BACKGROUND
the lap2-emerin-man <dig>  protein family comprises a group of inner nuclear membrane and nucleoplasmic proteins  <cit>  with important functions in various cellular processes, including nuclear envelope architecture  <cit> , dna replication  <cit> , cell cycle control  <cit> , chromatin organization  <cit>  and the regulation of gene expression and signaling pathways .
all proteins in this family share the lem domain, a ~ <dig> amino acid long bi-helical motif, which binds the conserved metazoan chromatin-associated protein barrier-to-autointegration factor  .
besides the lem domain, different additional motifs and functional domains are present in lem proteins, such as a transmembrane domain , a carboxy-terminal winged-helix domain , a lem-like motif , and ankyrin repeats in ankyrin repeats and lem-domain containing proteins   <dig> and  <dig> .
ankle <dig> has several unique features among the lem protein family members.
it lacks a transmembrane domain and shuttles between the cytoplasm and the nucleoplasm, and it contains a c-terminal giy-yig-type endonuclease domain  <cit> .
the giy-yig domain is the hallmark of a subgroup of the homing endonuclease superfamily  <cit> , represented mostly by group i and ii introns, archaeal introns and inteins that catalyze their transfer within genomes by introducing strand breaks in intron- and intein-lacking sequences  <cit> .
we have previously shown that the giy-yig domain of ankle <dig> has nuclease catalytic activity that cuts plasmid dna in vitro and induces dna damage in vivo  <cit> .
based on the findings in c. elegans that an inactivating mutation in the worm ankle <dig> ortholog lem <dig> leads to hypersensitivity of the worm mutants to various types of dna damage, including ionizing radiation, uv-c light and dna crosslinking agents, ankle1/lem <dig> was proposed to be involved in dna damage repair pathways  <cit> .
embryos from irradiated lem- <dig> mutant worms also suffer from severe defects during cell division, such as chromosome mis-segregation and anaphase bridges  <cit> .
in mammals, however ankle <dig> functions may be highly redundant, as ankle <dig> knockout mice and cells are normal and did not show an impaired dna damage response  <cit> .
the mammalian genome contains two genes encoding proteins with a giy-yig nuclease domain, ankle <dig>  <cit>  and slx <dig>  encoding slx <dig> resolvase  <cit> .
resolvases are dna-structure specific nucleases that process and cleave holliday junctions  .
hjs are intermediate structures of covalently linked homologous chromosomes during meiosis or of sister chromatids during homologous recombination-mediated double strand break  repair  <cit> .
hjs need to be eliminated before the end of mitosis or meiosis to assure proper chromosome segregation and to preserve genome stability.
homologous recombination followed by hj resolution can result either in crossover  events that are obligatory at a certain frequency in meiotic cells to allow exchange of genomic information, or non-crossover  events, which are preferred in mitotic cells because they avoid the loss of heterozygosity, a high risk factor for the development of cancer  <cit> .
two major mechanisms are responsible for hj processing in mitotic cells: the preferred dissolution by the so-called bloom helicase btr  complex that results in ncos  <cit> , and the resolution of hjs by structure specific endonucleases, such as the slx <dig> complex  or the canonical hj resolvase gen <dig>  that can lead to cos and ncos .
because dissolution is favored over resolution in mitosis, the actions of resolvases are strictly regulated in a spatio-temporal manner  <cit> .
in human cells, eme <dig>  a subunit of the slx <dig> complex is phosphorylated in prometaphase to stimulate interactions and activation of the mus81-eme1-slx1-slx <dig> complex  <cit> .
this ensures that resolvases eliminate only hjs, which were not processed by the btr complex in s and g <dig> phase.
the activity of the human gen <dig> resolvase is restricted to mitosis by nuclear exclusion during interphase through a leucine-rich nuclear export signal   <cit> .
based on the evidence of unprocessed chromatin structures in c. elegans lem- <dig> mutants and the presence of a giy-yig nuclease domain in ankle <dig>  we hypothesized that ankle <dig> may also function in dna damage repair, probably redundantly with other nucleases, and its nucleo-cytoplasmic shuttling and cellular localization may be tightly controlled during the cell cycle and upon dna damage.
hence, we wanted to identify the domains and motifs in ankle <dig> involved in its nucleo-cytoplasmic shuttling and test whether dna damage may alter ankle1’s cellular localization.
we identified one canonical nuclear localization sequence  and one nuclear export sequence  in ankle <dig> that mediate its translocation into and out of the nucleus, respectively.
however, no changes in shuttling and/or cellular localization were found upon induction of dna damage or during the cell cycle.
RESULTS
nucleocytoplasmic shuttling of ankle <dig> is mediated by active transport mechanisms
in a previous study, we showed that ankle <dig> is primarily localized in the cytoplasm in hela and b-cell derived ramos cell lines, but it accumulated in the nucleus upon inhibition of nuclear export by leptomycin  <cit> .
this suggested that ankle <dig> shuttles between the cytoplasm and the nucleus.
in this study, we wanted to identify the domains in ankle <dig> that mediate its shuttling, and test, whether nucleo-cytoplasmic shuttling of ankle <dig> and its cellular localization change upon dna damage or during the cell cycle.
therefore we expressed ankle <dig> in osteosarcoma u2os cells, which have intact prb and p <dig> pathways  <cit>  and have frequently been used in studies investigating dna damage repair pathways, including resolvase-mediated repair .
in agreement with previous studies  <cit> , ankle <dig> ectopically expressed in u2os cells localizes predominantly in the cytoplasm and accumulates in the nucleus upon pharmacological inhibition of the crm1-dependent nuclear export using the drug leptomycin b .
in silico analysis of the ankle <dig> primary sequence predicted the existence of at least two nuclear localization sequences  and two nuclear export signals  , suggesting that shuttling of ankle <dig> may be mediated by active nuclear import and export pathways.fig.
<dig> ankle <dig> shuttles between nucleus and cytoplasm.
a schematic representation of ankle1’s domain organization depicting predicted ankyrin repeats, the lem domain and a giy-yig nuclease domain.
putative nuclear export sequences  and nuclear localization sequences , identified in silico are indicated.
b immuno-fluorescence analysis of ectopic ankle1-v <dig> in u2os cells without or following a 3 h treatment with  <dig> nm leptomycin b, an inhibitor of crm1-mediated export.
cells were stained with antibodies to v <dig>  and dna with dapi.
scale bar: 10 μm
in order to get a first hint whether any of the nlss and ness are responsible for nucleo-cytoplasmic shuttling of ankle <dig>  we generated a series of truncated ankle <dig> constructs c-terminally fused to green fluorescence protein , and tested their subcellular localization by confocal fluorescence microscopy.
using this approach, we also wanted to test whether specific protein domains other than transport signal sequences influence ankle <dig> localization.
these include the ankyrin repeats, well known protein interaction domains found in many proteins  <cit> , the lem domain, known to mediate binding to chromatin via barrier-to-autointegration factor   <cit> , and the giy-yig domain, mediating nuclease activity  <cit>  .
ankle11– <dig>  which lacks the c-terminal domain including the giy-yig motif and the two nlss, is effectively exported to the cytoplasm in both u2os and hela cells .
additional deletion of the lem domain  did not alter the predominant cytoplasmic localization of the construct in the absence of leptomycin b. hence we concluded that neither the lem domain nor the giy-yig domain affect nuclear export .
the construct lacking the n-terminal ankyrin repeats   was efficiently excluded from the nucleus in both u2os and hela cells, indicating that neither ankyrin repeats nor nes <dig> are essential for nuclear export and/or cytoplasmic localization.
in agreement with this hypothesis, ankle11– <dig> containing only nes <dig> was not efficiently exported from the nucleus , while both ankle1158– <dig> and ankle1158– <dig> encompassing the central region containing only nes <dig> with or without the lem domain, respectively, localize exclusively to the cytoplasm .
in addition, leptomycin b-mediated inhibition of nuclear export caused efficient accumulation of most constructs in the nucleus, suggesting that a region including nes <dig> is sufficient for ankle <dig> export.
altogether these data suggest that ankle <dig> is actively exported from the nucleus predominantly through a nes2-mediated mechanism.
we assume that also its nuclear import is mediated by an active nls-mediated mechanism based on the following observations.
first, ankle1158– <dig>  containing nes <dig> and both predicted import sequences nls <dig> and nls <dig> efficiently accumulates in the nucleus in the presence of leptomycin b like the wild-type protein, although its molecular weight  does not allow efficient nuclear translocation by passive diffusion through nuclear pores  <cit> .
secondly, a 51 kda large construct containing the nuclear localization signal  is exclusively targeted to the nucleus , despite its size would allow some passive diffusion out of the nucleus.
we therefore conclude that ankle <dig> is shuttling between the nucleus and the cytoplasm through active import and export mechanisms, but its nuclear export seems to overrule nuclear transport, as at steady-state, ankle <dig> is not detected inside the nucleus.fig.
<dig> localization of ankle <dig> fragments containing different domains and export and import signals.
localization of gfp-tagged ankle <dig> truncation constructs ectopically expressed in u2os  and hela  cells was determined by confocal fluorescence microscopy.
molecular weights and schematic representations of domain organization of respective truncation protein constructs are indicated.
cells were fixed after 3 h of mock or leptomycin b treatment.
dna was counterstained with dapi.
scale bar: 10 μm
mutation analyses identify nes <dig> and nls <dig> as major transport-mediating signals
our experiments using truncated ankle <dig> constructs indicated that nls <dig> and nes <dig> are the predominant regulators of active import and export, respectively.
to test whether this hypothesis holds true also in the context of the full length protein, we analyzed the localization of full length ankle <dig> constructs carrying point mutations at conserved residues within the nuclear transport sequences nes <dig>  nes <dig>  nls <dig> and nls <dig> .
furthermore, in order to avoid potential effects of the bulky gfp tag we used the smaller v5-tag instead.
mutation of nes <dig>  caused only a subtle impairment of nuclear export , as indicated by the minor increase in the ratio of nucleoplasmic over cytoplasmic signal intensities of the mutant compared to wild-type control .
in contrast, substitution of conserved leucine residues within the nes <dig> impaired nuclear export of ankle <dig> and caused a significant increase in the steady-state fraction of the protein in the nucleus compared to the control .
given that nuclear export is predominant in defining the steady state localization of ankle <dig>  it was only possible to examine the consequences of nls <dig> and nls <dig> mutations following leptomycin b-mediated inhibition of nuclear export.
mutation of the positively charged residues within nls <dig> did not affect ankle1’s accumulation in the nucleus  under this condition, whereas mutation of nls <dig> impaired nuclear import nearly completely .
in summary, we concluded that nes <dig> and nls <dig> are the predominant signals mediating active nucleo-cytoplasmic shuttling of ankle <dig> fig.
<dig> mutation analyses identify nes <dig> and nls <dig> as the predominant sequences controlling nucleo-cytoplasmic shuttling of ankle <dig>  a, b, d, e u2os cells were transiently transfected with ankle1-v <dig> carrying point mutations in nls or nes sequences and either mock-treated or treated with leptomycin b for 3 h and processed for confocal immunofluorescence analyses using antibodies to v <dig> and dapi to detect dna.
scale bars: 10 μm.
c mean fluorescence intensities in nuclei and cytoplasm of cells expressing wild-type ankle1-v <dig>  ankle1-nes1mut-v <dig> or ankle1-nes2mut-v <dig> were measured in original unprocessed digital images prior to contrast/brightness adjustment and nucleus to cytoplasm signal ratios were calculated.
data were obtained from three independent experiments and analyzed using student’s t-test.
ankle1-nes <dig>  p =  <dig> ; ankle1-nes <dig>  p =  <dig> e-21; n = 50; 15– <dig> cells each from three independent experiments
in support of this, we also show by fusing wild-type and mutated versions of nls <dig> and nes <dig> sequences to gfp, that the wild-type, but not mutated sequences are sufficient to mediate active transport of heterologous proteins.
wild-type nes2-gfp, but not the mutated construct was efficiently excluded from the nucleus .
wild-type nls <dig> sequence mediated efficient accumulation of the fusion construct in the nucleus, whereas the mutated version was distributed throughout the cell .fig.
<dig> nes <dig> and nls <dig> are functional export and import signals in heterologous reporter constructs.
u2os cells were transiently transfected with gfp constructs fused to the wild-type or mutated versions of nes <dig> or nls <dig>  fixed and prepared for fluorescence microscopy .
representative images of at least three independent experiments are shown.
scale bars: 10 μm.
b, d mean fluorescence intensities in nuclei and cytoplasm of gfp-nes2wt, gfp-nes2mut , gfp-nls2wt or gfp-nls2mut  transfected cells were measured in original unprocessed digital images prior to contrast/brightness adjustment, and nucleus to cytoplasm signal ratios were calculated.
data were obtained from three independent experiments and analyses were done using student’s t-test.
gfp-nes2mut, p =  <dig> e-30; gfp-nls2mut, p =  <dig> e-18; n = 43; 13– <dig> cells each from three independent experiments
overall we show that ankle <dig> localization is determined predominantly by the activity of two transport signals, a nuclear export signal in the middle of the polypeptide and a c-terminal nuclear localization signal.
ankle <dig> does not change localization upon dna damage and during mitosis
it was previously reported that a c. elegans mutant for lem- <dig>  the worm ortholog of mammalian ankle <dig>  is hypersensitive to dna damage-causing agents  <cit> .
furthermore, ankle <dig> may be functionally related to slx <dig>  the only other known giy-yig-type endonuclease encoded in the mammalian genome, which is involved in homologous recombination-mediated repair of dna double strand breaks in a cell cycle-regulated manner.
thus, we speculated that ankle <dig> may have a similar function in dna damage repair and set out to test whether ankle <dig> may transiently accumulate in the nucleus and/or on chromatin upon induction of the dna damage response signaling or during the cell cycle.
however, as long-term expression of wild-type ankle <dig> causes cell death , we generated a stable u2os cell line ectopically expressing an endonuclease-defective, gfp-tagged version of ankle <dig> to address ankle <dig> dynamics during the cell cycle and upon induction of dna damage.
the catalytically dead ankle <dig> mutant shuttles between the cytoplasm and the nucleoplasm and has a predominant cytoplasmic localization at steady state like the wild-type protein .
however, unlike for wild-type ankle <dig>  leptomycin b-dependent accumulation of the ankle <dig> mutant in the nucleus did not induce dna damage .
to investigate potential changes in ankle <dig> localization upon induced dna damage, we treated cells with various chemical compounds, which cause different types of dna damage and elicite different dna repair pathways: bleomycin, a radio-mimetic drug inducing double strand breaks and mitomycin c, creating dna crosslinks, which both are mainly repaired by non homologous end joining  or hr, and uv irradiation that triggers the nucleotide excision repair  pathway .
drugs were applied for short time periods at high concentration to study immediate dna damage response  or at low dosage over a prolonged time period to cause constant dna damage to allow activation of downstream dna damage response signaling events .
however, although the different types of treatments induced dna damage response, as shown by the upregulation of the γh2a.x marker compared to untreated control samples , we did not observe a  change in the localization of ankle <dig> nuclease-dead mutant .
hence, ankle <dig> localization may not be affected upon induction of dna damage response signaling, or the catalytically dead mutant is unable to respond to induction of dna damage signaling.fig.
<dig> ankle <dig> does not relocalize to the nucleus in response to dna damage.
u2os cells stably expressing a catalytically inactive gfp-ankle version were treated with the dna damaging agents bleomycin or mitomycin c for 4 h or 48 h using high and low dosages, respectively: 3 μg/ml bleomycin, 1 μg/ml mitomycin c for 4 h , 1 μg/ml bleomycin or  <dig>  μg/ml mitomycin c for 48 h .
fixed cells were imaged using confocal microscopy and representative images of at least three independent experiments are shown.
scale bars: 10 μm
next we tested whether ankle <dig>  like the mammalian resolvases, may serve in dna repair pathways during late g <dig> or m phase of the cell cycle, as shown for the resolvases mus <dig>  slx <dig> and gen <dig>  <cit> .
we first analyzed ankle <dig> localization throughout the cell cycle and focused in particular on the potential association of ankle <dig> with condensed chromosomes during mitosis.
ankle <dig> is uniformly distributed throughout the cytoplasm in different stages of mitosis, both in fixed  and live cells with  or without dna damage induction .
unrepaired dna damage and persistent hjs were shown to lead to defects in chromosomal segregation during anaphase  <cit> , visible as small dna fibers connecting separated chromatids .
resolvases are known to be activated upon such mitotic defects and to localize to these anaphase bridges  <cit>  in order to resolve hjs and to allow mitotic progression without chromosomal mis-segregation.
we tested whether ankle <dig> shows a similar re-localization to anaphase bridges following treatment of cells with the replication inhibitor hydroxyurea to increase the frequency of such mitotic defects.
screening mitotic figures in three independent experiments did not reveal re-localization and/or accumulation of ankle1-dead mutants to anaphase bridges under the given experimental setup .fig.
<dig> ankle <dig> does not accumulate on mitotic dna bridges induced by hydroxyurea treatment.
u2os cells stably overexpressing inactive gfp-ankle <dig> were grown in the presence of 100 μm hydroxyurea for 18 h. cells were fixed and processed for confocal fluorescence microscopy.
representative image out of at least  <dig> mitotic cells from three independent experiments is shown.
arrows show dapi stained dna bridges.
scale bar: 5 μm
discussion
in this study we show that ankle <dig> endonuclease shuttles between the cytoplasm and the nucleus by an active nls-mediated nuclear import and a nes-mediated nuclear export, despite its predominant steady-state localization in the cytoplasm.
using various ankle <dig> deletion constructs and full length ankle <dig> with mutated, non-functional transport signals, we find that nucleo-cytoplasmic shuttling is predominantly achieved by the concerted actions of a c-terminal monopartite nls sequence  <cit>  and a canonical rev-type nes sequence  <cit>  in the central region of ankle <dig> polypeptide.
as ankle <dig> efficiently accumulates in the nucleus upon treatment with leptomycin b, a specific inhibitor of crm <dig>  <cit> , ankle <dig> is likely excluded from the nucleoplasm via a crm1-dependent nuclear export.
in silico analysis predicted two potential nuclear export sequences fitting the highly conserved leucine-rich nes sequence motif , one located within the ankyrin repeats of ankle <dig>  the other in the central region between the ankyrin repeats and the lem domain.
experimental testing showed that only the latter was sufficient to mediate efficient nuclear export of tested ankle <dig> constructs .
similarly, among the two predicted canonical monopartite nlss present in ankle <dig> polypeptide, nls <dig> at the very c-terminus was found to mediate nuclear import.
several observations speak in favor of an active nls- and nes-mediated transport of ankle <dig>  rather than a so-called “piggy-back” mechanism in which ankle <dig> is co-transported with other proteins: first, wild-type ankle <dig> but not ankle <dig> mutants with a mutated nonfunctional nes <dig> sequence were exported from the nucleus .
second, ankle <dig> mutants with a mutated nonfunctional nls <dig> sequence, unlike wild-type protein, were not imported into the nucleus following leptomycin b treatment .
third, both nls <dig> and nes <dig> sequences were able to mediate effective import and export, respectively of a heterologous protein .
despite the presence of an active nls sequence, ankle1’s steady state localization is predominantly cytoplasmic.
in fact we were unable to detect any signal above background in the nucleus by fluorescence microscopy.
this observation raises the question, whether additional factors may be involved favoring nuclear export over import or whether the predominant cytoplasmic localization is merely a consequence of a tightly regulated balance between import and export rates.
potential additional mechanisms involved in the regulation of ankle <dig> nucleo-cytoplasmic shuttling may involve posttranslational modifications of ankle <dig> or specific retention in the cytoplasm by interaction with cytoskeletal components, but currently there is no evidence for any of these pathways being involved.
what may be the physiological relevance of nucleo-cytoplasmic shuttling of ankle1?
constant shuttling of ankle <dig> across the nuclear envelope, while primarily localizing in the cytoplasm may prevent accidental damage in the genome caused by ankle1’s endonuclease activity.
this hypothesis is supported by previous findings showing that leptomycin b-mediated accumulation of ankle <dig> in the nucleus causes dna cleavage and cell death  <cit> .
this hypothesis also predicts that the localization of ankle <dig> has to be regulated in a tightly controlled manner dependent on dna damage and/or the cell cycle as shown for other nucleases:
dnasei, a nuclease involved in apoptosis, is excluded from the nucleus by association with cytoplasmic actin, and association is stabilized by cofilin and disrupted by n-gelsolin  <cit> .
a tight regulation of nucleases was also reported for hj-processing resolvases, including slx <dig>  the only other giy-yig domain-containing protein besides ankle <dig> in the human genome  <cit> .
dissolution of hjs by the btr complex is the preferred mechanism in mitotic cells, because it avoids co formation  <cit> , but hjs, which could not be processed or escaped the btr complex-mediated repair can be resolved by one of the three structure specific endonucleases: slx1-slx <dig>  mus81-eme <dig>  or gen <dig>  in order to allow the preferred processing of hjs by btr and use resolvases only as a backup mechanism in g <dig> and m-phase of the cell cycle, resolvase activity is tightly regulated throughout the cell cycle.
slx <dig> is active only in a complex with slx <dig>  slx <dig> forms homodimers in g <dig> and s-phase of the cell cycle  <cit> , and formation of the active slx <dig> complex  is only promoted in prometaphase by phosphorylation of eme <dig> by cyclin dependent kinases   <cit> .
gen <dig> is regulated at the level of nuclear exclusion via a leucine-rich nes sequence  <cit> , assuring that only breakdown of the nuclear envelope during mitosis allows gen <dig> to access unresolved dna bridges.
based on these recently reported findings on the regulation of resolvases we tested whether ankle <dig> localization may be changed transiently upon inducing dna damage or during the cell cycle.
however neither long-term treatment at low doses nor short treatments with higher doses of mitomycin c, uv-c and bleomycin changed ankle <dig> localization.
similarly, ankle <dig> did not change its localization during the cell cycle and did not associate with anaphase bridges  <cit>  in mitotic cells treated with hydroxyurea.
one caveat of these studies is that we had to use a catalytically inactive ankle <dig> mutant containing mutations in its giy-yig domain, as the wild-type protein causes cell death and precluded cell cycle dependent analyses.
as the catalytically dead mutant showed the same nucleo-cytoplasmic shuttling and steady state cytoplasmic localization, we consider it unlikely that the mutation in the giy-yig motive affects its cellular localization.
therefore, we concluded that ankle <dig> localization is neither affected by activated dna damage response signaling nor cell cycle stages in u2os cells.
alternatively the high redundancy of hj processing pathways may obstruct the analysis of specific ankle <dig> functions in hj processing under these conditions.
although data obtained in c. elegans  <cit>  and recent reports on a potential linkage of single nucleotide polymorphisms in the human ankle <dig> gene with an increased risk for certain cancers  <cit>  are consistent with a function of ankle <dig> in dna damage response pathways, we cannot exclude that it is involved in other cellular processes.
in this study, we elucidated one level in the regulation of ankle <dig> localization by identifying active nls and nes sequences.
CONCLUSIONS
this study identifies a centrally located rev-type crm1-dependent nes in the ankle <dig> polypeptide and a c-terminal canonical mono-partite nls, which together mediate shuttling of ankle <dig> between the cytoplasm and the nucleus and maintain a predominantly cytoplasmic localization at steady state.
induction of dna damage response signaling did not affect cellular localization or chromatin association of ankle <dig> leaving it unclear, if and how ankle <dig> localization may be regulated upon induction of dna damage response signaling.

 the lap2-emerin-man protein family comprises a group of inner nuclear membrane and nucleoplasmic proteins with important functions in various cellular processes, including nuclear envelope architecture, dna replication, cell cycle control, chromatin organization and the regulation of gene expression and signaling pathways. all proteins in this family share the lem domain, a ~ amino acid long bi-helical motif, which binds the conserved metazoan chromatin-associated protein barrier-to-autointegration factor. besides the lem domain, different additional motifs and functional domains are present in lem proteins, such as a transmembrane domain, a carboxy-terminal winged-helix domain, a lem-like motif, and ankyrin repeats in ankyrin repeats and lem-domain containing proteins and. ankle has several unique features among the lem protein family members. it lacks a transmembrane domain and shuttles between the cytoplasm and the nucleoplasm, and it contains a c-terminal giy-yig-type endonuclease domain. the giy-yig domain is the hallmark of a subgroup of the homing endonuclease superfamily, represented mostly by group i and ii introns, archaeal introns and inteins that catalyze their transfer within genomes by introducing strand breaks in intron- and intein-lacking sequences. we have previously shown that the giy-yig domain of ankle has nuclease catalytic activity that cuts plasmid dna in vitro and induces dna damage in vivo. based on the findings in c. elegans that an inactivating mutation in the worm ankle ortholog lem leads to hypersensitivity of the worm mutants to various types of dna damage, including ionizing radiation, uv-c light and dna crosslinking agents, ankle1/lem was proposed to be involved in dna damage repair pathways. embryos from irradiated lem- mutant worms also suffer from severe defects during cell division, such as chromosome mis-segregation and anaphase bridges. in mammals, however ankle functions may be highly redundant, as ankle knockout mice and cells are normal and did not show an impaired dna damage response. the mammalian genome contains two genes encoding proteins with a giy-yig nuclease domain, ankle and slx encoding slx resolvase. resolvases are dna-structure specific nucleases that process and cleave holliday junctions. hjs are intermediate structures of covalently linked homologous chromosomes during meiosis or of sister chromatids during homologous recombination-mediated double strand break repair. hjs need to be eliminated before the end of mitosis or meiosis to assure proper chromosome segregation and to preserve genome stability. homologous recombination followed by hj resolution can result either in crossover events that are obligatory at a certain frequency in meiotic cells to allow exchange of genomic information, or non-crossover events, which are preferred in mitotic cells because they avoid the loss of heterozygosity, a high risk factor for the development of cancer. two major mechanisms are responsible for hj processing in mitotic cells: the preferred dissolution by the so-called bloom helicase btr complex that results in ncos, and the resolution of hjs by structure specific endonucleases, such as the slx complex or the canonical hj resolvase gen that can lead to cos and ncos. because dissolution is favored over resolution in mitosis, the actions of resolvases are strictly regulated in a spatio-temporal manner. in human cells, eme a subunit of the slx complex is phosphorylated in prometaphase to stimulate interactions and activation of the mus81-eme1-slx1-slx complex. this ensures that resolvases eliminate only hjs, which were not processed by the btr complex in s and g phase. the activity of the human gen resolvase is restricted to mitosis by nuclear exclusion during interphase through a leucine-rich nuclear export signal. based on the evidence of unprocessed chromatin structures in c. elegans lem- mutants and the presence of a giy-yig nuclease domain in ankle we hypothesized that ankle may also function in dna damage repair, probably redundantly with other nucleases, and its nucleo-cytoplasmic shuttling and cellular localization may be tightly controlled during the cell cycle and upon dna damage. hence, we wanted to identify the domains and motifs in ankle involved in its nucleo-cytoplasmic shuttling and test whether dna damage may alter ankle1’s cellular localization. we identified one canonical nuclear localization sequence and one nuclear export sequence in ankle that mediate its translocation into and out of the nucleus, respectively. however, no changes in shuttling and/or cellular localization were found upon induction of dna damage or during the cell cycle.  nucleocytoplasmic shuttling of ankle is mediated by active transport mechanisms in a previous study, we showed that ankle is primarily localized in the cytoplasm in hela and b-cell derived ramos cell lines, but it accumulated in the nucleus upon inhibition of nuclear export by leptomycin. this suggested that ankle shuttles between the cytoplasm and the nucleus. in this study, we wanted to identify the domains in ankle that mediate its shuttling, and test, whether nucleo-cytoplasmic shuttling of ankle and its cellular localization change upon dna damage or during the cell cycle. therefore we expressed ankle in osteosarcoma u2os cells, which have intact prb and p pathways and have frequently been used in studies investigating dna damage repair pathways, including resolvase-mediated repair. in agreement with previous studies, ankle ectopically expressed in u2os cells localizes predominantly in the cytoplasm and accumulates in the nucleus upon pharmacological inhibition of the crm1-dependent nuclear export using the drug leptomycin b. in silico analysis of the ankle primary sequence predicted the existence of at least two nuclear localization sequences and two nuclear export signals, suggesting that shuttling of ankle may be mediated by active nuclear import and export pathways.fig. ankle shuttles between nucleus and cytoplasm. a schematic representation of ankle1’s domain organization depicting predicted ankyrin repeats, the lem domain and a giy-yig nuclease domain. putative nuclear export sequences and nuclear localization sequences, identified in silico are indicated. b immuno-fluorescence analysis of ectopic ankle1-v in u2os cells without or following a 3 h treatment with nm leptomycin b, an inhibitor of crm1-mediated export. cells were stained with antibodies to v and dna with dapi. scale bar: 10 μm in order to get a first hint whether any of the nlss and ness are responsible for nucleo-cytoplasmic shuttling of ankle we generated a series of truncated ankle constructs c-terminally fused to green fluorescence protein, and tested their subcellular localization by confocal fluorescence microscopy. using this approach, we also wanted to test whether specific protein domains other than transport signal sequences influence ankle localization. these include the ankyrin repeats, well known protein interaction domains found in many proteins, the lem domain, known to mediate binding to chromatin via barrier-to-autointegration factor, and the giy-yig domain, mediating nuclease activity. ankle11– which lacks the c-terminal domain including the giy-yig motif and the two nlss, is effectively exported to the cytoplasm in both u2os and hela cells. additional deletion of the lem domain did not alter the predominant cytoplasmic localization of the construct in the absence of leptomycin b. hence we concluded that neither the lem domain nor the giy-yig domain affect nuclear export. the construct lacking the n-terminal ankyrin repeats was efficiently excluded from the nucleus in both u2os and hela cells, indicating that neither ankyrin repeats nor nes are essential for nuclear export and/or cytoplasmic localization. in agreement with this hypothesis, ankle11– containing only nes was not efficiently exported from the nucleus, while both ankle1158– and ankle1158– encompassing the central region containing only nes with or without the lem domain, respectively, localize exclusively to the cytoplasm. in addition, leptomycin b-mediated inhibition of nuclear export caused efficient accumulation of most constructs in the nucleus, suggesting that a region including nes is sufficient for ankle export. altogether these data suggest that ankle is actively exported from the nucleus predominantly through a nes2-mediated mechanism. we assume that also its nuclear import is mediated by an active nls-mediated mechanism based on the following observations. first, ankle1158– containing nes and both predicted import sequences nls and nls efficiently accumulates in the nucleus in the presence of leptomycin b like the wild-type protein, although its molecular weight does not allow efficient nuclear translocation by passive diffusion through nuclear pores. secondly, a 51 kda large construct containing the nuclear localization signal is exclusively targeted to the nucleus, despite its size would allow some passive diffusion out of the nucleus. we therefore conclude that ankle is shuttling between the nucleus and the cytoplasm through active import and export mechanisms, but its nuclear export seems to overrule nuclear transport, as at steady-state, ankle is not detected inside the nucleus.fig. localization of ankle fragments containing different domains and export and import signals. localization of gfp-tagged ankle truncation constructs ectopically expressed in u2os and hela cells was determined by confocal fluorescence microscopy. molecular weights and schematic representations of domain organization of respective truncation protein constructs are indicated. cells were fixed after 3 h of mock or leptomycin b treatment. dna was counterstained with dapi. scale bar: 10 μm mutation analyses identify nes and nls as major transport-mediating signals our experiments using truncated ankle constructs indicated that nls and nes are the predominant regulators of active import and export, respectively. to test whether this hypothesis holds true also in the context of the full length protein, we analyzed the localization of full length ankle constructs carrying point mutations at conserved residues within the nuclear transport sequences nes nes nls and nls. furthermore, in order to avoid potential effects of the bulky gfp tag we used the smaller v5-tag instead. mutation of nes caused only a subtle impairment of nuclear export, as indicated by the minor increase in the ratio of nucleoplasmic over cytoplasmic signal intensities of the mutant compared to wild-type control. in contrast, substitution of conserved leucine residues within the nes impaired nuclear export of ankle and caused a significant increase in the steady-state fraction of the protein in the nucleus compared to the control. given that nuclear export is predominant in defining the steady state localization of ankle it was only possible to examine the consequences of nls and nls mutations following leptomycin b-mediated inhibition of nuclear export. mutation of the positively charged residues within nls did not affect ankle1’s accumulation in the nucleus under this condition, whereas mutation of nls impaired nuclear import nearly completely. in summary, we concluded that nes and nls are the predominant signals mediating active nucleo-cytoplasmic shuttling of ankle fig. mutation analyses identify nes and nls as the predominant sequences controlling nucleo-cytoplasmic shuttling of ankle a, b, d, e u2os cells were transiently transfected with ankle1-v carrying point mutations in nls or nes sequences and either mock-treated or treated with leptomycin b for 3 h and processed for confocal immunofluorescence analyses using antibodies to v and dapi to detect dna. scale bars: 10 μm. c mean fluorescence intensities in nuclei and cytoplasm of cells expressing wild-type ankle1-v ankle1-nes1mut-v or ankle1-nes2mut-v were measured in original unprocessed digital images prior to contrast/brightness adjustment and nucleus to cytoplasm signal ratios were calculated. data were obtained from three independent experiments and analyzed using student’s t-test. ankle1-nes p = ; ankle1-nes p =  e-21; n = 50; 15– cells each from three independent experiments in support of this, we also show by fusing wild-type and mutated versions of nls and nes sequences to gfp, that the wild-type, but not mutated sequences are sufficient to mediate active transport of heterologous proteins. wild-type nes2-gfp, but not the mutated construct was efficiently excluded from the nucleus. wild-type nls sequence mediated efficient accumulation of the fusion construct in the nucleus, whereas the mutated version was distributed throughout the cell.fig. nes and nls are functional export and import signals in heterologous reporter constructs. u2os cells were transiently transfected with gfp constructs fused to the wild-type or mutated versions of nes or nls fixed and prepared for fluorescence microscopy. representative images of at least three independent experiments are shown. scale bars: 10 μm. b, d mean fluorescence intensities in nuclei and cytoplasm of gfp-nes2wt, gfp-nes2mut, gfp-nls2wt or gfp-nls2mut transfected cells were measured in original unprocessed digital images prior to contrast/brightness adjustment, and nucleus to cytoplasm signal ratios were calculated. data were obtained from three independent experiments and analyses were done using student’s t-test. gfp-nes2mut, p =  e-30; gfp-nls2mut, p =  e-18; n = 43; 13– cells each from three independent experiments overall we show that ankle localization is determined predominantly by the activity of two transport signals, a nuclear export signal in the middle of the polypeptide and a c-terminal nuclear localization signal. ankle does not change localization upon dna damage and during mitosis it was previously reported that a c. elegans mutant for lem- the worm ortholog of mammalian ankle is hypersensitive to dna damage-causing agents. furthermore, ankle may be functionally related to slx the only other known giy-yig-type endonuclease encoded in the mammalian genome, which is involved in homologous recombination-mediated repair of dna double strand breaks in a cell cycle-regulated manner. thus, we speculated that ankle may have a similar function in dna damage repair and set out to test whether ankle may transiently accumulate in the nucleus and/or on chromatin upon induction of the dna damage response signaling or during the cell cycle. however, as long-term expression of wild-type ankle causes cell death, we generated a stable u2os cell line ectopically expressing an endonuclease-defective, gfp-tagged version of ankle to address ankle dynamics during the cell cycle and upon induction of dna damage. the catalytically dead ankle mutant shuttles between the cytoplasm and the nucleoplasm and has a predominant cytoplasmic localization at steady state like the wild-type protein. however, unlike for wild-type ankle leptomycin b-dependent accumulation of the ankle mutant in the nucleus did not induce dna damage. to investigate potential changes in ankle localization upon induced dna damage, we treated cells with various chemical compounds, which cause different types of dna damage and elicite different dna repair pathways: bleomycin, a radio-mimetic drug inducing double strand breaks and mitomycin c, creating dna crosslinks, which both are mainly repaired by non homologous end joining or hr, and uv irradiation that triggers the nucleotide excision repair pathway. drugs were applied for short time periods at high concentration to study immediate dna damage response or at low dosage over a prolonged time period to cause constant dna damage to allow activation of downstream dna damage response signaling events. however, although the different types of treatments induced dna damage response, as shown by the upregulation of the γh2a.x marker compared to untreated control samples, we did not observe a change in the localization of ankle nuclease-dead mutant. hence, ankle localization may not be affected upon induction of dna damage response signaling, or the catalytically dead mutant is unable to respond to induction of dna damage signaling.fig. ankle does not relocalize to the nucleus in response to dna damage. u2os cells stably expressing a catalytically inactive gfp-ankle version were treated with the dna damaging agents bleomycin or mitomycin c for 4 h or 48 h using high and low dosages, respectively: 3 μg/ml bleomycin, 1 μg/ml mitomycin c for 4 h, 1 μg/ml bleomycin or  μg/ml mitomycin c for 48 h. fixed cells were imaged using confocal microscopy and representative images of at least three independent experiments are shown. scale bars: 10 μm next we tested whether ankle like the mammalian resolvases, may serve in dna repair pathways during late g or m phase of the cell cycle, as shown for the resolvases mus slx and gen. we first analyzed ankle localization throughout the cell cycle and focused in particular on the potential association of ankle with condensed chromosomes during mitosis. ankle is uniformly distributed throughout the cytoplasm in different stages of mitosis, both in fixed and live cells with or without dna damage induction. unrepaired dna damage and persistent hjs were shown to lead to defects in chromosomal segregation during anaphase, visible as small dna fibers connecting separated chromatids. resolvases are known to be activated upon such mitotic defects and to localize to these anaphase bridges in order to resolve hjs and to allow mitotic progression without chromosomal mis-segregation. we tested whether ankle shows a similar re-localization to anaphase bridges following treatment of cells with the replication inhibitor hydroxyurea to increase the frequency of such mitotic defects. screening mitotic figures in three independent experiments did not reveal re-localization and/or accumulation of ankle1-dead mutants to anaphase bridges under the given experimental setup.fig. ankle does not accumulate on mitotic dna bridges induced by hydroxyurea treatment. u2os cells stably overexpressing inactive gfp-ankle were grown in the presence of 100 μm hydroxyurea for 18 h. cells were fixed and processed for confocal fluorescence microscopy. representative image out of at least mitotic cells from three independent experiments is shown. arrows show dapi stained dna bridges. scale bar: 5 μm discussion in this study we show that ankle endonuclease shuttles between the cytoplasm and the nucleus by an active nls-mediated nuclear import and a nes-mediated nuclear export, despite its predominant steady-state localization in the cytoplasm. using various ankle deletion constructs and full length ankle with mutated, non-functional transport signals, we find that nucleo-cytoplasmic shuttling is predominantly achieved by the concerted actions of a c-terminal monopartite nls sequence and a canonical rev-type nes sequence in the central region of ankle polypeptide. as ankle efficiently accumulates in the nucleus upon treatment with leptomycin b, a specific inhibitor of crm, ankle is likely excluded from the nucleoplasm via a crm1-dependent nuclear export. in silico analysis predicted two potential nuclear export sequences fitting the highly conserved leucine-rich nes sequence motif, one located within the ankyrin repeats of ankle the other in the central region between the ankyrin repeats and the lem domain. experimental testing showed that only the latter was sufficient to mediate efficient nuclear export of tested ankle constructs. similarly, among the two predicted canonical monopartite nlss present in ankle polypeptide, nls at the very c-terminus was found to mediate nuclear import. several observations speak in favor of an active nls- and nes-mediated transport of ankle rather than a so-called “piggy-back” mechanism in which ankle is co-transported with other proteins: first, wild-type ankle but not ankle mutants with a mutated nonfunctional nes sequence were exported from the nucleus. second, ankle mutants with a mutated nonfunctional nls sequence, unlike wild-type protein, were not imported into the nucleus following leptomycin b treatment. third, both nls and nes sequences were able to mediate effective import and export, respectively of a heterologous protein. despite the presence of an active nls sequence, ankle1’s steady state localization is predominantly cytoplasmic. in fact we were unable to detect any signal above background in the nucleus by fluorescence microscopy. this observation raises the question, whether additional factors may be involved favoring nuclear export over import or whether the predominant cytoplasmic localization is merely a consequence of a tightly regulated balance between import and export rates. potential additional mechanisms involved in the regulation of ankle nucleo-cytoplasmic shuttling may involve posttranslational modifications of ankle or specific retention in the cytoplasm by interaction with cytoskeletal components, but currently there is no evidence for any of these pathways being involved. what may be the physiological relevance of nucleo-cytoplasmic shuttling of ankle1? constant shuttling of ankle across the nuclear envelope, while primarily localizing in the cytoplasm may prevent accidental damage in the genome caused by ankle1’s endonuclease activity. this hypothesis is supported by previous findings showing that leptomycin b-mediated accumulation of ankle in the nucleus causes dna cleavage and cell death. this hypothesis also predicts that the localization of ankle has to be regulated in a tightly controlled manner dependent on dna damage and/or the cell cycle as shown for other nucleases: dnasei, a nuclease involved in apoptosis, is excluded from the nucleus by association with cytoplasmic actin, and association is stabilized by cofilin and disrupted by n-gelsolin. a tight regulation of nucleases was also reported for hj-processing resolvases, including slx the only other giy-yig domain-containing protein besides ankle in the human genome. dissolution of hjs by the btr complex is the preferred mechanism in mitotic cells, because it avoids co formation, but hjs, which could not be processed or escaped the btr complex-mediated repair can be resolved by one of the three structure specific endonucleases: slx1-slx mus81-eme or gen in order to allow the preferred processing of hjs by btr and use resolvases only as a backup mechanism in g and m-phase of the cell cycle, resolvase activity is tightly regulated throughout the cell cycle. slx is active only in a complex with slx slx forms homodimers in g and s-phase of the cell cycle, and formation of the active slx complex is only promoted in prometaphase by phosphorylation of eme by cyclin dependent kinases. gen is regulated at the level of nuclear exclusion via a leucine-rich nes sequence, assuring that only breakdown of the nuclear envelope during mitosis allows gen to access unresolved dna bridges. based on these recently reported findings on the regulation of resolvases we tested whether ankle localization may be changed transiently upon inducing dna damage or during the cell cycle. however neither long-term treatment at low doses nor short treatments with higher doses of mitomycin c, uv-c and bleomycin changed ankle localization. similarly, ankle did not change its localization during the cell cycle and did not associate with anaphase bridges in mitotic cells treated with hydroxyurea. one caveat of these studies is that we had to use a catalytically inactive ankle mutant containing mutations in its giy-yig domain, as the wild-type protein causes cell death and precluded cell cycle dependent analyses. as the catalytically dead mutant showed the same nucleo-cytoplasmic shuttling and steady state cytoplasmic localization, we consider it unlikely that the mutation in the giy-yig motive affects its cellular localization. therefore, we concluded that ankle localization is neither affected by activated dna damage response signaling nor cell cycle stages in u2os cells. alternatively the high redundancy of hj processing pathways may obstruct the analysis of specific ankle functions in hj processing under these conditions. although data obtained in c. elegans and recent reports on a potential linkage of single nucleotide polymorphisms in the human ankle gene with an increased risk for certain cancers are consistent with a function of ankle in dna damage response pathways, we cannot exclude that it is involved in other cellular processes. in this study, we elucidated one level in the regulation of ankle localization by identifying active nls and nes sequences.  this study identifies a centrally located rev-type crm1-dependent nes in the ankle polypeptide and a c-terminal canonical mono-partite nls, which together mediate shuttling of ankle between the cytoplasm and the nucleus and maintain a predominantly cytoplasmic localization at steady state. induction of dna damage response signaling did not affect cellular localization or chromatin association of ankle leaving it unclear, if and how ankle localization may be regulated upon induction of dna damage response signaling. 
hence, we aimed at identifying the domains mediating ankle shuttling and investigating whether cellular localization is affected during dna damage response. the predominant localization of ankle in the cytoplasm, however, does not change upon induction of several dna damage response pathways throughout the cell cycle. we identified the domains mediating nuclear import and export of ankle ankle1’s cellular localization was not affected following dna damage. unlike most other lem proteins, ankle is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. sequence analysis predicts the presence of two canonical nuclear import and export signals in ankle immunofluorescence microscopy of cells expressing wild-type and various mutated ankle1-fusion proteins revealed a c-terminally located classical monopartite nuclear localization signal and a centrally located crm1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of ankle these sequences are also functional in heterologous proteins. ankyrin repeats and lem domain containing protein belongs to the lem protein family, whose members share a chromatin-interacting lem motif. we hypothesized that ankle may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of ankle activity. resolvase activity is regulated in a spatial and temporal manner during the cell cycle. the mammalian genome encodes only one other giy-yig domain protein, termed slx slx has been described as a resolvase that processes holliday junctions during homologous recombination-mediated dna double strand break repair.
 ankyrin repeats and lem domain containing protein belongs to the lem protein family, whose members share a chromatin-interacting lem motif. unlike most other lem proteins, ankle is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. it contains a giy-yig-type nuclease domain, but its function is unknown. the mammalian genome encodes only one other giy-yig domain protein, termed slx slx has been described as a resolvase that processes holliday junctions during homologous recombination-mediated dna double strand break repair. resolvase activity is regulated in a spatial and temporal manner during the cell cycle. we hypothesized that ankle may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of ankle activity. hence, we aimed at identifying the domains mediating ankle shuttling and investigating whether cellular localization is affected during dna damage response.  sequence analysis predicts the presence of two canonical nuclear import and export signals in ankle immunofluorescence microscopy of cells expressing wild-type and various mutated ankle1-fusion proteins revealed a c-terminally located classical monopartite nuclear localization signal and a centrally located crm1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of ankle these sequences are also functional in heterologous proteins. the predominant localization of ankle in the cytoplasm, however, does not change upon induction of several dna damage response pathways throughout the cell cycle.  we identified the domains mediating nuclear import and export of ankle ankle1’s cellular localization was not affected following dna damage. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ankle1lem proteinnucleaseresolvasenuclear transportnuclear exporthttp://dx.doi.org/ /501100002428austrian science fundp b09dk w1220zlopasa livija foisner roland http://dx.doi.org/http://dx.doi.org/ /501100003494herzfelder'sche familienstiftungissue-copyright-statement© the author 2016 