1889-1900. PubMed ID: 11102382

Lorenzen, J. A., et al. (2001). Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin. Development 128(8): 1403-14. 11262240

Mazumdar, A. and Mazumdar, M. (2002). How one becomes many: blastoderm cellularization in Drosophila melanogaster. Bioessays 24: 1012-1022. PubMed ID: 12386932

Nachury, M. V. et al. (2001). Importin-β is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104: 95-106. PubMed ID: 11163243

Pal, M., Nagy, O. and Deak, P. (2007a). Characterization of the APC10/Doc1 subunit of the anaphase promoting complex in Drosophila melanogaster. Acta Biol. Hung. 58: 51-64. PubMed ID: 18297794

Pal, M., et al. (2007b). Structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex in Drosophila melanogaster. J. Cell. Sci. 120: 3238-3248. PubMed ID: 17878237

Piekny, A. J. and Glotzer, M. (2008). Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis. Curr. Biol. 18(1): 30-6. PubMed ID: 18158243

Ratan, R., Mason, D. A., Sinnot, B., Goldfarb, D. S. and Fleming, R. J. (2008). Drosophila importin α1 performs paralog-specific functions essential for gametogenesis. Genetics 178(2): 839-50. PubMed ID: 18245351

Sabri, N., et al. (2007). Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport. J. Cell Biol. 178(4): 557-65. PubMed ID: 17682050

Sillje, H. H., Nagel, S., Korner, R. and Nigg, E. A. (2006). HURP is a Ran-importin beta-regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes. Curr. Biol. 16: 731-742. PubMed ID: 16631580

Silverman-Gavrila, R. V. and Wilde, A. (2006). Ran is required before metaphase for spindle assembly and chromosome alignment and after metaphase for chromosome segregation and spindle midbody organization. Mol. Biol. Cell 17(4): 2069-80. PubMed ID: 16481399

Silverman-Gavrila. R. V., Hales. K. G. and Wilde, A. (2008). Anillin-mediated targeting of peanut to pseudocleavage furrows is regulated by the GTPase Ran. Mol. Biol. Cell 19(9): 3735-44. PubMed ID: 18579688

Szabad, J., et al. (1989). Isolation and characterization of dominant female sterile mutations of Drosophila melanogaster II. Mutations on the second chromosome. Genetics 122: 823-835. PubMed ID: 2503422

Timinszky, G. et al. (2002). The importin-β P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope. J. Cell Sci. 115(8): 1675-1687. PubMed ID: 11950886

Tirian, L., et al. (2000). The Ketel(D) dominant-negative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation. Genetics 156: 1901-1912. PubMed ID: 11102383

Tirian, L., Timinszky, G. and Szabad, J. (2003). P446L-importin-beta inhibits nuclear envelope assembly by sequestering nuclear envelope assembly factors to the microtubules. Eur. J. Cell Biol. 82: 351-359. PubMed ID: 12924630

Villányi, Z., Debec, A., Timinszky, G., Tirián, L. and Szabad, J. (2008a). Long persistence of importin-β explains extended survival of cells and zygotes that lack the encoding gene. Mech. Dev. 125(3-4): 196-206. PubMed ID: 18221858

Villanyi, Z., Papp, B., Szikora, S., Boros, I. and Szabad, J. (2008b). The DRE motif is a key component in the expression regulation of the importin-beta encoding Ketel gene in Drosophila. Mech. Dev. 125(9-10): 822-31. PubMed ID: 18656533

Walther, T.C., et al. (2001). The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins. EMBO J. 20: 5703-5714. PubMed ID: 11598013

Wehman, A. M., Staub, W. and Baier, H. (2007). The anaphase-promoting complex is required in both dividing and quiescent cells during zebrafish development. Dev. Biol. 303: 144-156. PubMed ID: 17141209

Wiese, C., et al. (2001). Role of importin-beta in coupling Ran to downstream targets in microtubule assembly. Science 291: 653-656. PubMed ID: 11229403

Wozniak, R. W., Rout, M. P. and Aitchison, J. D. (1998). Karyopherins and kissing cousins. Trends Cell Biol. 8: 184-8. PubMed ID: 9695836

Zhang, C. and Clarke, P. R. (2000). Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts. Science 288: 1429-1432. PubMed ID: 10827954

Zhang, C., et al. (2002). Role of importin-beta in the control of nuclear envelope assembly by Ran. Curr. Biol. 12: 498-502. PubMed ID: 11909537

Xylourgidis, N., Roth, P., Sabri, N., Tsarouhas, V. and Samakovlis, C. (2006). The nucleoporin Nup214 sequesters CRM1 at the nuclear rim and modulates NFkappaB activation in Drosophila. J. Cell Sci. 119: 4409-4419. PubMed ID: 17032737

date revised: 20 June 2023

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