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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 N-terminal domains are required for NPC and kinetochore localization.(A) Still images from time-lapse recording of embryo carrying a GFP insertion into the endogenous mel-28 locus. Time is indicated relative to anaphase onset (min:sec). (B) Metaphase plate of early embryo expressing GFP::MEL-28 (green in merge) analyzed by immunofluorescence with a specific antibody against HCP-3/CENP-A (red in merge) and Hoechst (blue in merge) to visualize chromosomes. MEL-28 localized to kinetochores, which appear as lines on both sides of the chromosomes. (C) Cropped images from embryos expressing different MEL-28 truncations fused to GFP. Except GFP::MEL-28 and GFP::MEL-28Δ1140–1186 embryos, all embryos also expressed un-tagged endogenous MEL-28. Purple boxes in MEL-28 cartoons indicate a putative coiled-coil domain (aa. 1127–1160) whereas yellow (aa. 1630–1642) and orange (aa. 1746–1758) boxes indicate AT-hook sequences: their homology to the consensus AT-hook sequence is low and high, respectively. (D) Cropped images from metaphase embryos expressing GFP::MEL-28 or GFP::MEL-28Δ1140–1186. Images were processed identically to facilitate visualization of full-length GFP::MEL-28 associated with the mitotic spindle. Signal intensities in boxed areas were quantified in raw images, normalized and plotted (n = 5, GFP::MEL-28; n = 2, GFP::MEL-28Δ1140–1186). * p<0.05 by unpaired two-tailed t-test. Scale bars, 5 μm.
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pgen.1006131.g002: MEL-28 N-terminal domains are required for NPC and kinetochore localization.(A) Still images from time-lapse recording of embryo carrying a GFP insertion into the endogenous mel-28 locus. Time is indicated relative to anaphase onset (min:sec). (B) Metaphase plate of early embryo expressing GFP::MEL-28 (green in merge) analyzed by immunofluorescence with a specific antibody against HCP-3/CENP-A (red in merge) and Hoechst (blue in merge) to visualize chromosomes. MEL-28 localized to kinetochores, which appear as lines on both sides of the chromosomes. (C) Cropped images from embryos expressing different MEL-28 truncations fused to GFP. Except GFP::MEL-28 and GFP::MEL-28Δ1140–1186 embryos, all embryos also expressed un-tagged endogenous MEL-28. Purple boxes in MEL-28 cartoons indicate a putative coiled-coil domain (aa. 1127–1160) whereas yellow (aa. 1630–1642) and orange (aa. 1746–1758) boxes indicate AT-hook sequences: their homology to the consensus AT-hook sequence is low and high, respectively. (D) Cropped images from metaphase embryos expressing GFP::MEL-28 or GFP::MEL-28Δ1140–1186. Images were processed identically to facilitate visualization of full-length GFP::MEL-28 associated with the mitotic spindle. Signal intensities in boxed areas were quantified in raw images, normalized and plotted (n = 5, GFP::MEL-28; n = 2, GFP::MEL-28Δ1140–1186). * p<0.05 by unpaired two-tailed t-test. Scale bars, 5 μm.

Mentions: The overall architecture of MEL-28/ELYS is similar throughout the metazoa (see schematic representations in Figs 2C and 7B). All metazoan MEL-28/ELYS homologs include an N-terminal β-propeller domain, a central α-helical domain, and a C-terminal domain that includes at least one AT hook. Crystal structure determination of the N-terminal domain of mammalian ELYS showed that it forms a seven bladed β-propeller structure with an extra loop decorating each of the propeller blades [15]. In human cells, the N-terminal 1018 amino acids of ELYS (which includes the β-propeller domain and the central α-helical domain but not the C-terminal AT hook) is sufficient to localize the protein to NPCs [15]. Mutational disruption of the conserved loop on blade 6 of the β-propeller domain (“loop2”) prevents the 1–1018 aa. fragment from localizing to the nuclear rim.


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 N-terminal domains are required for NPC and kinetochore localization.(A) Still images from time-lapse recording of embryo carrying a GFP insertion into the endogenous mel-28 locus. Time is indicated relative to anaphase onset (min:sec). (B) Metaphase plate of early embryo expressing GFP::MEL-28 (green in merge) analyzed by immunofluorescence with a specific antibody against HCP-3/CENP-A (red in merge) and Hoechst (blue in merge) to visualize chromosomes. MEL-28 localized to kinetochores, which appear as lines on both sides of the chromosomes. (C) Cropped images from embryos expressing different MEL-28 truncations fused to GFP. Except GFP::MEL-28 and GFP::MEL-28Δ1140–1186 embryos, all embryos also expressed un-tagged endogenous MEL-28. Purple boxes in MEL-28 cartoons indicate a putative coiled-coil domain (aa. 1127–1160) whereas yellow (aa. 1630–1642) and orange (aa. 1746–1758) boxes indicate AT-hook sequences: their homology to the consensus AT-hook sequence is low and high, respectively. (D) Cropped images from metaphase embryos expressing GFP::MEL-28 or GFP::MEL-28Δ1140–1186. Images were processed identically to facilitate visualization of full-length GFP::MEL-28 associated with the mitotic spindle. Signal intensities in boxed areas were quantified in raw images, normalized and plotted (n = 5, GFP::MEL-28; n = 2, GFP::MEL-28Δ1140–1186). * p<0.05 by unpaired two-tailed t-test. Scale bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1006131.g002: MEL-28 N-terminal domains are required for NPC and kinetochore localization.(A) Still images from time-lapse recording of embryo carrying a GFP insertion into the endogenous mel-28 locus. Time is indicated relative to anaphase onset (min:sec). (B) Metaphase plate of early embryo expressing GFP::MEL-28 (green in merge) analyzed by immunofluorescence with a specific antibody against HCP-3/CENP-A (red in merge) and Hoechst (blue in merge) to visualize chromosomes. MEL-28 localized to kinetochores, which appear as lines on both sides of the chromosomes. (C) Cropped images from embryos expressing different MEL-28 truncations fused to GFP. Except GFP::MEL-28 and GFP::MEL-28Δ1140–1186 embryos, all embryos also expressed un-tagged endogenous MEL-28. Purple boxes in MEL-28 cartoons indicate a putative coiled-coil domain (aa. 1127–1160) whereas yellow (aa. 1630–1642) and orange (aa. 1746–1758) boxes indicate AT-hook sequences: their homology to the consensus AT-hook sequence is low and high, respectively. (D) Cropped images from metaphase embryos expressing GFP::MEL-28 or GFP::MEL-28Δ1140–1186. Images were processed identically to facilitate visualization of full-length GFP::MEL-28 associated with the mitotic spindle. Signal intensities in boxed areas were quantified in raw images, normalized and plotted (n = 5, GFP::MEL-28; n = 2, GFP::MEL-28Δ1140–1186). * p<0.05 by unpaired two-tailed t-test. Scale bars, 5 μm.
Mentions: The overall architecture of MEL-28/ELYS is similar throughout the metazoa (see schematic representations in Figs 2C and 7B). All metazoan MEL-28/ELYS homologs include an N-terminal β-propeller domain, a central α-helical domain, and a C-terminal domain that includes at least one AT hook. Crystal structure determination of the N-terminal domain of mammalian ELYS showed that it forms a seven bladed β-propeller structure with an extra loop decorating each of the propeller blades [15]. In human cells, the N-terminal 1018 amino acids of ELYS (which includes the β-propeller domain and the central α-helical domain but not the C-terminal AT hook) is sufficient to localize the protein to NPCs [15]. Mutational disruption of the conserved loop on blade 6 of the β-propeller domain (“loop2”) prevents the 1–1018 aa. fragment from localizing to the nuclear rim.

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