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The nucleoporin ELYS/Mel28 regulates nuclear envelope subdomain formation in HeLa cells.

Clever M, Funakoshi T, Mimura Y, Takagi M, Imamoto N - Nucleus (2012)

Bottom Line: The depletion of the LBR affected neither the behavior of emerin nor Lap2α indicating that the recruitment of the LBR to mitotic chromosomes is not involved in formation of the core region.The depletion of ELYS/Mel28 also accelerates the entry into cytokinesis after recruitment of emerin to chromosomes.Our data show that ELYS/Mel28 plays a role in NE subdomain formation in late mitosis.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Riken Advanced Science Institute, Saitama, Japan.

ABSTRACT
In open mitosis the nuclear envelope (NE) reassembles at the end of each mitosis. This process involves the reformation of the nuclear pore complex (NPC), the inner and outer nuclear membranes, and the nuclear lamina. In human cells cell cycle-dependent NE subdomains exist, characterized as A-type lamin-rich/NPC-free or B-type lamin-rich/NPC-rich, which are initially formed as core or noncore regions on mitotic chromosomes, respectively. Although postmitotic NE formation has been extensively studied, little is known about the coordination of NPC and NE assembly. Here, we report that the nucleoporin ELYS/Mel28, which is crucial for postmitotic NPC formation, is essential for recruiting the lamin B receptor (LBR) to the chromosomal noncore region. Furthermore, ELYS/Mel28 is responsible for focusing of A-type lamin-binding proteins like emerin, Lap2α and the barrier-to-autointegration factor (BAF) at the chromosomal core region. ELYS/Mel28 biochemically interacts with the LBR in a phosphorylation-dependent manner. Recruitment of the LBR depends on the nucleoporin Nup107, which interacts with ELYS/Mel28 but not on nucleoporin Pom121, suggesting that the specific molecular interactions with ELYS/Mel28 are involved in the NE assembly at the noncore region. The depletion of the LBR affected neither the behavior of emerin nor Lap2α indicating that the recruitment of the LBR to mitotic chromosomes is not involved in formation of the core region. The depletion of ELYS/Mel28 also accelerates the entry into cytokinesis after recruitment of emerin to chromosomes. Our data show that ELYS/Mel28 plays a role in NE subdomain formation in late mitosis.

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The targeting of the LBR depends on ELYS/Mel28 and the Nup107–160 complex but not on the nucleoporin Pom121. Immunofluorescence (IF) staining of mitotic cells depleted of (A) ELYS/Mel28 for 50 h, (B) Pom121 for 50 h, (C) or Nup107 after double transfection for 75–80 h. Two different siRNA oligos were used to for ELYS/Mel28 depletion (I and II). (D) The depletion efficiency of ELYS/Mel28, Pom121 and Nup107-Venus, observed in (A–C) respectively, was evaluated by comparing the IF signal intensities of the control cells and the depleted cells. (E) Endogenous Pom121, ELYS/Mel28, and LBR were detected by IF staining after LBR-depletion with RNAi (LBR kd). (F) Endogenous Lamin B, LBR, and Nup62 were detected by IF staining in ELYS/Mel28-depleted cells. Scale bars = 10 µm. The pictures are projections of image stacks (distance = 0.2 µm; three images).
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Figure 2: The targeting of the LBR depends on ELYS/Mel28 and the Nup107–160 complex but not on the nucleoporin Pom121. Immunofluorescence (IF) staining of mitotic cells depleted of (A) ELYS/Mel28 for 50 h, (B) Pom121 for 50 h, (C) or Nup107 after double transfection for 75–80 h. Two different siRNA oligos were used to for ELYS/Mel28 depletion (I and II). (D) The depletion efficiency of ELYS/Mel28, Pom121 and Nup107-Venus, observed in (A–C) respectively, was evaluated by comparing the IF signal intensities of the control cells and the depleted cells. (E) Endogenous Pom121, ELYS/Mel28, and LBR were detected by IF staining after LBR-depletion with RNAi (LBR kd). (F) Endogenous Lamin B, LBR, and Nup62 were detected by IF staining in ELYS/Mel28-depleted cells. Scale bars = 10 µm. The pictures are projections of image stacks (distance = 0.2 µm; three images).

Mentions: Transfection with two different siRNA oligos for ELYS/Mel28, which depleted ELYS/Mel28 by about 70–80% (Fig. 2A and D, andFig. S2B) severely impaired the recruitment of the endogenous LBR to mitotic chromosomes. As postmitotic NPC formation was inhibited, the Pom121 signal was absent from mitotic chromosomes and accumulated in the cytoplasm in ELYS/Mel28-depleted cells, as reported previously.42 The depletion of Pom121 by siRNA did not affect the recruitment and localization of the LBR to chromosomes (Fig. 2B and D; Fig. S2C), indicating that ELYS/Mel28 had a primary effect on LBR recruitment.


The nucleoporin ELYS/Mel28 regulates nuclear envelope subdomain formation in HeLa cells.

Clever M, Funakoshi T, Mimura Y, Takagi M, Imamoto N - Nucleus (2012)

The targeting of the LBR depends on ELYS/Mel28 and the Nup107–160 complex but not on the nucleoporin Pom121. Immunofluorescence (IF) staining of mitotic cells depleted of (A) ELYS/Mel28 for 50 h, (B) Pom121 for 50 h, (C) or Nup107 after double transfection for 75–80 h. Two different siRNA oligos were used to for ELYS/Mel28 depletion (I and II). (D) The depletion efficiency of ELYS/Mel28, Pom121 and Nup107-Venus, observed in (A–C) respectively, was evaluated by comparing the IF signal intensities of the control cells and the depleted cells. (E) Endogenous Pom121, ELYS/Mel28, and LBR were detected by IF staining after LBR-depletion with RNAi (LBR kd). (F) Endogenous Lamin B, LBR, and Nup62 were detected by IF staining in ELYS/Mel28-depleted cells. Scale bars = 10 µm. The pictures are projections of image stacks (distance = 0.2 µm; three images).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: The targeting of the LBR depends on ELYS/Mel28 and the Nup107–160 complex but not on the nucleoporin Pom121. Immunofluorescence (IF) staining of mitotic cells depleted of (A) ELYS/Mel28 for 50 h, (B) Pom121 for 50 h, (C) or Nup107 after double transfection for 75–80 h. Two different siRNA oligos were used to for ELYS/Mel28 depletion (I and II). (D) The depletion efficiency of ELYS/Mel28, Pom121 and Nup107-Venus, observed in (A–C) respectively, was evaluated by comparing the IF signal intensities of the control cells and the depleted cells. (E) Endogenous Pom121, ELYS/Mel28, and LBR were detected by IF staining after LBR-depletion with RNAi (LBR kd). (F) Endogenous Lamin B, LBR, and Nup62 were detected by IF staining in ELYS/Mel28-depleted cells. Scale bars = 10 µm. The pictures are projections of image stacks (distance = 0.2 µm; three images).
Mentions: Transfection with two different siRNA oligos for ELYS/Mel28, which depleted ELYS/Mel28 by about 70–80% (Fig. 2A and D, andFig. S2B) severely impaired the recruitment of the endogenous LBR to mitotic chromosomes. As postmitotic NPC formation was inhibited, the Pom121 signal was absent from mitotic chromosomes and accumulated in the cytoplasm in ELYS/Mel28-depleted cells, as reported previously.42 The depletion of Pom121 by siRNA did not affect the recruitment and localization of the LBR to chromosomes (Fig. 2B and D; Fig. S2C), indicating that ELYS/Mel28 had a primary effect on LBR recruitment.

Bottom Line: The depletion of the LBR affected neither the behavior of emerin nor Lap2α indicating that the recruitment of the LBR to mitotic chromosomes is not involved in formation of the core region.The depletion of ELYS/Mel28 also accelerates the entry into cytokinesis after recruitment of emerin to chromosomes.Our data show that ELYS/Mel28 plays a role in NE subdomain formation in late mitosis.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Riken Advanced Science Institute, Saitama, Japan.

ABSTRACT
In open mitosis the nuclear envelope (NE) reassembles at the end of each mitosis. This process involves the reformation of the nuclear pore complex (NPC), the inner and outer nuclear membranes, and the nuclear lamina. In human cells cell cycle-dependent NE subdomains exist, characterized as A-type lamin-rich/NPC-free or B-type lamin-rich/NPC-rich, which are initially formed as core or noncore regions on mitotic chromosomes, respectively. Although postmitotic NE formation has been extensively studied, little is known about the coordination of NPC and NE assembly. Here, we report that the nucleoporin ELYS/Mel28, which is crucial for postmitotic NPC formation, is essential for recruiting the lamin B receptor (LBR) to the chromosomal noncore region. Furthermore, ELYS/Mel28 is responsible for focusing of A-type lamin-binding proteins like emerin, Lap2α and the barrier-to-autointegration factor (BAF) at the chromosomal core region. ELYS/Mel28 biochemically interacts with the LBR in a phosphorylation-dependent manner. Recruitment of the LBR depends on the nucleoporin Nup107, which interacts with ELYS/Mel28 but not on nucleoporin Pom121, suggesting that the specific molecular interactions with ELYS/Mel28 are involved in the NE assembly at the noncore region. The depletion of the LBR affected neither the behavior of emerin nor Lap2α indicating that the recruitment of the LBR to mitotic chromosomes is not involved in formation of the core region. The depletion of ELYS/Mel28 also accelerates the entry into cytokinesis after recruitment of emerin to chromosomes. Our data show that ELYS/Mel28 plays a role in NE subdomain formation in late mitosis.

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