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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

MEL-28 is essential for female meiosis.(A) GFP::MEL-28 (green in merged images) was expressed in oocytes and accumulated at kinetochores of meiotic chromosomes (visualized with mCherry::HisH2B; magenta in merge). Shown are the four most proximal oocytes where position -1 is immediately next to the spermatheca. The -1 oocyte was observed every two minutes until germinal vesicle breakdown. (B) GFP::MEL-28 associated with chromosomes throughout meiosis I and II and accumulated at the NE at pronuclear formation. (C) Chromosomes (magenta) and meiotic spindles (green) were observed in utero. Anaphase I and II were characterized by abundant microtubules between segregating chromosomes in control mel-28/+ animals (top) whereas chromosomes failed to segregate in homozygous mel-28 mutants (bottom). White arrows point to segregating chromosomes. Red arrowheads and white asterisks mark sperm and somatic nuclei, respectively, outside the fertilized oocyte; yellow arrowheads indicate polar bodies. Time is indicated relative to germinal vesicle breakdown (min:sec). Scale bars, 5 μm.
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pgen.1006131.g001: MEL-28 is essential for female meiosis.(A) GFP::MEL-28 (green in merged images) was expressed in oocytes and accumulated at kinetochores of meiotic chromosomes (visualized with mCherry::HisH2B; magenta in merge). Shown are the four most proximal oocytes where position -1 is immediately next to the spermatheca. The -1 oocyte was observed every two minutes until germinal vesicle breakdown. (B) GFP::MEL-28 associated with chromosomes throughout meiosis I and II and accumulated at the NE at pronuclear formation. (C) Chromosomes (magenta) and meiotic spindles (green) were observed in utero. Anaphase I and II were characterized by abundant microtubules between segregating chromosomes in control mel-28/+ animals (top) whereas chromosomes failed to segregate in homozygous mel-28 mutants (bottom). White arrows point to segregating chromosomes. Red arrowheads and white asterisks mark sperm and somatic nuclei, respectively, outside the fertilized oocyte; yellow arrowheads indicate polar bodies. Time is indicated relative to germinal vesicle breakdown (min:sec). Scale bars, 5 μm.

Mentions: MEL-28 strongly accumulated on condensed oocyte chromosomes (S1C Fig; [9, 18]). Moreover, we noted during our initial studies of mel-28 mutant or RNAi-treated embryos that formation and migration of the maternal pronucleus was often more severely affected than the paternal pronucleus [9, 10]. Based on these observations we speculated that MEL-28 might have important functions in meiosis. C. elegans oocytes are arranged in a linear fashion in the proximal part of the gonad, where each oocyte is numbered relative to the spermatheca (-1, -2, -3, etc.) [19]. The -1 oocyte completes maturation including germinal vesicle breakdown immediately before ovulation and fertilization triggers rapid progression through meiosis I and II. To examine these processes we performed live in utero recordings of animals expressing GFP::MEL-28 and mCherry::HisH2B. In the -4 oocyte, MEL-28 localized to the NE and was absent from condensed chromosomes (Fig 1A). In the -3 and -2 oocytes MEL-28 gradually moved away from the NE and accumulated uniformly on meiotic chromosomes. Later, in the -1 oocyte MEL-28 redistributed to cover the surface of meiotic chromosomes (Fig 1A; S1 Video), in some cases completely enclosing the chromosomes and in other cases similar to the “cup-shaped” localization of kinetochore proteins, such as KNL-1 and KNL-3 [20]. The association of MEL-28 with chromosomes persisted throughout meiosis I and II until pronuclear formation ~30 minutes after germinal vesicle breakdown (Fig 1B; S1 Video). The localization pattern of MEL-28 suggested a possible role during segregation of meiotic chromosomes, similar to the situation in mitosis [9, 10]. We therefore analyzed mel-28(t1684) embryos expressing GFP::β-tubulin and mCherry::HisH2B. mel-28(t1684) encodes a premature termination codon at aa. 766 and behaves like a strong loss-of-function of MEL-28, presumably due to nonsense-mediated mRNA decay [10]. Maternal contribution enables homozygous mel-28(t1684) hermaphrodites to develop until adulthood but they produce only unviable embryos (hereafter referred to as mel-28 embryos, whereas embryos produced by heterozygous siblings are referred to as control or mel-28/+ embryos) with severe NE assembly defects [10]. Strikingly, in mel-28 embryos chromosomes failed to segregate in anaphase I (n = 5/6 embryos) and anaphase II (n = 4/6) and, consequently, mel-28 embryos had either no (n = 4/6) or a single (n = 2/6) polar body, whereas control embryos had two polar bodies (n = 6/6; Fig 1C; S2 Video). In addition, chromosomes in mel-28 embryos were not organized in a pronucleus but appeared scattered in the cytoplasm (Fig 1C; 36:00). To our knowledge, this is the first report describing the involvement of MEL-28/ELYS in meiosis, expanding previously described MEL-28 functions and establishing an important role in chromosome segregation during both meiosis and mitosis.


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)

MEL-28 is essential for female meiosis.(A) GFP::MEL-28 (green in merged images) was expressed in oocytes and accumulated at kinetochores of meiotic chromosomes (visualized with mCherry::HisH2B; magenta in merge). Shown are the four most proximal oocytes where position -1 is immediately next to the spermatheca. The -1 oocyte was observed every two minutes until germinal vesicle breakdown. (B) GFP::MEL-28 associated with chromosomes throughout meiosis I and II and accumulated at the NE at pronuclear formation. (C) Chromosomes (magenta) and meiotic spindles (green) were observed in utero. Anaphase I and II were characterized by abundant microtubules between segregating chromosomes in control mel-28/+ animals (top) whereas chromosomes failed to segregate in homozygous mel-28 mutants (bottom). White arrows point to segregating chromosomes. Red arrowheads and white asterisks mark sperm and somatic nuclei, respectively, outside the fertilized oocyte; yellow arrowheads indicate polar bodies. Time is indicated relative to germinal vesicle breakdown (min:sec). Scale bars, 5 μm.
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Related In: Results  -  Collection

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pgen.1006131.g001: MEL-28 is essential for female meiosis.(A) GFP::MEL-28 (green in merged images) was expressed in oocytes and accumulated at kinetochores of meiotic chromosomes (visualized with mCherry::HisH2B; magenta in merge). Shown are the four most proximal oocytes where position -1 is immediately next to the spermatheca. The -1 oocyte was observed every two minutes until germinal vesicle breakdown. (B) GFP::MEL-28 associated with chromosomes throughout meiosis I and II and accumulated at the NE at pronuclear formation. (C) Chromosomes (magenta) and meiotic spindles (green) were observed in utero. Anaphase I and II were characterized by abundant microtubules between segregating chromosomes in control mel-28/+ animals (top) whereas chromosomes failed to segregate in homozygous mel-28 mutants (bottom). White arrows point to segregating chromosomes. Red arrowheads and white asterisks mark sperm and somatic nuclei, respectively, outside the fertilized oocyte; yellow arrowheads indicate polar bodies. Time is indicated relative to germinal vesicle breakdown (min:sec). Scale bars, 5 μm.
Mentions: MEL-28 strongly accumulated on condensed oocyte chromosomes (S1C Fig; [9, 18]). Moreover, we noted during our initial studies of mel-28 mutant or RNAi-treated embryos that formation and migration of the maternal pronucleus was often more severely affected than the paternal pronucleus [9, 10]. Based on these observations we speculated that MEL-28 might have important functions in meiosis. C. elegans oocytes are arranged in a linear fashion in the proximal part of the gonad, where each oocyte is numbered relative to the spermatheca (-1, -2, -3, etc.) [19]. The -1 oocyte completes maturation including germinal vesicle breakdown immediately before ovulation and fertilization triggers rapid progression through meiosis I and II. To examine these processes we performed live in utero recordings of animals expressing GFP::MEL-28 and mCherry::HisH2B. In the -4 oocyte, MEL-28 localized to the NE and was absent from condensed chromosomes (Fig 1A). In the -3 and -2 oocytes MEL-28 gradually moved away from the NE and accumulated uniformly on meiotic chromosomes. Later, in the -1 oocyte MEL-28 redistributed to cover the surface of meiotic chromosomes (Fig 1A; S1 Video), in some cases completely enclosing the chromosomes and in other cases similar to the “cup-shaped” localization of kinetochore proteins, such as KNL-1 and KNL-3 [20]. The association of MEL-28 with chromosomes persisted throughout meiosis I and II until pronuclear formation ~30 minutes after germinal vesicle breakdown (Fig 1B; S1 Video). The localization pattern of MEL-28 suggested a possible role during segregation of meiotic chromosomes, similar to the situation in mitosis [9, 10]. We therefore analyzed mel-28(t1684) embryos expressing GFP::β-tubulin and mCherry::HisH2B. mel-28(t1684) encodes a premature termination codon at aa. 766 and behaves like a strong loss-of-function of MEL-28, presumably due to nonsense-mediated mRNA decay [10]. Maternal contribution enables homozygous mel-28(t1684) hermaphrodites to develop until adulthood but they produce only unviable embryos (hereafter referred to as mel-28 embryos, whereas embryos produced by heterozygous siblings are referred to as control or mel-28/+ embryos) with severe NE assembly defects [10]. Strikingly, in mel-28 embryos chromosomes failed to segregate in anaphase I (n = 5/6 embryos) and anaphase II (n = 4/6) and, consequently, mel-28 embryos had either no (n = 4/6) or a single (n = 2/6) polar body, whereas control embryos had two polar bodies (n = 6/6; Fig 1C; S2 Video). In addition, chromosomes in mel-28 embryos were not organized in a pronucleus but appeared scattered in the cytoplasm (Fig 1C; 36:00). To our knowledge, this is the first report describing the involvement of MEL-28/ELYS in meiosis, expanding previously described MEL-28 functions and establishing an important role in chromosome segregation during both meiosis and mitosis.

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