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Open Access Highly Accessed Research article

Crosstalk between the actin cytoskeleton and Ran-mediated nuclear transport

Svetlana Minakhina, Ron Myers, Marina Druzhinina and Ruth Steward*

Author affiliations

Waksman Institute, Department of Molecular Biology and Biochemistry, NJ Cancer Center, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA

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Citation and License

BMC Cell Biology 2005, 6:32  doi:10.1186/1471-2121-6-32

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2121/6/32


Received:16 December 2004
Accepted:24 August 2005
Published:24 August 2005

© 2005 Minakhina et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Transport of macromolecules into and out of the nucleus is a highly regulated process. The RanGTP/RanGDP gradient controls the trafficking of molecules exceeding the diffusion limit of the nuclear pore across the nuclear envelope.

Results

We found genetic interaction between genes establishing the Ran gradient, nuclear transport factor 2 (ntf-2), Ran GTPase activating protein (Sd), and the gene encoding Drosophila Profilin, chickadee (chic). The severe eye phenotype caused by reduction of NTF2 is suppressed by loss of function mutations in chic and gain of function mutations in Sd (RanGAP). We show that in chic mutants, as in Sd-RanGAP, nuclear export is impaired.

Conclusion

Our data suggest that Profilin and the organization of the actin cytoskeleton play an important role in nuclear trafficking.

Background

In eukaryotic cells the nuclear envelope serves as a barrier between the nucleus and cytoplasm. The transport of molecules between the nucleus and cytoplasm occurs through nuclear pore complexes (NPCs). Although some small molecules (<40 kDa) diffuse through the pore, most proteins and RNAs require facilitated transport and special receptors called importins and exportins. This facilitated transport further depends on the small Ras family GTPase Ran (for review see [1,2]).

Similar to other GTPases, Ran is regulated by conformational changes driven by GTP hydrolysis and nucleotide exchange. GDP to GTP exchange happens in the nucleus and is catalyzed by the chromatin-associated protein RCC1 [3]. The GTPase activating protein RanGAP controls RanGTP hydrolysis in the cytoplasm. Spatial partition of these two processes generates a RanGTP concentration gradient across the nuclear pore. This gradient is thought to guide the directionality of nuclear transport.

Protein cargo containing nuclear localization signals (NLS) is recognized by importins and translocates through nuclear pores into the nuclei, where binding to RanGTP causes release of the cargo. In the nuclei, proteins containing nuclear export signals (NESs) form export complexes together with RanGTP. These complexes are then exported, and upon RanGTP hydrolysis in the cytoplasm, the cargo is released. RanGDP nuclear reentry is mediated by the nuclear transport factor 2 (NTF2, [4,5]).

NTF2 was originally identified by its ability to stimulate protein import into nuclei in permeabilized mammalian cells [6]. NTF2 was further shown to have a critical role in actively replenishing the nuclear stock of Ran [4,7,8]. NTF2 catalyzes RanGDP nuclear import, and the concentration of nuclear RanGTP is ultimately increased because of the activity of the RCC1 exchange factor.

Although the RanGTP gradient is required for both import and export, components of the pathway that regulate the gradient have an effect on directionality of cargo nuclear transport. For example, decrease in NTF2 has a primary effect on nuclear import. Conditional alleles of yeast ntf-2 show defects in protein nuclear import [9]. Also, depletion of NTF2 using antibodies inhibits nuclear import of NLS-containing proteins in HeLa cells [10]. In Drosophila, partial loss of function of ntf-2 affects nuclear import of Rel proteins in immune response and some loss of function alleles show a strong eye phenotype [11,12]. It has been shown in vertebrates that both low levels as well as increased levels of NTF2 impair nuclear import [13-15].

In contrast to NTF2, the intracellular localization of RanGAP appears to be critical for nuclear export [16]. In interphase cells RanGAP is localized to the cytoplasm and a large fraction of the cytoplasmic protein is modified by the ubiquitin related protein SUMO and localized to fibers of the NPCs [17,18]. This strategic position of RanGAP is thought to control the steep concentration gradient of RanGTP across the nuclear envelope.

Mislocalization of RanGAP can reduce nuclear RanGTP levels and lead to reduction of NES-mediated nuclear export, as happens in Segregation distortion (Sd) mutants in Drosophila [16]. In Sd mutants Sd-RanGAP, an enzymatically active protein refractory to SUMO modification, is expressed in addition to wild-type RanGAP. In these mutants RanGAP is found in higher levels in the cytoplasm and is also detected in nuclei. Sd-RanGAP presumably catalyzes hydrolysis of RanGTP in the nuclei thereby interfering with cargo export. A similar effect can be caused by over-expression of wild-type RanGAP [19].

Ran, in addition to functioning in nuclear-cytoplasmic transport, controls mitotic spindle formation. RanGTP stimulates polymerization of microtubules and RanGAP is found to associate with mitotic spindles [20,21]. Ran also functions in nuclear envelope and nuclear pore assembly [22,23]. These functions require the activities of RanGAP and RCC1, but the contribution of NTF2 is so far unclear.

In a genetic screen we identified mutants in Drosophila Profilin as modifiers of the partial loss of function eye phenotype of ntf-2. We find that Profilin is essential for normal nuclear export. This is surprising because the main function of Profilin is to control actin polymerization. RanGAP controls nuclear export and we find that gain of function mutants in Sd-RanGAP also suppress the ntf-2 eye phenotype. Our studies suggest a close connection between the organization of the actin cytoskeleton and nuclear transport.

Results

NTF2 and eye development

ntf-2 is an X-linked essential gene. Depending on the allele, animals die between the 2nd larval instar and the pupal stage. Some alleles have an adult survival rate of 8–15% of expected (Table 1), and all survivors show a small eye phenotype, strongly reduced numbers of ommatidia [11,12,24]. The eye phenotype varies from 30% of normal size to a more severe phenotype displaying one or two small patches of 10–40 ommatidia (Fig. 1A).

Table 1. Genetic interactions between ntf-2, chic, and Sd. The genetic crosses performed (top of the table). #s in parenthesis indicate columns in the table. The progeny resulting from the cross of ntf-2/FM7 females to males carrying the suppressing chromosome were counted. Four alleles of ntf-2 are listed in column 1. The genotype of the suppressing chromosomes are presented in the column 2. The numbers of trans-heterozygous females (ntf-2/+; Su/+; column 3), of non-mutant males (FM7/Y; Su/+, column 4), and of potentially suppressed mutant males (ntf-2/Y; Su/+) are presented. The ntf-2/+; Su/+ males were divided into two columns (5 and 6) depending on their eye phenotype and the viability of these males is indicated (100% X ntf-2 males/ntf-2/+ females). The number of progeny of the cross shown on the top and marked by * are not shown.

thumbnailFigure 1. The ntf-2 eye phenotype is rescued by mutants in Profilin (chic). (A) Wild-type eye, a representative ntf-2 eye and the phenotype of a ntf-2 eye suppressed by chic/+. Note that the antennae (arrow) are normal in mutant animals. (B) Wild-type and ntf-2 eye-antennal discs. The antennal discs (ant) are normal in wild-type and mutant, while the ntf-2 eye disc (eye) shows abnormal growth and patterning. Size bar represents 10 μm.

The mutant eye-imaginal discs are smaller than wild-type and are often abnormally shaped (Fig. 1B). Overall, the structure of the mutant eye discs is perturbed and the organization of the actin cytoskeleton is strongly altered (compare Fig. 2A and 2B,C). Only few disorganized, irregularly spaced rabdomere-like structures are apparent in the posterior compartment of the eye disc (arrow in Fig. 2A–C).

thumbnailFigure 2. The ntf-2 eye dics are disorganized. Wild-type eye disc (A, D; arrowhead indicates morphogenetic furrow, arrow indicates rabdomeres). In ntf-2 mutants (B, C, E, F) the furrow fails to move and fewer rabdomeres are formed; the organization of the actin cytoskeleton (green) and distribution of RanGAP (red) look abnormal. Squares are magnified in panels D, E, F. In all Figures DNA is shown in blue and the size bar represents 10 μm.

A deficiency screen to identify dominant suppressors of ntf-2

We took advantage of the partial loss of function eye phenotype of ntf-2 alleles to identify genes functioning with ntf-2, and performed a dominant suppressor screen of the eye phenotype. Males from 2nd and 3rd chromosomal deficiency stocks (deficiency/balancer) uncovering 70% to 80% of the two autosomes, or about 60% of the Drosophila genome, were crossed with ntf-2P7/FM7 females (Table 1 top). In the next generation the number of surviving ntf-2 males also carrying a deletion was counted and the survivors monitored for their eye phenotype. For our screen we set up 136 individual crosses, many of them repeatedly in order to obtain at least 150 adult progeny to screen for the eye phenotype. We only identified deletions and rearrangements in four regions of the second chromosome that showed suppression (Table 1). The suppression was confirmed using a second ntf-2 (P49) allele.

DNA rearrangements affecting regions 22A and 60B-D showed different results with the two ntf-2 alleles tested and were not pursued. Df(2l)cl-h2 (25D-F) appeared to rescue both viability and the eye phenotype, but the gene responsible for the suppression could not be identified. Df(2L)GpdhA (25D-26A) rescued the eye phenotype, but not viability. To identify the gene(s) responsible for the suppression of the eye phenotype we tested mutations in several genes that are uncovered by Df(2L)GpdhA and are available from the Drosophila stock center.

Mutants in one gene, chickadee (chic), encoding Drosophila Profilin [25], uncovered by Df(2L)GpdhA, showed suppression of the ntf-2 eye phenotype. We tested several loss-of-function alleles of chic, including a complete lethal null allele (chic221) and other partially viable alleles, that are either female, or male and female sterile. All chic alleles were crossed with at least 2 ntf-2 alleles, except chic221 that was tested with 4 different ntf-2 alleles. The suppression of the eye phenotype was observed in all crosses and the majority of surviving trans-heterozygous males showed suppression of the ntf-2 eye phenotype, restoration of wild-type eyes (Fig. 1A). The percent of males with wild-type eyes varied in different allele combinations. Surprisingly, the eye phenotype was usually either small or wild-type and virtually no eyes of intermediate size were observed.

Mutations in chic (Profilin) affect nuclear export

To investigate the cause underlying the suppression of the ntf-2 phenotype and possible function of Profilin in nuclear transport, we used a reporter gene approach. We assayed nuclear transport using UAS-NLS-NES reporter constructs C-terminally tagged with GFP in different mutant backgrounds. One construct contains a wild-type NLS and NES (UAS-NLS-NES-GFP), the other a wild-type NLS but a mutant NES that is not recognized by the nuclear export machinery (UAS-NLS-NESP12-GFP; [16,26]). Expression of the transgenes was driven by a heatshock-GAL4 driver, and the distribution of GFP was analyzed in salivary glands. As previously shown, the activity of the wild-type NES is stronger then that of the NLS [26]. Hence, in wild-type the NLS-NES-GFP is usually localized in the cytoplasm (Fig. 3B). In contrast, NLS-NESP12-GFP has impaired nuclear export and strongly accumulates in nuclei (Fig. 3A). In homozygous chic01320 and the hetero-allelic combination chic2/chic221, the distribution of the GFP reporter is altered. In contrast to the cytoplasmic distribution of NLS-NES-GFP in wild-type, in the chic mutant salivary glands the GFP reporter is found predominantly in the nucleus (Fig. 3D,F). The localization of NLS-NESP12-GFP is similar in chic and wild-type (Fig. 3C,E), indicating that NLS-mediated import is not affected.

thumbnailFigure 3. Chic impairs nuclear export. Localization of GFP reporter appended with wild-type NLS and NES (B, D, F) or mutant NESP12 (A, C, E) in salivary gland cells. In wild-type (A) and chic mutants (C, E) NLS-NESP12-GFP is predominantly localized to the nucleus. In wild-type salivary glands NLS-NES+-GFP is mostly found in the cytoplasm (B). In contrast, in chic mutants, chic2/chic221 and chic01320 homozygotes, NLS-NES+-GFP is predominantly nuclear (D, F).

Sd (RanGAP) suppresses the ntf-2 phenotype

RanGAP functions in nuclear export of cargo and in Sd-RanGAP mutants the NLS-NES-GFP is found in the nucleus and NLS-NESP12-GFP is distributed the same as in wild-type [16,19]. This failure of exporting NLS-NES-GFP in Sd-RanGAP mutants is reminiscent of what we observe in chic alleles (Fig. 3).

Given the similarity in nuclear export phenotypes in Sd and chic mutants, we tested if Sd would also suppress the eye phenotype of ntf-2 alleles. We crossed the Sd (Sd72, [27]) chromosome with two ntf-2 alleles and found that the eye phenotype was suppressed in both of them. To confirm that the SD-RanGAP mutation, and not other genes on the Sd chromosome, is responsible for the suppression, we expressed a mutated Sd-RanGAP transgene (UAS-Sd-RanGAP12A-6; [16]) driven by hsp70-GAL4 or arm-GAL4 in ntf-2P7 and ntf-2P49 males and observed similar levels of suppression as seen with Sd72 (Table 1).

The genetic interaction between Sd-RanGAP and ntf-2 is not altogether surprising because both RanGAP and NTF2 are known to function in the formation of the RanGTP-GDP gradient. To investigate if RanGAP is affected in ntf-2 mutants we studied the distribution of RanGAP in eye discs.

In wild-type cells Ran-Gap is present in low levels in the cytoplasm and forms a clearly visible punctuated circle around the nucleus (Fig. 2A,D). The punctuate pattern of RanGAP is due to its association with nuclear pores [17,18]. This distribution is different in ntf-2 discs. Patches of cells are observed in which RanGAP aggregates in small or large clumps near the nuclei (Fig 2E,F), but in other cells the distribution of the protein looks relatively normal. This observation suggests, that the clumping of RanGAP is an effect of the abnormal organization of the cells within the ntf-2 disc. The cells with clumped RanGAP are usually in close proximity to cells with high levels of F-actin.

Lack of Profilin also affects RanGAP distribution

To investigate a connection between Profilin, RanGAP, and actin, we next asked whether the function of Profilin or actin polymerization might have an effect on RanGAP localization. We generated clones in eye discs of null alleles of the two genes chic (chic221) and, as a control, act up (acuE636). Acu participates in actin de-polymerization, the opposite function of Profilin [28].

In chic clones RanGAP protein is increased around the nuclear envelope and its distribution is uneven and patchy on the nuclear envelope surface (Fig. 4D arrows). In wild-type even, punctuated circles are observed (arrowheads). This abnormal distribution was found in 100% of examined clones (more then 50). In chic clones the level of F-actin was reduced as previously shown [28]. In the acu control clones high levels of F-actin are detected as expected (results not shown, [28]), but the distribution of RanGAP is not significantly changed (Fig. 4E).

thumbnailFigure 4. Localization of RanGAP on chic nuclear envelopes is irregular. chic221 eye disc clones show only minor changes in distribution of (A) Lamin (red) and (B) Nups (red). But RanGAP localization (C, D red) is strongly altered appearing more clustered and patchy (arrows) than in wild-type cells, where RanGAP forms uniform dotted rings around nuclei (arrowheads). act up loss of function does not affect RanGAP (red) in acuE636 eye disc clones (E). Mutant clones are marked by the absence of green. The borders of some clones are highlighted with dashed lines.

To test whether this patchy protein distribution of RanGAP on nuclear pores of chic22 cells is caused by problems in nuclear envelope assembly, we analyzed the distribution of Lamin and nuclear pore proteins (Nups) in chic221 clones (Fig. 4A,B). The distribution of both Lamin and Nups is affected in about 30% of clones. This is likely due to the mislocalization of RanGAP. It has been shown previously that RanGTPase functions in nuclear pore and envelope formation [22,23].

The staining experiments show higher levels of RanGAP around nuclei in chic eye disc clones. We investigated if this is due to overall higher levels of RanGAP in mutant cells. The chic alleles used in the clonal analysis are homozygous lethal therefore we prepared extracts from wild-type and mutant 1st instar larvae. In western blots from extracts of chic221 (lethal at first and early second larval instar) and chic01320 (viable and female sterile) larvae, the amount of RanGAP present in mutants is not dramatically changed compared to wild-type (results not shown). This may be because RanGAP and Profilin are maternally contributed and therefore at these early stages a difference in levels is not detected. We then dissected eye-antennal discs from normal larvae and larvae with chic clones (see experiments shown in Figure 4). The dissected tissues also contained some brain material because eye-antennal discs are next to the brain hemispheres and are difficult to separate. In two separate experiments we see an increase of 30–50% in the intensity of the RanGAP band in extracts from discs carrying chic221 somatic clones compared to normal eye discs from chic221/+ larvae. The intensity of the RanGAP bands were normalized to that of the control Bic-D band and equals 2.6 for discs with clones and 1.8 for wild-type discs (Fig. 5).

thumbnailFigure 5. RanGAP levels in chic mutants. Western blot of extracts from eye-antennal discs and some brain lobes. Compare the amount of RanGAP in chic221/+ tissue without somatic clones and chic221/+ with somatic clones; chic null tissue, represents 10–30% of eye discs cells and 2–5% of antennae and brain cells. The extracts were loaded at two different concentrations to allow comparison of levels of protein.

Discussion

NTF2 regulates nuclear import in every cell of the organism. Some ntf-2 alleles can produce male sterile but female fertile adults, that all have a striking eye phenotype. This phenotype appears to be caused by lower levels of NTF2 and not an altered protein since alleles showing the eye phenotype have a P-element insertion in the 5' UTR [11,12,24]. The difference in response of tissues to the lower levels of NTF2 is surprising. For instance, the wild-type and mutant antennal discs and antennae appear normal (Fig. 1A arrows), while the eye disc of the mutant has a strongly modified appearance.

We used partial loss of function ntf-2 alleles to screen for dominant suppressors of the eye phenotype. Using deletions uncovering more then half of the genome, we identified four regions that can function in the control of the RanGTP-RanGDP balance. While unable to identify specific genes responsible for the dominant interaction in three of these regions, we did find one suppressor, chic, encoding Profilin.

We tested several alleles of chic with several alleles of ntf-2 and observed suppression of the eye phenotype in all combinations. Reducing Profilin suppresses the effects of lower than normal amounts of NTF2. In all cases we observe either small eyes or completely restored eyes similar to wild-type. We detected no intermediate phenotypes, suggesting that a threshold level exists for each protein.

The ntf-2 eye phenotype can also be suppressed by a gain of function mutation in Sd (RanGAP). RanGAP regulates the Ran-GTP-to-Ran-GDP balance and is involved in nuclear export. NTF2 controls nuclear import of RanGDP and thus the nuclear trafficking of cargo. Because of the low viability of ntf-2 mutants we cannot obtain ntf-2 flies expressing a driver and NLS-NES-GFP reporter genes. Hence, the nuclear transport phenotype of ntf-2 alleles cannot be determined as we did for chic mutants, and was done for Sd mutants [16]. Previous investigations in several organisms indicate that in ntf-2 mutants, the Ran gradient or its formation is changed affecting cargo import [13-15].

Why lowering the level of Profilin that functions in actin polymerization suppresses the ntf-2 phenotype is not immediately apparent, but there are several possible explanations. Lower levels of Profilin may result in reduction of the abnormal actin polymerization in ntf-2 mutant eye discs (see Fig. 2). But our finding that the ntf-2 eye phenotype is suppressed by the over-expression of RanGAP suggests that the disorganized appearance of F-actin is an indirect result of abnormal nuclear trafficking. Therefore lowering Profilin seems to also affect the abnormal nuclear trafficking inherent to ntf-2 eye discs. This supposition is bolstered by our finding that Profilin is essential for normal nuclear export. Our results are consistent with F-actin being regulated by nuclear transport, and in turn, Profilin and Actin controlling aspects of nuclear trafficking.

Unpolymerized actin is found on NPC-attached nucleoplasmic filaments. It has been shown to function in the nuclear export of proteins and RNA [29]. Unpolymerized actin also associates with Profilin and is exported from the nuclei in a Ran-dependant manner [30]. We do not think that these processes have a primary role in our mutant phenotypes because staining of ntf-2 eye discs and chic clones with anti-actin antibody display no obvious difference in the distribution of non-polymerized actin (results not shown). Nevertheless, these processes have to be considered as part of the crosstalk between the actin cytockeleton and Ran-mediated nuclear trafficking.

That Profilin controls the localization of RanGAP is evident from the abnormal distribution of the protein in chic clones. The uneven distribution of RanGAP at the nuclear envelope is not due simply to higher levels of protein. In Sd transgenic lines that express wild-type or mutant RanGAP, higher levels of protein are found uniformly distributed in the cytoplasm and nucleus [19]. In chic mutant cells, the RanGAP level is about doubled, but the protein distribution is different than that observed in the over-expressing lines.

Methods

Drosophila stocks and suppressor screen

The w118 stock was used as wild-type stock (WT) in all experiments. All fly stocks were obtained from the Bloomington Stock Center [24], except UAS-RanGAP12A-6, UAS-NLS-NESP12-GFP and UAS-NLS-NES-GFP transgenic flies that were sent by Barry Ganetzky and Edwin Chan [16,31].

The following ntf-2 stocks were used for suppression experiments: w ntf-2P7/FM7i, P{w [+mC]=ActGFP}JMR3, y[1] f[1], w ntf-2P49/FM7c, w ntf-2G0086/FM7c, ntf-2G0337/FM7c. All alleles carry different P-element insertions in the 5' UTR of the gene, 102–112 bases upstream from the start of transcription that cause reduced viability and the eye phenotype (see Table 1, [11,12,24]).

Suppressor screen

To identify suppressors of ntf-2, ntf-2P7/FM7 and ntf-2P49/FM7 virgins were crossed with deficiency males. In the next generation the ntf-2 males not carrying a balancer chromosomes were analyzed. We tested 66 deficiencies on the second chromosome and about 70 deficiencies on the third chromosome, and about 25 existing mutations and P-element insertions mapping to the 22–30 cytological region of chromosome two. All were obtained from the Bloomington Stock center.

Alleles of chickadee, chicK1332, chic221, chic01320, chic2, In(2R)SD72, In(2R)NS, Sd[72], GAL4 drivers and Balancer chromosomes were obtained from Bloomington Stock Center. UAS-RanGAP12A-6, UAS-NLS-NESP12-GFP and UAS-NLS-NES-GFP transgenic flies were kindly sent to us by Barry Ganetzky and Edwin Chan [16].

Nuclear transport assay

To identify mutant animals at larval stages we used the GFP marked balancers FM7i P{w [+mC]=ActGFP}JMR3 and CyO, P{w [+mW.hs]=Ubi-GFP.S65T}PAD1. Mutant 2nd instar larvae were distinguished from their heterozygous siblings by the absence of green fluorescent protein. The expression of GFP-reporter genes were induced by 1 h incubation at 36°C. To diminish the possible effect of heating on nuclear trafficking we waited for 24 h before dissecting and fixing salivary glands. Localization of GFP was observed and fluorescence images obtained using a Zeizz Axioplan 2 (Zeiss) microscope and Image Pro Plus software.

Eye disc clones

FRT40 chic221 and FRT40 acuE636 chromosomes were obtained from Jessica Treisman. Clones in eye discs were generated as described [28].

Antibodies

Rabbit polyclonal antibody against Drosophila RanGAP was provided by Sinthia Stabber and Barry Ganetzki [16], mab414 recognizing nucleoporins with FXF repeats [32,33] was obtained from BAbCO (Richmond, CA), and anti-Lamin antibody was a gift from Paul Fischer, Stonybrook. Western analysis was performed as previously described [16,17].

Immunostaining

For antibody staining 3rd instar larvae were inverted in phosphate-buffered saline (PBS) and immediately fixed in 4% paraformaldehyde in PBS with 2% DMSO for 40 min and washed several times in PBT (PBS, 0.1% Triton X-100). Then tissues were blocked for 2 hours in PBS containing 1% bovine serum albumin (BSA) and 1% Triton X-100. Antibody incubations were done in PBT with 1% BSA overnight at 4°C. Anti-RanGAP rabbit serum was used at a 1:1000 or 1:800 dilution, and anti-RanGAP-1 monoclonal antibody was used at a 1:400 dilution, mab414 was used at a 1:300 dilution, anti-Lamin – 1:30. Secondary antibodies were used at a dilution of 1:500. Cy-3 conjugated anti-mouse and anti-rabbit IgG were purchased from Jackson Immuno Research Laboratories Inc. (West Grove, PA). F-actin was visualized by incubation with Alexa488 Phalloidin at 1:80 for 2 hours. DNA was stained using Hoechst 33258 (Molecular Probes, Eugene, OR). Samples were mounted in Vectashield (Vector Laboratories) and examined with a Leica DM IRBE (Leica) laser scanning confocal microscope. The images were analyzed with Leica Microsystems software and further processed using Adobe PhotoShop.

Authors' contributions

SM participated in the design of the study, and was involved in all experiments presented. RM carried out the suppressor screen and characterized chic as a suppressor of ntf-2. MD carried out the immunoassays. RS conceived the study, worked on the genetic screens. SM and RS wrote the paper. All authors read and approved the final manuscript.

Acknowledgements

We thank Sinthia Stabber, Edwin Chan, Barry Ganetzki, Jessica Treisman, and Paul Fischer for transgenic flies and antibodies. We are grateful to Ursula Stochaj, Frank Solomon, and Chris Rongo for helpful suggestion on the manuscript. We also thank Le Nguyen, Ananya Bhattacharya, Sarika Patel and Sini Abraham for initial work and technical help. This work was supported by a grant from the NIH and by the W. Horace Goldsmith Foundation.

References

  1. Weis K: Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle.

    Cell 2003, 112(4):441-451. PubMed Abstract | Publisher Full Text OpenURL

  2. Gorlich D, Kutay U: Transport between the cell nucleus and the cytoplasm.

    Annu Rev Cell Dev Biol 1999, 15:607-660. PubMed Abstract | Publisher Full Text OpenURL

  3. Bischoff FR, Ponstingl H: Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide.

    Proc Natl Acad Sci U S A 1991, 88(23):10830-10834. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  4. Ribbeck K, Lipowsky G, Kent HM, Stewart M, Gorlich D: NTF2 mediates nuclear import of Ran.

    Embo J 1998, 17(22):6587-6598. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  5. Paschal BM, Gerace L: Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.

    J Cell Biol 1995, 129(4):925-937. PubMed Abstract | Publisher Full Text OpenURL

  6. Moore MS, Blobel G: Purification of a Ran-interacting protein that is required for protein import into the nucleus.

    Proc Natl Acad Sci U S A 1994, 91(21):10212-10216. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  7. Smith A, Brownawell A, Macara IG: Nuclear import of Ran is mediated by the transport factor NTF2.

    Curr Biol 1998, 8(25):1403-1406. PubMed Abstract | Publisher Full Text OpenURL

  8. Gorlich D, Seewald MJ, Ribbeck K: Characterization of Ran-driven cargo transport and the RanGTPase system by kinetic measurements and computer simulation.

    Embo J 2003, 22(5):1088-1100. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  9. Corbett AH, Silver PA: The NTF2 gene encodes an essential, highly conserved protein that functions in nuclear transport in vivo.

    J Biol Chem 1996, 271(31):18477-18484. PubMed Abstract | Publisher Full Text OpenURL

  10. Steggerda SM, Black BE, Paschal BM: Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran.

    Mol Biol Cell 2000, 11(2):703-719. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  11. Bhattacharya A, Steward R: The Drosophila homolog of NTF-2, the nuclear transport factor-2, is essential for immune response.

    EMBO Rep 2002, 3(4):378-383. PubMed Abstract | Publisher Full Text OpenURL

  12. Peter A, Schottler P, Werner M, Beinert N, Dowe G, Burkert P, Mourkioti F, Dentzer L, He Y, Deak P, Benos PV, Gatt MK, Murphy L, Harris D, Barrell B, Ferraz C, Vidal S, Brun C, Demaille J, Cadieu E, Dreano S, Gloux S, Lelaure V, Mottier S, Galibert F, Borkova D, Minana B, Kafatos FC, Bolshakov S, Siden-Kiamos I, Papagiannakis G, Spanos L, Louis C, Madueno E, de Pablos B, Modolell J, Bucheton A, Callister D, Campbell L, Henderson NS, McMillan PJ, Salles C, Tait E, Valenti P, Saunders RD, Billaud A, Pachter L, Klapper R, Janning W, Glover DM, Ashburner M, Bellen HJ, Jackle H, Schafer U: Mapping and identification of essential gene functions on the X chromosome of Drosophila.

    EMBO Rep 2002, 3(1):34-38. PubMed Abstract | Publisher Full Text OpenURL

  13. Feldherr C, Akin D, Moore MS: The nuclear import factor p10 regulates the functional size of the nuclear pore complex during oogenesis.

    J Cell Sci 1998, 111 ( Pt 13):1889-1896. PubMed Abstract | Publisher Full Text OpenURL

  14. Hu W, Jans DA: Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2.

    J Biol Chem 1999, 274(22):15820-15827. PubMed Abstract | Publisher Full Text OpenURL

  15. Tachibana T, Hieda M, Sekimoto T, Yoneda Y: Exogenously injected nuclear import factor p10/NTF2 inhibits signal-mediated nuclear import and export of proteins in living cells.

    FEBS Lett 1996, 397(2-3):177-182. PubMed Abstract | Publisher Full Text OpenURL

  16. Kusano A, Staber C, Ganetzky B: Nuclear mislocalization of enzymatically active RanGAP causes segregation distortion in Drosophila.

    Dev Cell 2001, 1(3):351-361. PubMed Abstract | Publisher Full Text OpenURL

  17. Mahajan R, Delphin C, Guan T, Gerace L, Melchior F: A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.

    Cell 1997, 88(1):97-107. PubMed Abstract | Publisher Full Text OpenURL

  18. Matunis MJ, Coutavas E, Blobel G: A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

    J Cell Biol 1996, 135(6 Pt 1):1457-1470. PubMed Abstract | Publisher Full Text OpenURL

  19. Kusano A, Staber C, Ganetzky B: Segregation distortion induced by wild-type RanGAP in Drosophila.

    Proc Natl Acad Sci U S A 2002, 99(10):6866-6870. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  20. Ohba T, Nakamura M, Nishitani H, Nishimoto T: Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran.

    Science 1999, 284(5418):1356-1358. PubMed Abstract | Publisher Full Text OpenURL

  21. Wilde A, Lizarraga SB, Zhang L, Wiese C, Gliksman NR, Walczak CE, Zheng Y: Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities.

    Nat Cell Biol 2001, 3(3):221-227. PubMed Abstract | Publisher Full Text OpenURL

  22. Walther TC, Askjaer P, Gentzel M, Habermann A, Griffiths G, Wilm M, Mattaj IW, Hetzer M: RanGTP mediates nuclear pore complex assembly.

    Nature 2003, 424(6949):689-694. PubMed Abstract | Publisher Full Text OpenURL

  23. Zhang C, Goldberg MW, Moore WJ, Allen TD, Clarke PR: Concentration of Ran on chromatin induces decondensation, nuclear envelope formation and nuclear pore complex assembly.

    Eur J Cell Biol 2002, 81(11):623-633. PubMed Abstract | Publisher Full Text OpenURL

  24. The FlyBase C: The FlyBase database of the Drosophila genome projects and community literature.

    Nucleic Acids Research 2003, 31:172-175 http://flybase.org/. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  25. Cooley L, Verheyen E, Ayers K: chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis.

    Cell 1992, 69(1):173-184. PubMed Abstract | Publisher Full Text OpenURL

  26. Stade K, Ford CS, Guthrie C, Weis K: Exportin 1 (Crm1p) is an essential nuclear export factor.

    Cell 1997, 90(6):1041-1050. PubMed Abstract | Publisher Full Text OpenURL

  27. Powers PA, Ganetzky B: On the components of segregation distortion in Drosophila melanogaster. V. Molecular analysis of the Sd locus.

    Genetics 1991, 129(1):133-144. PubMed Abstract | Publisher Full Text OpenURL

  28. Benlali A, Draskovic I, Hazelett DJ, Treisman JE: act up controls actin polymerization to alter cell shape and restrict Hedgehog signaling in the Drosophila eye disc.

    Cell 2000, 101(3):271-281. PubMed Abstract | Publisher Full Text OpenURL

  29. Hofmann W, Reichart B, Ewald A, Muller E, Schmitt I, Stauber RH, Lottspeich F, Jockusch BM, Scheer U, Hauber J, Dabauvalle MC: Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

    J Cell Biol 2001, 152(5):895-910. PubMed Abstract | Publisher Full Text OpenURL

  30. Stuven T, Hartmann E, Gorlich D: Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes.

    Embo J 2003, 22(21):5928-5940. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  31. Kusano A, Staber C, Chan HY, Ganetzky B: Closing the (Ran)GAP on segregation distortion in Drosophila.

    Bioessays 2003, 25(2):108-115. PubMed Abstract | Publisher Full Text OpenURL

  32. Davis LI, Blobel G: Identification and characterization of a nuclear pore complex protein.

    Cell 1986, 45(5):699-709. PubMed Abstract | Publisher Full Text OpenURL

  33. Aris JP, Blobel G: Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins.

    J Cell Biol 1989, 108(6):2059-2067. PubMed Abstract | Publisher Full Text OpenURL