InteractiveFly: GeneBrief

Pendulin: Biological Overview | References

Gene name - Pendulin

Synonyms - imp-α2, importin α2

Cytological map position - 31A1-31A2

Function - signaling

Keywords - adaptor in the Ran-GTP nuclear transport cycle that binds a cargo protein to the nuclear import receptor Fs(2)Ket - Nanos inhibits translation of maternal importin-α2 mRNA thus regulating the maternal-zygotic transition - regulates Piwi nuclear transport which in turn transcriptionally regulates transposons - involved in centrosome duplication, mitotic spindle dynamics, nuclear envelope assembly, ring canal formation in the female germline, geotaxic behaviour and perception of pain

Symbol - Pen

FlyBase ID: FBgn0267727

Genetic map position - chr2L:10,056,900-10,060,095

NCBI classification - Karyopherin (importin) alpha

Cellular location - cytoplasmic and nuclear

NCBI links: EntrezGene, Nucleotide, Protein
Recent literature
Serway, C. N., Dunkelberger, B. S., Del Padre, D., Nolan, N. W. C., Georges, S., Freer, S., Andres, A. J. and de Belle, J. S. (2020). Importin-alpha2 mediates brain development, learning and memory consolidation in Drosophila. J Neurogenet: 1-14. PubMed ID: 31965871
Neuronal development and memory consolidation are conserved processes that rely on nuclear-cytoplasmic transport of signaling molecules to regulate gene activity and initiate cascades of downstream cellular events. Surprisingly, few reports address and validate this widely accepted perspective. This study shows that Importin-alpha2 (Imp-alpha2), a soluble nuclear transporter that shuttles cargoes between the cytoplasm and nucleus, is vital for brain development, learning and persistent memory in Drosophila melanogaster. Mutations in importin-alpha2 (imp-alpha2, known as Pendulin or Pen and homologous with human KPNA2) are alleles of mushroom body miniature B (mbmB), a gene known to regulate aspects of brain development and influence adult behavior in flies. Mushroom bodies (MBs), paired associative centers in the brain, are smaller than normal due to defective proliferation of specific intrinsic Kenyon cell (KC) neurons in mbmB mutants. Extant KCs projecting to the MB beta-lobe terminate abnormally on the contralateral side of the brain. mbmB adults have impaired olfactory learning but normal memory decay in most respects, except that protein synthesis-dependent long-term memory (LTM) is abolished. This observation supports an alternative mechanism of persistent memory in which mutually exclusive protein-synthesis-dependent and -independent forms rely on opposing cellular mechanisms or circuits. A testable model of Imp-alpha2 and nuclear transport roles in brain development and conditioned behavior is proposed. Based on molecular characterization, it is suggested that mbmB is hereafter referred to as imp-alpha2(mbmB).

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. This study shows that Nos represses somatic gene expression by inhibiting translation of maternal importin-alpha2 (impalpha2) mRNA. Mis-expression of Impalpha2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, it is proposed that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a 'double-lock' mechanism to inhibit somatic gene expression in germline progenitors (Asaoka, 2019).

How germ cell fate is established and maintained is a century-old question in developmental, cellular, and reproductive biology. Metazoan species have two distinct modes of germline specification. In some species, germline progenitors are characterized by inheritance of a specialized ooplasm, or the germ plasm, which contains maternal factors necessary and sufficient for germline development. In other species, germline progenitors are specified by inductive signals from surrounding tissues. Irrespective of the mode of germline specification, transcriptional repression of somatic genes is common in germline progenitors, implying that this phenomenon is critical for separation of the germline from the soma (Asaoka, 2019).

In Drosophila, the germ plasm is localized in the posterior pole of cleavage embryos (stage 1-2), and is partitioned into germline progenitors called pole cells (stage 3-4). In pole cells of blastoderm embryos (stage 4-5), the genes required for somatic differentiation are transcriptionally repressed by two maternal proteins in the germ plasm, Polar granule component (Pgc) and Nanos (Nos). Pgc is a Drosophila-specific peptide that suppresses RNA polymerase II-dependent transcription in pole cells by inhibiting the function of positive transcriptional elongation factor b (P-TEFb, a dimer of Cyclin dependent kinase 9 and Cyclin T). By contrast, Nos is an evolutionarily conserved protein that plays an essential role in germline development in various animals. For example, in Drosophila, pole cells lacking Nos (nos pole cells) can adopt a somatic, rather than a germline, fate. Furthermore, depletion of Nos is reported to show ectopic expression of somatic genes, such as fushi tarazu (ftz), even-skipped (eve), and the sex-determination gene Sex lethal (Sxl), in pole cells. Thus, maternal Nos is required in pole cells for repression of somatic genes and establishment of the germ/soma dichotomy. However, the mechanism by which Nos represses somatic gene expression remains unknown (Asaoka, 2019).

Nos acts as a translational repressor of mRNAs that harbor a discrete sequence motif called Nanos Response Element (NRE) in the 3' UTR. NRE contains an evolutionarily conserved Pumilio (Pum)-binding sequence, UGU trinucleotide. In abdominal patterning, Pum represses translation of maternal hunchback (hb) mRNA by binding to NREs in its 3' UTR and recruiting Nos to the RNA/protein complex. Deletion of the NREs from hb mRNA causes its ectopic translation in the posterior half of embryos, which in turn suppresses abdomen formation. Furthermore, deletion of NREs causes hb translation in pole cells, suggesting that NRE-dependent translational repression occurs in pole cells. Indeed, Nos represses translation of head involution defective (hid) mRNA in pole cells in an NRE-like-sequence-dependent manner. In addition, Nos and Pum repress Cyclin B translation in pole cells by binding to a discrete sequence containing two UGU trinucleotides (Cyclin B NRE) These findings led to a speculation that Nos, along with Pum, represses somatic gene expression in pole cells by suppressing translation of mRNAs containing NRE or UGU in their 3' UTRs (Asaoka, 2019).

This study reports that, in pole cells, Nos, along with Pum, represses translation of importin-α2 (impα2)/Pendulin/oho31/CG4799 mRNA, which contains an NRE-like sequence in its 3' UTR. The impα2 mRNA encodes a Drosophila Importin-α homologue that plays a critical role in nuclear import of karyophilic proteins. Nos inhibits expression of a somatic gene, ftz, in pole cells by repressing Impα2-dependent nuclear import of the transcriptional activator, Ftz-F1. Based on these observations, it is proposed that Nos-dependent inhibition of nuclear import of transcriptional activators and Pgc-dependent global transcriptional silencing act as a 'double-lock' mechanism to repress somatic gene expression in pole cells (Asaoka, 2019).

Maternally supplied impα2 mRNA is distributed throughout cleavage embryos. When embryos develop to the blastoderm stage, impα2 mRNA is degraded in the somatic region, but not in pole cells, resulting in enrichment of impα2 mRNA in pole cells. However, this study found that expression of Impα2 protein was at background levels in pole cells. Because impα2 mRNA contains a sequence very similar to the NRE (hereafter, NRE-like sequence) in its 3' UTR, it was assumed that impα2 mRNA is a target of Nos/Pum-dependent translational repression in pole cells. To investigate this possibility, the expression of the Impα2 protein was first monitored in pole cells of embryos lacking maternal Nos or Pum (nos or pum embryos, respectively). In these pole cells, expression of Impα2 protein was higher than in those of control (nos/+) embryos. Because neither nos nor pum mutation affected the impα2 mRNA level in pole cells, these observations show that Nos and Pum repress protein expression from the impα2 mRNA in pole cells (Asaoka, 2019).

Whether this repression is mediated by the NRE-like sequence in the impα2 3' UTR was investigated. To this end, impα2 mRNA, with or without the NRE-like sequence (impα2 WT and impα2 ΔNRE, respectively), was maternally supplied to embryos, and their protein expression was examined in pole cells at the blastoderm stage. Because a triple Myc tag sequence was inserted at the C-terminal end of the coding sequence, protein expression from these mRNAs could be monitored using an anti-Myc antibody. When impα2 WT mRNA was supplied to normal (y w) embryos, the tagged protein was expressed at low levels in the soma, but was barely detectable in pole cells. By contrast, the tagged protein from impα2 ΔNRE mRNA was detected in normal pole cells. Similar protein expression was observed in pole cells lacking Nos (nos pole cells), when impα2 WT mRNA was supplied, as well as when impα2 ΔNRE mRNA was supplied. Because the frequency of tagged protein expression from impα2 ΔNRE mRNA did not increase in cells lacking Nos, these results indicate that the NRE-like sequence mediates Nos-dependent repression of Impα2 protein expression in pole cells (Asaoka, 2019).

The NRE-like sequence of impα2 mRNA contains two UGU trinucleotides. The UGU trinucleotide is a core sequence of an RNA motif (Nos-Pum SEQRS motif: 5'-HWWDUGUR) that was highly enriched in a SEQRS (in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes) analysis of the Nos-Pum-RNA ternary complex. Hence, it was asked whether Pum and Nos form a ternary complex with impα2 mRNA in an NRE-like sequence-dependent manner. To address this question, electrophoretic mobility shift assay (EMSA) was performed using the Pum RNA-binding domain and the Nos protein containing Zn finger motifs and C-terminal region, which are reported to form a Nos-Pum-target RNA ternary complex in vitro. Nos and Pum together, but neither alone, formed a complex with impα2 RNA containing an NRE-like sequence (WT), whereas alteration of the NRE-like sequence (mut) abolished this interaction. These results demonstrate that Nos and Pum are able to interact with the impα2 3' UTR in an NRE-like sequence-dependent manner. The observations described above led to a conclusion that Nos, along with Pum, directly represses impα2 translation in pole cells (Asaoka, 2019).

Impα2 is a Drosophila homologue of Importin-α that mediates nuclear import of karyophilic proteins with classical nuclear localization signal (NLS). It was predicted that ectopic production of Impα2 in nos pole cells would cause aberrant nuclear import of NLS-containing karyophilic proteins. To explore this possibility, this study focused on a transcriptional activator, Ftz-F1, which contains a classical NLS and is expressed throughout early embryos, including pole cells. In normal embryos, Ftz-F1 was enriched in the cytoplasm of pole cells, although it was in the nuclei of somatic cells. In the absence of maternal Nos, the percentage of embryos with Ftz-F1 signal accumulating in pole-cell nuclei was higher than in normal embryos. Furthermore, the nuclear/cytoplasmic ratio of Ftz-F1 signal intensities in nos pole cells was higher than in normal pole cells. To determine whether this aberrant concentration of Ftz-F1 was caused by mis-expression of Impα2, this study expressed Impα2 ectopically in pole cells of normal embryos. To this end, impα2 mRNA in which the 3' UTR was replaced with the nos 3' UTR, was maternally supplied under the control of the nos promoter; the mRNA was localized to the germ plasm and pole cells under the control of the nos 3' UTR. The percentage of these embryos (impα2-nos3'UTR embryos) with Ftz-F1 in pole-cell nuclei and the nuclear/cytoplasmic ratio of Ftz-F1 intensities in their pole cells were higher than those of normal pole cells. These observations suggest that mis-expression of Impα2 in pole cells caused by depletion of maternal Nos results in aberrant nuclear import of Ftz-F1 (Asaoka, 2019).

Depletion of maternal Nos results in ectopic expression of the somatic genes ftz, eve and Sxl in pole cells. Because Ftz-F1 is required for proper expression of ftz in the soma, it was asked whether mis-expression of Impα2 causes ectopic expression of ftz in pole cells. In normal embryos, ftz mRNA was expressed in seven stripes of somatic cells, but never expressed in pole cells. By contrast, in impα2-nos3'UTR embryos, ftz mRNA was rarely detectable in pole cells. It is assumed that this low frequency of ftz expression was due to Pgc-mediated silencing of global mRNA transcription. To test this idea, Impα2 was expressed in pole cells of embryos lacking maternal Pgc (pgc impα2-nos3'UTR embryos); the frequency of ftz expression was drastically increased, compared to those of impα2-nos3'UTR embryos and the embryos lacking Pgc (pgc embryos). A similar situation was observed in embryos lacking both Pgc and Nos activities (pgc nos embryos). The percentage of embryos expressing ftz in pole cells was 82.8%, an increase relative to 35.8% in pgc embryos. Furthermore, ectopic ftz expression in pgc nos pole cells was suppressed by injecting double-stranded RNA (dsRNA) against impα2. Therefore, it is concluded that ectopic expression of ftz in pole cells is cooperatively repressed by Nos-dependent suppression of Impα2 production and Pgc (Asaoka, 2019).

In addition to ftz expression, eve was expressed ectopically in pole cells of pgc impα2-nos3'UTR embryos. Ectopic eve mRNA and its protein expression were significantly higher in pgc impα2-nos3'UTR pole cells than pgc or impα2-nos3'UTR pole cells (S3 Fig). Expression of the sex-determination gene Sxl was examined in early pole cells, because Sxl is also repressed by nos in both male and female pole cells. In males, Sxl mRNA expression was rarely detectable in pole cells of nos, impα2-nos3'UTR, pgc, and pgc impα2-nos3'UTR embryos. By contrast, in females, the percentage of embryos expressing Sxl mRNA in pole cells was significantly higher in pgc impα2-nos3'UTR embryos than in impα2-nos3'UTR, and pgc embryos. These results indicate that eve and Sxl, like ftz, are cooperatively repressed in pole cells by Impα2 depletion and Pgc-dependent transcriptional silencing. Because there is no evidence for the involvement of Ftz-F1 in eve and Sxl expression, it is likely that Impα2 mediates nuclear import of other transcriptional activator(s) for eve and/or Sxl in pole cells (Asaoka, 2019).

Nos is required in pole cells for mitotic quiescence, repression of apoptosis, and proper migration to embryonic gonads. Hence, it was asked whether mis-expression of Impα2 causes defects in these processes. First, using an antibody against a phosphorylated form of histone H3 (PH3), a marker of mitosis, whether pole cells enter mitosis in stage 7-9 embryos was investigated. Premature mitosis was detected in pole cells of nos embryos, as described previously, but never in pole cells of impα2-nos3'UTR or pgc impα2-nos3'UTR embryos. Second, using an antibody against cleaved Caspase-3, a marker of apoptosis, whether pole cells enter apoptosis in stage 10-16 embryos was investigated. Pole cells never expressed the apoptotic marker in impα2-nos3'UTR embryos, whereas in pgc impα2-nos3'UTR embryos, 20.4% of pole cells expressed the apoptotic marker. The latter was statistically indistinguishable from pgc pole cells, which have been reported to enter apoptosis. These data indicate that mis-expression of Impα2 does not affect apoptosis of pole cells even in the absence of pgc function. Last, whether mis-expression of Impα2 affects pole cell migration was investigated. The ability of pole cells to migrate properly into the embryonic gonads was never impaired in impα2-nos3'UTR embryos, and the percentage of pole cells entering the gonads in pgc impα2-nos3'UTR embryos was statistically indistinguishable from that of pgc pole cells, which has been reported to exhibit migration defect. These observations indicate that mis-expression of Impα2 does not induce premature mitosis, apoptosis, or mis-migration of pole cells. This can be partly explained by the facts that Cyclin B and hid mRNAs are the targets for Nos-dependent translational repression regulating mitosis and apoptosis in pole cells, respectively (Asaoka, 2019).

During the course of the experiments described above, it was observed that impα2-nos3'UTR interacts genetically with the pgc mutation to cause dysgenic gametogenesis. Because almost all of the ovaries in females derived from pgc mothers mated with y w males were agametic, the effect of impα2-nos3'UTR in pgc/+ background was examined. The percentage of dysgenic ovaries in pgc/+ impα2-nos3'UTR females derived from pgc/+ impα2-nos3'UTR mothers mated with y w males was significantly higher than those in pgc/+ and impα2-nos3'UTR females. In the dysgenic ovaries, almost all of the egg chambers fail to complete the vitellogenic stage, and consequently only a few mature oocytes were present. Furthermore, the percentages of dysgenic and agametic testes in pgc impα2-nos3'UTR males derived from pgc impα2-nos3'UTR mothers mated with y w males were higher than those in pgc and impα2-nos3'UTR males. In these testes, the abundance of Vasa-positive germline cells was reduced (dysgenic) or absent (agametic). Because dysgenic and agametic gonads were barely detectable in females and males derived from reciprocal crosses, the data suggest that mis-expression of Impα2 from maternal transcript, concomitant with maternal pgc depletion in pole cells, causes defects in gametogenesis. However, it cannot be tested whether concomitant depletion of maternal Nos and Pgc causes a similar phenotype because nos pole cells degenerate before adulthood, even when apoptosis in these cells is genetically repressed (Asaoka, 2019).

Expression of Importin-α subtypes is spatio-temporally regulated in the soma during development in multiple animal species, including Drosophila, and they control nuclear transport of unique karyophilic proteins to activate different sets of somatic genes. Drosophila genome contains three Importin-α family genes: impα1, 2, and 3. impα1/Kap-α1/CG8548 mRNA is not detectable in pole cells during early embryogenesis, and its protein product is ubiquitously expressed at a very low level throughout embryogenesis. By contrast, maternal impα3/Kap-α3/CG9423 mRNA is detectable in germ plasm during pole cell formation, and production of Impα3 protein is upregulated during the blastoderm stage. Because Impα3 production was independent of maternal nos activity, it is likely that Nos-dependent repression of Impα2 production is solely responsible for suppression of somatic gene expression in pole cells. By contrast, pole cells become transcriptionally active during gastrulation, when Impα2 is undetectable in these pole cells. Thus, the onset of zygotic transcription in pole cells may require Impα3-dependent nuclear import of transcription factors, in addition to the disappearance of Pgc and the alteration in chromatin-based regulation. After gastrulation, maternal impα2 mRNA is rapidly degraded in pole cells, and neither impα2 mRNA nor protein is detectable in the germline before adulthood. This suggests that maternal impα2 is dispensable for germline development, and that maternal impα2 mRNA partitioned into early pole cells must be silenced by Nos and Pum in order to suppress mis-expression of somatic genes (Asaoka, 2019).

Depletion of maternal Nos activities caused mis-expression of ftz in pole cells. Although ftz expression was barely observed in pole cells lacking only maternal Nos, it was partially derepressed in pole cells in the absence of Pgc alone, probably because a trace amount of Ftz-F1 enters pole cell nuclei even in the absence of the impα2 translation. Therefore, it is proposed that a subset of somatic genes, including ftz and eve, are repressed in pole cells by two distinct mechanisms: Nos-dependent repression of nuclear import of transcriptional activators and Pgc-dependent silencing of mRNA transcription. Pgc inhibits P-TEFb-dependent phosphorylation of Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of RNA polymerase II, a modification that is critical for transcriptional elongation; thus, mRNA transcription in pole cells is globally suppressed by Pgc. By contrast, Nos inhibits transcription of particular genes by repressing Impα2-dependent nuclear import of the corresponding transcriptional activators (Asaoka, 2019).

Nos is evolutionarily conserved and expressed in the germline progenitors of various animal species. In C. elegans, nos-1 and -2 are essential for rapid turnover of maternal lin-15B mRNA, which encodes a transcription factor that would otherwise cause inappropriate transcriptional activation in primordial germ cells. In the germline progenitors of Xenopus embryos, Nos-1, along with Pum, destabilizes maternal VegT mRNA and represses its translation to inhibit somatic (endodermal) gene expression, which is activated by VegT protein. Furthermore, in the germline progenitors (small micromeres) of sea urchin embryos, Nos silences maternal mRNA encoding a deadenylase, CNOT6, to stabilize other maternal mRNAs inherited into small micromeres. This study demonstrated that Nos inhibits translation of maternal impα2 mRNA in pole cells in order to suppress nuclear import of a transcriptional activator for somatic gene expression. Based on these observations, it is proposed that Nos silences maternal transcripts that are inherited into germline progenitors but deter the proper germline development. In addition to Nos-dependent silencing of maternal transcripts, transient suppression of RNA polymerase II elongation is observed during germline development of a wide range of animals, including Drosophila, C. elegans, Xenopus, and an ascidian, Halocynthia roretzi. Therefore, it is proposed that the 'double-lock' mechanism achieved by Nos and global suppression of RNA polymerase II activity plays an evolutionarily widespread role in germline development (Asaoka, 2019).

Piwi nuclear localization and its regulatory mechanism in Drosophila ovarian somatic cells

In Drosophila ovarian somatic cells (OSCs), Piwi represses transposons transcriptionally to maintain genome integrity. Piwi nuclear localization requires the N terminus and PIWI-interacting RNA (piRNA) loading of Piwi. However, the underlying mechanism remains unknown. This study shows that Importinα (Impα) plays a pivotal role in Piwi nuclear localization and that Piwi has a bipartite nuclear localization signal (NLS). Impα2 and Impα3 are highly expressed in OSCs, whereas Impα1 is the least expressed. Loss of Impα2 or Impα3 forces Piwi to be cytoplasmic, which is rectified by overexpression of any Impα members. Extension of Piwi-NLS with an additional Piwi-NLS leads Piwi to be imported to the nucleus in a piRNA-independent manner, whereas replacement of Piwi-NLS with SV40-NLS fails. Limited proteolysis analysis suggests that piRNA loading onto Piwi triggers conformational change, exposing the N terminus to the environment. These results suggest that Piwi autoregulates its nuclear localization by exposing the NLS to Impα upon piRNA loading (Yashiro, 2018).

Drosophila Importin-alpha2 is involved in synapse, axon and muscle development

Nuclear import is required for communication between the cytoplasm and the nucleus and to enact lasting changes in gene transcription following stimuli. Binding to an Importin-alpha molecule in the cytoplasm is often required to mediate nuclear entry of a signaling protein. As multiple isoforms of Importin-alpha exist, some may be responsible for the entry of distinct cargoes rather than general nuclear import. Indeed, in neuronal systems, Importin-alpha isoforms can mediate very specific processes such as axonal tiling and communication of an injury signal. To study nuclear import during development, the expression and function of Importin-alpha2 was examined in Drosophila melanogaster. Importin-alpha2 was found to be expressed in the nervous system where it was required for normal active zone density at the NMJ and axonal commissure formation in the central nervous system. Other aspects of synaptic morphology at the NMJ and the localization of other synaptic markers appeared normal in importin-alpha2 mutants. Importin-alpha2 also functioned in development of the body wall musculature. Mutants in importin-alpha2 exhibited errors in muscle patterning and organization that could be alleviated by restoring muscle expression of Importin-alpha2. Thus, Importin-alpha2 is needed for some processes in the development of both the nervous system and the larval musculature (Mosca, 2010b).

The nuclear import of Frizzled2-C by Importins-beta11 and alpha2 promotes postsynaptic development

Synapse-to-nucleus signaling is critical for synaptic development and plasticity. In Drosophila, the ligand Wingless causes the C terminus of its Frizzled2 receptor (Fz2-C) to be cleaved and translocated from the postsynaptic density to nuclei. The mechanism of nuclear import is unknown and the developmental consequences of this translocation are uncertain. This study found that Fz2-C localization to muscle nuclei required the nuclear import factors Importin-beta11 and Importin-alpha2 and that this pathway promoted the postsynaptic development of the subsynaptic reticulum (SSR), an elaboration of the postsynaptic plasma membrane. importin-beta11 (imp-beta11) and dfz2 mutants had less SSR, and some boutons lacked the postsynaptic marker Discs Large. These developmental defects in imp-beta11 mutants could be overcome by expression of Fz2-C fused to a nuclear localization sequence that can bypass Importin-beta11. Thus, Wnt-activated growth of the postsynaptic membrane is mediated by the synapse-to-nucleus translocation and active nuclear import of Fz2-C via a selective Importin-beta11/alpha2 pathway (Mosca, 2010a).

Drosophila importin α1 performs paralog-specific functions essential for gametogenesis

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

The animal importin α-gene family is diverse, having undergone multiple rounds of duplications and lineage-specific expansions. Most animal importin α's belong to one of three conserved clades, referred to here as α1, α2, and α3. Animal α1 genes are more similar to plant and fungal α1-like genes than to animal α2 or α3 genes, which arose from an animal α1-like progenitor to perform specialized roles in animal development and differentiation (Goldfarb, 2004). The evolution and maintenance of α1, α2, and α3 genes among animals is likely due to their specialized roles in conserved aspects of animal development. Although importin α1, α2, and α3 proteins are coexpressed in many adult tissues, they exhibit complex temporal and spatial expression patterns during development (see Kamei, 1999; Hogarth, 2006). Animal importin α1's, α2's, and α3's all mediate the import of classical NLS-containing cargoes and, in addition, each paralog is specialized to bind and mediate the import of distinct repertoires of NLS cargoes. Importin α's may also mediate the import of a few cargoes independent of β1. Finally, importin α's play specialized roles in cell processes other than nuclear transport (Ratan, 2008).

The proliferation of the importin α-gene family in animals may have been driven in part by the specialized needs of gametogenesis (Goldfarb, 2004; Hogarth, 2005). Importin α1, α2, and α3 proteins are differentially expressed during spermatogenesis in both Drosophila (Giarre, 2002) and mouse (Hogarth, 2006). Two of the three Caenorhabditis elegans importin α's are expressed exclusively in the germline and are both required for gametogenesis (Geles, 2001; Geles, 2002). Likewise, Drosophila lacking importin α2 (Dα2) develop normally to adulthood but have serious defects in gametogenesis (Gorjanacz, 2002; Mason, 2002; Mason, 2003). The spermatogenesis defect of Dα2 null flies is due to the loss of an activity shared by all three paralogs, since Dα1, Dα2, and Dα3 transgenes rescued the defect. In contrast, the role of Dα2 in oogenesis is unique since only Dα2 transgenes could rescue the phenotype. Like the C. elegans importin α3, Dα3 is required for somatic development and differentiation (Mason, 2003). Dα3 may also be required for gametogenesis but mutant animals die as larvae. Therefore, in addition to shared housekeeping roles in classical nuclear transport, importin α2's and α3's each have unique roles in animal-specific processes such as gametogenesis. What remains is to describe the consequences of mutating the single Drosophila importin α1 (Dα1) (Ratan, 2008).

This paper describes the isolation and characterization of a Dα1 null mutation. Like Dα2 mutant flies, Dα1 null flies develop to adulthood with severe defects in gametogenesis. Spermatogenesis in Dα1 null flies is arrested and males are completely sterile. Oogenesis is morphologically less severely affected, but virtually all Dα1 null females are sterile. In addition, overexpression of Dα1 results in defects in tergite development and viability that are enhanced by mutations in the importin α-recycling factor CAS and suppressed by mutations in importin β. This is the first genetic analysis of an animal importin α1 mutant and completes an analysis on the null phenotypes of the conventional Drosophila α1, α2, and α3 gene family (Ratan, 2008).

Cargo adapters such as importin α may have evolved to provide a greater range of control over nuclear transport in response to variable environmental conditions. The evolution of multiple importin α-genes would seem to extend the utility of these adapters by allowing the independent control of distinct sets of cargo repertoires. A genetic approach was taken in Drosophila to analyze the in vivo function of the conserved family of animal importin α1's, α2's, and α3's. In addition to binding unique repertoires of NLS cargoes, all three types likely share housekeeping duties in cNLS cargo import. The contribution of individual importin α's to redundant activities is influenced by their differing temporal and spatial expression patterns in various cells and tissues. In this study the first animal importin α1 mutant is described (Ratan, 2008).

The key finding here is that Dα1 mutant flies develop (almost) normally to adulthood but both males and females are sterile due to defects in gametogenesis. Dα1 null flies also exhibit a minor wing defect, so Dα1's nonredundant activities extend in this small way to somatic development. In contrast to Dα1 and Dα2, Dα3 is required for somatic development and Dα3 mutants arrest as larvae. Interestingly, Dα1 and Dα2 mutants display distinct phenotypes in gametogenesis. Spermatogenesis is more severely affected than oogenesis in Dα1 mutants, while Dα2 mutants have more severe defects in oogenesis (Mason, 2002). Dα2 is not absolutely essential for spermatogenesis -- some motile sperm and viable progeny are produced by mutant males -- and the defect can be rescued by Dα1, Dα2, or Dα3 transgenes. In contrast, no viable sperm are produced in Dα1 mutants, and only a Dα1 transgene can rescue the defect. Therefore, Dα1 serves a paralog-specific role in spermatogenesis that is distinct from the role of Dα2 in this process (Ratan, 2008).

Dα1 and Dα2 are both required for gametogenesis and have no significant roles in somatic development. It seems likely, therefore, that the evolutionary expansion of the importin α-gene family occurred to serve the uniquely complex processes of spermatogenesis and oogenesis, both of which involve the differentiation of germ-line stem cells using analogous signaling pathways (Gilboa, 2004). Dα1 plays an especially important paralog-specific role in spermatogenesis. All three importin α's are expressed in the fly testes, although in distinct, partially overlapping patterns that correspond to different stages of spermatogenesis, which include stem cell division, spermatogonial divisions, growth, meiotic divisions, and spermatid differentiation (reviewed in Gilboa, 2004; Hogarth, 2005). The expression of Dα1 overlaps with Dα2 expression during meiosis, and later with Dα3 during differentiation and individualization (Giarre, 2002). Dα1 is expressed at low levels in testes until the growth stage, when it appears cytoplasmic. Dα1 levels rise during meiosis when it accumulates in spermatid nuclei. Dα1 levels are lower during differentiation and, by the time spermatid heads become aligned toward the wall of the testes, are equally distributed between the nucleus and cytoplasm. Dα1 was not detectable in sperm with elongated heads. The defects exhibited by Dα1 and Dα2 mutants are manifested at different stages of sperm differentiation, although the timing and nature of these defects do not necessarily correspond to when and where during spermatogenesis these factors are actually required (Ratan, 2008).

The oogenesis defects of Dα1 and Dα2 mutant flies are also distinct from one another, and both phenotypes are due to paralog-specific activities. The cause of the severe Dα2 mutant phenotype (deflated oocytes) is likely related to the Dα2-dependent targeting of Kelch to ring canals, through which nurse cell cytoplasm is dumped into the developing oocyte (Gorjanacz, 2002). Kelch localization and dumping appear normal in Dα1 mutant females. Giarre (2002) reported that Dα1 expression in ovaries is weaker than that of Dα2 or Dα3, and is, therefore, unlikely to play a major role. This prediction is partially supported by the finding that the ovaries and eggs of Dα1 null flies are only mildly defective. Still, Dα1 must have an important role in oogenesis since almost all mutant females are sterile. It remains possible that the female sterility is due to a behavioral phenotype in egg laying or mating or some other defect that was too subtle to notice (Ratan, 2008).

The finding that two of the three conventional Drosophila importin α's are specialized to serve important roles in gametogenesis has a strong parallel in C. elegans (reviewed in Goldfarb, 2004; Hogarth, 2005). The C. elegans genome encodes three importin α's, IMA-1, IMA-2, and IMA-3, two of which (IMA-1 and IMA-2) localize exclusively to the germ line and are required for gametogenesis (Geles, 2001; Geles, 2002). Therefore, two of the importin α's in both fly and worm are required for gametogenesis. IMA-3, a conventional α3 type, is expressed in both somatic and germ-line cells, and like Dα3, is required for somatic development (Geles, 2001). Although IMA-1 and IMA-2 are highly divergent and dissimilar to any of the conventional types, their exclusive expression in the germ line and important role in gametogenesis suggest they may be functional homologs of Dα1 and Dα2. Also, like Dα2, IMA-2 displays cell cycle-dependent shifts between the nucleus and cytoplasm in the gonads, and both accumulate around chromosomes at the onset of nuclear envelope breakdown (Geles, 2002). Taken together, these results suggest the possibility that the special needs of gametogenesis may have driven the early expansion and specialization of the metazoan animal importin α-gene family. The complex temporal expression patterns of the five mouse importin α1's, α2's, and α3's in testes indicate that this role likely extends to mammalian spermatogenesis, which, in many ways, is similar to spermatogenesis in flies (Ratan, 2008).

Because importin α1's are very similar both by sequence and gene structure to ancestral plant and fungal α1-like genes, it was originally expected that the loss of Dα1 would cause defects in the nuclear transport of many important proteins with catastrophic consequences. Therefore, it was initially surprising to find that Dα1 null flies developed normally to adulthood with only a slight wing defect. Phenotypically, then, Dα1 is more similar to Dα2, whose loss also primarily affects gametogenesis. At gene structure and primary sequence levels Dα2 is more similar to Dα3. Thus the evolutionary history of the three genes does not predict the nature of their mutant phenotypes. It is hypothesized that the ancient and essential role the importin α's play in cNLS cargo import is redundantly supported in somatic tissues by the partially overlapping coexpression of the three paralogous proteins. The loss of any one is apparently masked by the activity of one or both of the others. Most of the phenotypes that appear in single gene mutants are likely due to paralog-specific functions that were divided among the genes following the duplications that gave rise to the extant importin α-gene family. An exception is the spermatogenesis defect of Dα2 mutant flies that is rescued by any of the three paralogs (Mason, 2002). It is established that importin α1's each have both shared and distinct cargo repertoires. The simplest explanations for the paralog-specific phenotypes associated with Dα1, Dα2, and Dα3 mutants invoke deficiencies in the nuclear import of their distinct NLS cargoes (Ratan, 2008).

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

The defect in tergite development observed in Dα1-overexpressing flies may lend insight into the mechanisms underlying the deleterious effects of excess nuclear importin α. Development of the tergites involves a tightly coordinated process of epithelial cell sheet replacement during which the adult tergites arise from histoblast nests that proliferate and spread to replace larval epidermal cells during pupal morphogenesis. The tergite defects observed in Dα1-expressing abdomens may be attributable either to the failure of the adult histoblast nests to proliferate or spread correctly or to a failure of the larval epidermal cells to undergo apoptosis since both of these processes are thought to be codependent. The genetic interactions between Dcas and Dα1 may be especially relevant to understanding the tergite phenotypes associated with Dα1 overexpression. Expression of CAS antisense RNA in MCF-7 breast carcinoma cells, which likely leads to increased nuclear levels of importin α, inhibits apoptosis. It is possible, then, that elevated levels of nuclear importin α inhibit apoptosis in these cells. By analogy, it is possible that elevated levels of nuclear Dα1 interfere with the apoptosis of larval epidermal cells, the persistence of which might impair the ability of the adult cuticle to properly proliferate and spread. Consistent with this hypothesis, blocking cell death in the larval epidermal cells of the abdomen result in defects in spreading of the histoblast nests and resulted clefts in the abdominal cuticle. Alternatively, these tergite phenotypes may be caused by defects in tergite development since thermocautery of histoblast nests also produces similar tergite defects. Nonetheless, it is intriguing to speculate that the regulated subcellular localization of importin α-proteins affects susceptibility to proapoptotic signals (Ratan, 2008).

This analysis of Dα1 complements a previous analyses of Dα2 and Dα3 (Mason, 2002; Mason, 2003). It is now possible to now say that two of the three conserved Drosophila importin α-genes are required almost exclusively for gametogenesis (Dα1 and Dα2), and only one (Dα3) is required for general viability. The larger picture emerges of a gene family that likely arose by gene duplication to serve the newly evolving requirements of gametogenesis. Following their initial establishment, each of the three paralogous genes was available to evolve specialized (derived) roles and, in mammals, undergo further gene duplications and specializations. It is curious that Dα1, which is more similar to ancient plant and fungal importin α1-like genes than to Dα2 or Dα3, exhibits paralog-specific phenotypes only in derived processes such as gametogenesis and wing development. It is hypothesized that α1 genes are not functionally constrained; rather, ancestral α2/α3 genes simply diverged. Why ancestral α2 and α3 genes evolved more rapidly remains a mystery, although important clues no doubt lie among their largely unexplored NLS cargo repertoires. It will be extremely interesting to learn if these roles and relationships are conserved in the more complex mouse and human importin α-gene family (Ratan, 2008).

Domains of Importin-alpha2 required for ring canal assembly during Drosophila oogenesis

Null-mutation in Drosophila importin-α2, such as the deficiency imp-α2(D14), causes recessive female sterility with the formation of dumpless eggs. In imp-α2(D14) the transfer of nurse cell components to the oocyte is interrupted and the Kelch protein, an oligomeric ring canal actin organizer, is normally produced but fails to associate with the ring canals resulting in their occlusion. To define domains regulating Kelch deposition on ring canals site-directed mutagenesis was performed on protein binding domains and putative phosphorylation sites of Imp-α2. Phenotypic analysis of the mutant transgenes in imp-α2(D14) revealed that mutations affecting the Imp-beta binding-domain, the dimerization domain, and specific serine residues of putative phosphorylation sites led to a normal or nearly normal oogenesis but arrested early embryonic development, whereas mutations in the nuclear localization signal (NLS) and CAS/exportin binding domains resulted in ring canal occlusion and a drastic nuclear accumulation of the mutant proteins. Deletion of the Imp-beta binding domain also gave rise to a nuclear localization of the mutant protein, which partially retained its function in ring canal assembly. Thus, it is proposed that mutations in NLS and CAS binding domains affect the deposition of Kelch onto the ring canals and prevent the association of Imp-α2 with a negative regulator of Kelch function (Gorjanacz, 2006).

Drosophila melanogaster importin α1 and α3 can replace importin α2 during spermatogenesis but not oogenesis.

Importin α's mediate the nuclear transport of many classical nuclear localization signal (cNLS)-containing proteins. Multicellular animals contain multiple importin α genes, most of which fall into three conventional phylogenetic clades, designated α1, α2, and α3. Using degenerate PCR, Drosophila importin α1, α2, and α3 genes were cloned, demonstrating that the complete conventional importin α gene family arose prior to the split between invertebrates and vertebrates. The genetic interactions among conventional importin α genes were analyzed by studying their capacity to rescue the male and female sterility of importin α2 null flies. The sterility of α2 null males was rescued to similar extents by importin α1, α2, and α3 transgenes, suggesting that all three conventional importin α's are capable of performing the important role of importin α2 during spermatogenesis. In contrast, sterility of α2 null females was rescued only by importin α2 transgenes, suggesting that it plays a paralog-specific role in oogenesis. Female infertility was also rescued by a mutant importin α2 transgene lacking a site that is normally phosphorylated in ovaries. These rescue experiments suggest that male and female gametogenesis have distinct requirements for importin α2 (Mason, 2002).

Patterns of importin α expression during Drosophila spermatogenesis

Importin-α proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-α genes are present in the genome of Drosophila. To gain further insights into their function their expression during spermatogenesis was analyzed by using antibodies raised against each of the three Importin-α proteins identified in Drosophila, namely, Imp-α1, Imp-α2, and Imp-α3. Each Imp-α was found to be expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-α2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-α2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-α2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-α1 and -α3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-α1 expression extends up to the onset of the elongation phase, whereas that of Imp-α3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-α1 and -α3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-α1 and -α3 are nuclear. These data indicate that each Imp-α protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes (Giarre, 2002).

Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation

Drosophila Pendulin exhibits dynamic intracellular localization and plays a role in the control of cell proliferation. Pendulin is a new member of a superfamily of proteins that contains Armadillo (Arm) repeats and displays extensive sequence similarity with the Srp1 protein from yeast, with RAG-1 interacting proteins from humans, and with the importin protein from Xenopus. Almost the entire polypeptide chain of Pendulin is composed of degenerate tandem repeats of approximately 42 amino acids each. A short NH2-terminal domain contains adjacent consensus sequences for nuclear localization and cdc2 kinase phosphorylation. The subcellular distribution of Pendulin is dependent on cell cycle phase. During interphase, Pendulin protein is found exclusively in the cytoplasm of embryonic cells. At the transition between G2 and M-phase, Pendulin rapidly translocates into the nuclei, where it is distributed throughout the nucleoplasm and the areas around the chromosomes. In the larval CNS, Pendulin is predominantly expressed in the dividing neuroblasts, where it undergoes the same cell cycle-dependent redistribution as in embryos. Pendulin is encoded by the oho31 locus and is expressed both maternally and zygotically. Recessive lethal mutations in the oho31 gene result in a massive decrease or loss of zygotic Pendulin expression. Hematopoietic cells of mutant larvae overproliferate and form melanotic tumors, suggesting that Pendulin normally acts as a blood cell tumor suppressor. In contrast, growth and proliferation in imaginal tissues are reduced and irregular, resulting in abnormal development of imaginal discs and the CNS of the larvae. This phenotype shows that Pendulin is required for normal growth regulation. Based on the structure of the protein, it is proposed that Pendulin may serve as an adaptor molecule to form complexes with other proteins. The sequence similarity with importin indicates that Pendulin may play a role in the nuclear import of karyophilic proteins and some of these may be required for the normal transmission and function of proliferative signals in the cells (Kussel, 1995).

Functions of Importinα orthologs in other species

A fast-evolving X-linked duplicate of importin-α2 is overexpressed in sex-ratio drive in Drosophila neotestacea

Selfish genetic elements that manipulate gametogenesis to achieve a transmission advantage are known as meiotic drivers. Sex-ratio X-chromosomes (SR) are meiotic drivers that prevent the maturation of Y-bearing sperm in male carriers to result in the production of mainly female progeny. The spread of an SR chromosome can affect host genetic diversity and genome evolution, and can even cause host extinction if it reaches sufficiently high prevalence. Meiotic drivers have evolved independently many times, though only in a few cases is the underlying genetic mechanism known. This study use a combination of transcriptomics and population genetics to identify widespread expression differences between the standard (ST) and sex-ratio (SR) X-chromosomes of the fly Drosophila neotestacea. The X-chromosome was found to be enriched for differentially expressed transcripts, and many of these X-linked differentially expressed transcripts had elevated Ka /Ks values between ST and SR, indicative of potential functional differences. A set of candidate transcripts was identified, including a testis-specific, X-linked duplicate of the nuclear transport gene importin-α2 that is overexpressed in SR. Suggestions were found of positive selection in the lineage leading to the duplicate, and its molecular evolutionary patterns are consistent with relaxed purifying selection in ST. As these patterns are consistent with involvement in the mechanism of drive in this species, this duplicate is a strong candidate worthy of further functional investigation. Nuclear transport may be a common target for genetic conflict, as the mechanism of the autosomal Segregation Distorter drive system in D. melanogaster involves the same pathway (Pieper, 2018).


Search PubMed for articles about Drosophila Pendulin

Asaoka, M., Hanyu-Nakamura, K., Nakamura, A. and Kobayashi, S. (2019). Maternal Nanos inhibits Importin-alpha2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline. PLoS Genet 15(5): e1008090. PubMed ID: 31091233

Geles, K. G. and Adam, S. A. (2001). Germline and developmental roles of the nuclear transport factor importin alpha3 in C. elegans. Development 128: 1817-1830. PubMed ID: 11311162

Geles, K. G., Johnson, J. J., Jong S. and Adam, S. A. (2002). A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis. Mol. Biol. Cell 13: 3138-3147. PubMed ID: 12221121

Giarre, M., et al. (2002). Patterns of importin alpha expression during Drosophila spermatogenesis. J. Struct. Biol. 140 279-290. PubMed ID: 12490175

Gilboa, L. and Lehmann, R. (2004). How different is Venus from Mars? The genetics of germ-line stem cells in Drosophila females and males. Development 131: 4895-4905. PubMed ID: 15459096

Goldfarb, D. S., et al. (2004). Importin α: a multipurpose nuclear transport receptor. Trends Cell Biol. 14: 505-514. PubMed ID: 15350979

Gorjanacz, M., et al. (2002). Importin-alpha 2 is critically required for the assembly of ring canals during Drosophila oogenesis. Dev. Biol. 251 271-282. PubMed ID: 12435357

Gorjanacz, M., Torok, I., Pomozi, I., Garab, G., Szlanka, T., Kiss, I. and Mechler, B. M. (2006). Domains of Importin-alpha2 required for ring canal assembly during Drosophila oogenesis. J Struct Biol 154(1): 27-41. PubMed ID: 16458020

Hogarth, C., Itman, C., Jans, D. A. and Loveland, K. L. (2005). Regulated nucleocytoplasmic transport in spermatogenesis: A driver of cellular differentiation? BioEssays 27: 1011-1025. PubMed ID: 16163727

Hogarth, C. A., Calanni, S., Jans, D. A. and Loveland, K. L. (2006). Importin alpha mRNAs have distinct expression profiles during spermatogenesis. Dev. Dyn. 235: 253-262. PubMed ID: 16261624

Kamei, Y., Yuba, S., Nakayma, T. and Yoneda, Y. (1999). Three distinct classes of the alpha-subunit of the nuclear pore-targeting complex (importin-alpha) are differentially expressed in adult mouse tissues. J. Histochem. Cytochem. 47: 363-372. PubMed ID: 10026238

Kussel, P. and Frasch, M. (1995). Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation. J Cell Biol 129(6): 1491-1507. PubMed ID: 7790350

Mason, D. A., Fleming, R. J. and Goldfarb, D. S. (2002). Drosophila melanogaster importin α1 and α3 can replace importin α2 during spermatogenesis but not oogenesis. Genetics 161: 157-170. PubMed ID: 12019231

Mason, D. A., Mathe, E., Fleming, R. J. and Goldfarb, D.S. (2003). The Drosophila melanogaster importin α3 locus encodes an essential gene required for the development of both larval and adult tissues. Genetics 165: 1943-1958. PubMed ID: 14704178

Mosca, T. J. and Schwarz, T. L. (2010a). The nuclear import of Frizzled2-C by Importins-beta11 and alpha2 promotes postsynaptic development. Nat Neurosci 13(8): 935-943. PubMed ID: 20601947

Mosca, T. J. and Schwarz, T. L. (2010b). Drosophila Importin-alpha2 is involved in synapse, axon and muscle development. PLoS One 5(12): e15223. PubMed ID: 21151903

Pieper, K. E., Unckless, R. L. and Dyer, K. A. (2018). A fast-evolving X-linked duplicate of importin-α2 is overexpressed in sex-ratio drive in Drosophila neotestacea. Mol Ecol. PubMed ID: 30411843

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

Yashiro, R., Murota, Y., Nishida, K. M., Yamashiro, H., Fujii, K., Ogai, A., Yamanaka, S., Negishi, L., Siomi, H. and Siomi, M. C. (2018). Piwi Nuclear Localization and Its Regulatory Mechanism in Drosophila Ovarian Somatic Cells. Cell Rep 23(12): 3647-3657. PubMed ID: 29925005

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date revised: 25 June 2019

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