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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 loop2 is required during meiosis and mitosis.(A) Still images from time-lapse recordings of control (left) and mel-28 (right) embryos expressing MEL-28loop2mut::GFP. Note the presence of two polar bodies in the left embryo but only a single polar body in the right embryo (yellow arrowheads). Concordantly, two oocyte-derived pronuclei were observed in the right embryo (white arrowheads). Red arrowheads indicate sperm-derived chromosomes. Whole-embryo images are max projections; inserts are single confocal sections. Scale bars, 5 μm. (B) Frequency of embryos with a single or two polar bodies. ** p<0.01 by Fisher exact test. (C) Timing from P0 division to P1 division is significantly delayed in mel-28 embryos expressing MEL-28loop2mut::GFP. *** p<0.001 by unpaired two-tailed t-test.
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pgen.1006131.g003: MEL-28 loop2 is required during meiosis and mitosis.(A) Still images from time-lapse recordings of control (left) and mel-28 (right) embryos expressing MEL-28loop2mut::GFP. Note the presence of two polar bodies in the left embryo but only a single polar body in the right embryo (yellow arrowheads). Concordantly, two oocyte-derived pronuclei were observed in the right embryo (white arrowheads). Red arrowheads indicate sperm-derived chromosomes. Whole-embryo images are max projections; inserts are single confocal sections. Scale bars, 5 μm. (B) Frequency of embryos with a single or two polar bodies. ** p<0.01 by Fisher exact test. (C) Timing from P0 division to P1 division is significantly delayed in mel-28 embryos expressing MEL-28loop2mut::GFP. *** p<0.001 by unpaired two-tailed t-test.

Mentions: Bilokapic and Schwartz identified through protein crystallization and sequence alignments two conserved loops (loop1 and loop2) on the surface of the β-propeller of ELYS [15]. When they substituted 5 aa. within loop2 the structural fold of the β-propeller was maintained but NPC localization of the N-terminal half of ELYS (aa. 1–1018) fused to GFP was abrogated in HeLa cells. To test the relevance of loop2 in the context of full-length protein we introduced the equivalent aa. substitutions in MEL-28 (D409S/Y412S/R415A/V416S/P417G; MEL-28loop2mut; Fig 3A). In mel-28/+ embryos MEL-28loop2mut::GFP localized normally during interphase and mitosis (Fig 3A, left panels; compare with wild type GFP::MEL-28 in Fig 2A; S4 Video; S3 Fig), suggesting that loop2 residues are not essential for association of full-length MEL-28 with NPCs or kinetochores. However, MEL-28loop2mut::GFP was not able to substitute for endogenous MEL-28: mel-28 embryos expressing MEL-28loop2mut::GFP were unviable (Table 1) and had frequent meiosis defects as evidenced by failure in polar body extrusion and presence of multiple female pronuclei (Fig 3A, right panels; S4 Video; Fig 3B). Moreover, pronuclei were abnormally small, contained less MEL-28loop2mut::GFP and did not position properly. In 83% of mel-28; MEL-28loop2mut::GFP embryos (n = 10/12) female and male pronuclei did not meet before the first mitotic division. Instead, only the male pronucleus was positioned between the centrosomes, whereas female pronuclei exhibited shorter migration and remained in the anterior of the embryo. During mitosis chromosomes failed to congress to the metaphase plate (Fig 3A; 0:00) and severe segregation defects were observed (Fig 3A; 20:00–31:45). We also noticed alterations in cell cycle timing, in particular for the posterior P1 blastomere at the two-cell stage. In mel-28; GFP::MEL-28 and mel-28/+; MEL-28loop2mut::GFP embryos the cell cycle of P1 lasted ~1075 sec, whereas it lasted ~1513 sec (41% delay) in mel-28 embryos expressing MEL-28loop2mut::GFP (Fig 3C). Other frequent defects included cleavage furrow regression (37%; n = 6/16) and abnormal positioning of cells within the eggshell (53%; n = 8/15).


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 loop2 is required during meiosis and mitosis.(A) Still images from time-lapse recordings of control (left) and mel-28 (right) embryos expressing MEL-28loop2mut::GFP. Note the presence of two polar bodies in the left embryo but only a single polar body in the right embryo (yellow arrowheads). Concordantly, two oocyte-derived pronuclei were observed in the right embryo (white arrowheads). Red arrowheads indicate sperm-derived chromosomes. Whole-embryo images are max projections; inserts are single confocal sections. Scale bars, 5 μm. (B) Frequency of embryos with a single or two polar bodies. ** p<0.01 by Fisher exact test. (C) Timing from P0 division to P1 division is significantly delayed in mel-28 embryos expressing MEL-28loop2mut::GFP. *** p<0.001 by unpaired two-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920428&req=5

pgen.1006131.g003: MEL-28 loop2 is required during meiosis and mitosis.(A) Still images from time-lapse recordings of control (left) and mel-28 (right) embryos expressing MEL-28loop2mut::GFP. Note the presence of two polar bodies in the left embryo but only a single polar body in the right embryo (yellow arrowheads). Concordantly, two oocyte-derived pronuclei were observed in the right embryo (white arrowheads). Red arrowheads indicate sperm-derived chromosomes. Whole-embryo images are max projections; inserts are single confocal sections. Scale bars, 5 μm. (B) Frequency of embryos with a single or two polar bodies. ** p<0.01 by Fisher exact test. (C) Timing from P0 division to P1 division is significantly delayed in mel-28 embryos expressing MEL-28loop2mut::GFP. *** p<0.001 by unpaired two-tailed t-test.
Mentions: Bilokapic and Schwartz identified through protein crystallization and sequence alignments two conserved loops (loop1 and loop2) on the surface of the β-propeller of ELYS [15]. When they substituted 5 aa. within loop2 the structural fold of the β-propeller was maintained but NPC localization of the N-terminal half of ELYS (aa. 1–1018) fused to GFP was abrogated in HeLa cells. To test the relevance of loop2 in the context of full-length protein we introduced the equivalent aa. substitutions in MEL-28 (D409S/Y412S/R415A/V416S/P417G; MEL-28loop2mut; Fig 3A). In mel-28/+ embryos MEL-28loop2mut::GFP localized normally during interphase and mitosis (Fig 3A, left panels; compare with wild type GFP::MEL-28 in Fig 2A; S4 Video; S3 Fig), suggesting that loop2 residues are not essential for association of full-length MEL-28 with NPCs or kinetochores. However, MEL-28loop2mut::GFP was not able to substitute for endogenous MEL-28: mel-28 embryos expressing MEL-28loop2mut::GFP were unviable (Table 1) and had frequent meiosis defects as evidenced by failure in polar body extrusion and presence of multiple female pronuclei (Fig 3A, right panels; S4 Video; Fig 3B). Moreover, pronuclei were abnormally small, contained less MEL-28loop2mut::GFP and did not position properly. In 83% of mel-28; MEL-28loop2mut::GFP embryos (n = 10/12) female and male pronuclei did not meet before the first mitotic division. Instead, only the male pronucleus was positioned between the centrosomes, whereas female pronuclei exhibited shorter migration and remained in the anterior of the embryo. During mitosis chromosomes failed to congress to the metaphase plate (Fig 3A; 0:00) and severe segregation defects were observed (Fig 3A; 20:00–31:45). We also noticed alterations in cell cycle timing, in particular for the posterior P1 blastomere at the two-cell stage. In mel-28; GFP::MEL-28 and mel-28/+; MEL-28loop2mut::GFP embryos the cell cycle of P1 lasted ~1075 sec, whereas it lasted ~1513 sec (41% delay) in mel-28 embryos expressing MEL-28loop2mut::GFP (Fig 3C). Other frequent defects included cleavage furrow regression (37%; n = 6/16) and abnormal positioning of cells within the eggshell (53%; n = 8/15).

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