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Identification of Conserved MEL-28/ELYS Domains with Essential Roles in Nuclear Assembly and Chromosome Segregation.

Gómez-Saldivar G, Fernandez A, Hirano Y, Mauro M, Lai A, Ayuso C, Haraguchi T, Hiraoka Y, Piano F, Askjaer P - PLoS Genet. (2016)

Bottom Line: The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes.We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation.Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Developmental Biology (CABD), CSIC/Junta de Andalucia/Universidad Pablo de Olavide, Seville, Spain.

ABSTRACT
Nucleoporins are the constituents of nuclear pore complexes (NPCs) and are essential regulators of nucleocytoplasmic transport, gene expression and genome stability. The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes. Here we present a systematic functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28's conserved AT-hook domain do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression in a DNA damage checkpoint-dependent manner. Our analyses also uncover a novel meiotic role of MEL-28. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

No MeSH data available.


Related in: MedlinePlus

The AT-hook domain of MEL-28 is required for nuclear growth, chromosome segregation and cell cycle timing.(A) Still images from time-lapse recordings of control (middle) and mel-28 (right) embryos expressing GFP::MEL-281-1629 as well as a mel-28 embryo expressing GFP::MEL-28 (left). Note defective chromosome segregation in the right embryo. Scale bars, 5 μm. Nuclear growth (B) and distribution of GFP fusion protein between nucleoplasm and cytoplasm (C) was specifically reduced in mel-28 embryos expressing GFP::MEL-281-1629. Measurements were performed on fully-grown P1 nuclei. (D) Asynchrony between division of AB and P1 blastomeres was significantly delayed in mel-28 embryos expressing GFP::MEL-281-1629; this delay was partially reduced by depletion of ATL-1. (E) Four-cell stage embryos from mel-28 mutants expressing either GFP::MEL-28 or GFP::MEL-281-1629 were analyzed with Hoechst (blue in merge), a specific antibody against NPP-10C/NUP96 (green in merge) and mAb414 recognizing multiple nups (red in merge). Scale bars, 5 μm. *** p<0.001 by unpaired two-tailed t-test.
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pgen.1006131.g006: The AT-hook domain of MEL-28 is required for nuclear growth, chromosome segregation and cell cycle timing.(A) Still images from time-lapse recordings of control (middle) and mel-28 (right) embryos expressing GFP::MEL-281-1629 as well as a mel-28 embryo expressing GFP::MEL-28 (left). Note defective chromosome segregation in the right embryo. Scale bars, 5 μm. Nuclear growth (B) and distribution of GFP fusion protein between nucleoplasm and cytoplasm (C) was specifically reduced in mel-28 embryos expressing GFP::MEL-281-1629. Measurements were performed on fully-grown P1 nuclei. (D) Asynchrony between division of AB and P1 blastomeres was significantly delayed in mel-28 embryos expressing GFP::MEL-281-1629; this delay was partially reduced by depletion of ATL-1. (E) Four-cell stage embryos from mel-28 mutants expressing either GFP::MEL-28 or GFP::MEL-281-1629 were analyzed with Hoechst (blue in merge), a specific antibody against NPP-10C/NUP96 (green in merge) and mAb414 recognizing multiple nups (red in merge). Scale bars, 5 μm. *** p<0.001 by unpaired two-tailed t-test.

Mentions: Comparing the behavior of GFP::MEL-281239-1601 and GFP::MEL-281188-1784 indicated that MEL-28’s two AT hooks are not required for chromatin association, at least during mitosis (Fig 5). Moreover, in vitro binding experiments found no difference in chromatin affinity between recombinant peptides that contained either the C-terminal 128 aa of Xenopus ELYS including the single ELYS AT hook or a variant with mutated AT hook although the former was more efficient in competition assays [4]. In agreement with the competition assay, it was independently demonstrated that the same 128-aa. peptide efficiently binds nucleosome beads but not when the AT hook is mutated [13]. However, both studies concluded that the 128-aa. peptide contains residues outside the AT hook important for chromatin and nucleosome interaction. We attempted to address this in further detail, but we were unable to detect expression of a construct encoding the C-terminal 161 aa. of MEL-28 fused to GFP (S4B Fig; GFP::MEL-281624-1784). A shorter 48-aa. fragment containing a single AT hook localized similarly to free GFP (S4A Fig GFP::MEL-281740-1784). As a complementary approach, we examined the consequences of deleting the AT hooks from full-length MEL-28. We first compared mel-28/+ embryos expressing GFP::MEL-281-1629 (GFP::MEL-28ΔAT) with mel-28 embryos expressing full-length MEL-28 fused to GFP. Time-lapse confocal microscopy demonstrated that the mel-28/+; GFP::MEL-281-1629 embryos developed normally and the fluorescent protein localized similarly to GFP::MEL-28 (Fig 6A; compare left and middle panels; S9 and S10 Videos). In the absence of endogenous MEL-28, GFP::MEL-281-1629 still accumulated at the periphery of interphase nuclei and to kinetochores of mitotic chromosomes (Fig 6A; right panels; S10 Video). This was in contrast to the severe phenotypes observed in MEL-28loop2mut::GFP embryos (Fig 3A) and suggested that MEL-28’s function in post-mitotic nuclear assembly is not strictly dependent on the AT hook domain. However, mel-28; GFP::MEL-281-1629 embryos were unviable (Table 1) and displayed several defects. Most prominently, daughter nuclei were often (n = 5/7) trapped at the cleavage furrow during cytokinesis of the anterior AB blastomere of two-cell stage embryos (Fig 6A, right panels; 27:31–34:30). More direct evidence for chromosome segregation failure was obtained by immunofluorescence analysis of four-cell stage embryos, which also demonstrated that NPP-10C/NUP96 and other Nups accumulated at the NE of mel-28; GFP::MEL-281-1629 embryos, albeit in an irregular pattern (Fig 6E). In addition, nuclear growth was significantly reduced in GFP::MEL-281-1629 embryos (Fig 6A, third row; Fig 6B), consistent with defects in NPC-mediated nucleocytoplasmic transport [26]. While nuclei from mel-28; GFP::MEL-28 and mel-28/+; GFP::MEL-281-1629 grew to the same size (363.8 ± 19 μm3 and 363.3 ± 63 μm3; respectively), the maximum volume of P1 nuclei was reduced by 32% in mel-28; GFP::MEL-281-1629 embryos (346.6 ± 44 μm3). We also noticed that the nucleoplasmic pool of GFP::MEL-281-1629 was strongly diminished in mel-28 embryos compared to GFP::MEL-28 in mel-28 embryos and GFP::MEL-281-1629 in mel-28/+ embryos (Fig 6A and 6C). Whereas the ratio between nucleoplasmic and cytoplasmic GFP signal was similar between mel-28; GFP::MEL-28 and mel-28/+; GFP::MEL-281-1629 embryos (5.60 ± 1.29 and 4.72 ± 0.99; respectively), the ratio was 87% lower in mel-28; GFP::MEL-281-1629 embryos (0.76 ± 0.18). These data are compatible with a model in which GFP::MEL-281-1629 has reduced affinity for interphase chromatin and therefore accumulates at NPCs: in mel-28/+ embryos interaction of GFP::MEL-281-1629 with endogenous MEL-28 accumulates the former in the nucleoplasm, potentially interacting with chromatin.


Identification of Conserved MEL-28/ELYS Domains with Essential Roles in Nuclear Assembly and Chromosome Segregation.

Gómez-Saldivar G, Fernandez A, Hirano Y, Mauro M, Lai A, Ayuso C, Haraguchi T, Hiraoka Y, Piano F, Askjaer P - PLoS Genet. (2016)

The AT-hook domain of MEL-28 is required for nuclear growth, chromosome segregation and cell cycle timing.(A) Still images from time-lapse recordings of control (middle) and mel-28 (right) embryos expressing GFP::MEL-281-1629 as well as a mel-28 embryo expressing GFP::MEL-28 (left). Note defective chromosome segregation in the right embryo. Scale bars, 5 μm. Nuclear growth (B) and distribution of GFP fusion protein between nucleoplasm and cytoplasm (C) was specifically reduced in mel-28 embryos expressing GFP::MEL-281-1629. Measurements were performed on fully-grown P1 nuclei. (D) Asynchrony between division of AB and P1 blastomeres was significantly delayed in mel-28 embryos expressing GFP::MEL-281-1629; this delay was partially reduced by depletion of ATL-1. (E) Four-cell stage embryos from mel-28 mutants expressing either GFP::MEL-28 or GFP::MEL-281-1629 were analyzed with Hoechst (blue in merge), a specific antibody against NPP-10C/NUP96 (green in merge) and mAb414 recognizing multiple nups (red in merge). Scale bars, 5 μm. *** p<0.001 by unpaired two-tailed t-test.
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pgen.1006131.g006: The AT-hook domain of MEL-28 is required for nuclear growth, chromosome segregation and cell cycle timing.(A) Still images from time-lapse recordings of control (middle) and mel-28 (right) embryos expressing GFP::MEL-281-1629 as well as a mel-28 embryo expressing GFP::MEL-28 (left). Note defective chromosome segregation in the right embryo. Scale bars, 5 μm. Nuclear growth (B) and distribution of GFP fusion protein between nucleoplasm and cytoplasm (C) was specifically reduced in mel-28 embryos expressing GFP::MEL-281-1629. Measurements were performed on fully-grown P1 nuclei. (D) Asynchrony between division of AB and P1 blastomeres was significantly delayed in mel-28 embryos expressing GFP::MEL-281-1629; this delay was partially reduced by depletion of ATL-1. (E) Four-cell stage embryos from mel-28 mutants expressing either GFP::MEL-28 or GFP::MEL-281-1629 were analyzed with Hoechst (blue in merge), a specific antibody against NPP-10C/NUP96 (green in merge) and mAb414 recognizing multiple nups (red in merge). Scale bars, 5 μm. *** p<0.001 by unpaired two-tailed t-test.
Mentions: Comparing the behavior of GFP::MEL-281239-1601 and GFP::MEL-281188-1784 indicated that MEL-28’s two AT hooks are not required for chromatin association, at least during mitosis (Fig 5). Moreover, in vitro binding experiments found no difference in chromatin affinity between recombinant peptides that contained either the C-terminal 128 aa of Xenopus ELYS including the single ELYS AT hook or a variant with mutated AT hook although the former was more efficient in competition assays [4]. In agreement with the competition assay, it was independently demonstrated that the same 128-aa. peptide efficiently binds nucleosome beads but not when the AT hook is mutated [13]. However, both studies concluded that the 128-aa. peptide contains residues outside the AT hook important for chromatin and nucleosome interaction. We attempted to address this in further detail, but we were unable to detect expression of a construct encoding the C-terminal 161 aa. of MEL-28 fused to GFP (S4B Fig; GFP::MEL-281624-1784). A shorter 48-aa. fragment containing a single AT hook localized similarly to free GFP (S4A Fig GFP::MEL-281740-1784). As a complementary approach, we examined the consequences of deleting the AT hooks from full-length MEL-28. We first compared mel-28/+ embryos expressing GFP::MEL-281-1629 (GFP::MEL-28ΔAT) with mel-28 embryos expressing full-length MEL-28 fused to GFP. Time-lapse confocal microscopy demonstrated that the mel-28/+; GFP::MEL-281-1629 embryos developed normally and the fluorescent protein localized similarly to GFP::MEL-28 (Fig 6A; compare left and middle panels; S9 and S10 Videos). In the absence of endogenous MEL-28, GFP::MEL-281-1629 still accumulated at the periphery of interphase nuclei and to kinetochores of mitotic chromosomes (Fig 6A; right panels; S10 Video). This was in contrast to the severe phenotypes observed in MEL-28loop2mut::GFP embryos (Fig 3A) and suggested that MEL-28’s function in post-mitotic nuclear assembly is not strictly dependent on the AT hook domain. However, mel-28; GFP::MEL-281-1629 embryos were unviable (Table 1) and displayed several defects. Most prominently, daughter nuclei were often (n = 5/7) trapped at the cleavage furrow during cytokinesis of the anterior AB blastomere of two-cell stage embryos (Fig 6A, right panels; 27:31–34:30). More direct evidence for chromosome segregation failure was obtained by immunofluorescence analysis of four-cell stage embryos, which also demonstrated that NPP-10C/NUP96 and other Nups accumulated at the NE of mel-28; GFP::MEL-281-1629 embryos, albeit in an irregular pattern (Fig 6E). In addition, nuclear growth was significantly reduced in GFP::MEL-281-1629 embryos (Fig 6A, third row; Fig 6B), consistent with defects in NPC-mediated nucleocytoplasmic transport [26]. While nuclei from mel-28; GFP::MEL-28 and mel-28/+; GFP::MEL-281-1629 grew to the same size (363.8 ± 19 μm3 and 363.3 ± 63 μm3; respectively), the maximum volume of P1 nuclei was reduced by 32% in mel-28; GFP::MEL-281-1629 embryos (346.6 ± 44 μm3). We also noticed that the nucleoplasmic pool of GFP::MEL-281-1629 was strongly diminished in mel-28 embryos compared to GFP::MEL-28 in mel-28 embryos and GFP::MEL-281-1629 in mel-28/+ embryos (Fig 6A and 6C). Whereas the ratio between nucleoplasmic and cytoplasmic GFP signal was similar between mel-28; GFP::MEL-28 and mel-28/+; GFP::MEL-281-1629 embryos (5.60 ± 1.29 and 4.72 ± 0.99; respectively), the ratio was 87% lower in mel-28; GFP::MEL-281-1629 embryos (0.76 ± 0.18). These data are compatible with a model in which GFP::MEL-281-1629 has reduced affinity for interphase chromatin and therefore accumulates at NPCs: in mel-28/+ embryos interaction of GFP::MEL-281-1629 with endogenous MEL-28 accumulates the former in the nucleoplasm, potentially interacting with chromatin.

Bottom Line: The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes.We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation.Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Developmental Biology (CABD), CSIC/Junta de Andalucia/Universidad Pablo de Olavide, Seville, Spain.

ABSTRACT
Nucleoporins are the constituents of nuclear pore complexes (NPCs) and are essential regulators of nucleocytoplasmic transport, gene expression and genome stability. The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes. Here we present a systematic functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28's conserved AT-hook domain do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression in a DNA damage checkpoint-dependent manner. Our analyses also uncover a novel meiotic role of MEL-28. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

No MeSH data available.


Related in: MedlinePlus