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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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Figure 1. 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 1: Figure 1. 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: To examine the function of ELYS/Mel28 in postmitotic NE reformation, we followed recruitment and localization of INM proteins to mitotic chromosomes using live imaging with HeLa cells stably expressing the yellow fluorescent protein (YFP) derivative Venus fused to the LBR and the super enhanced cyan fluorescent protein (SECFP) fused to emerin (Fig. 1A). We compared control cells (control: either untreated or transfected with control oligo; see also Fig. S1) with cells depleted of ELYS/Mel28 (ELYS/Mel28 kd) by RNA interference (RNAi). The position of each cell observed with live imaging was marked and checked for an efficient depletion of ELYS/Mel28 by immediate immunofluorescence (IF) staining with ELYS/Mel28 antibody after live observation (see legend of Fig. 1). Comparing the signal intensities of ELYS/Mel28 by IF staining (Fig. 1C and D) in control cells and cells depleted of ELYS/Mel28 showed an average depletion efficiency of 80%, which is in agreement with the results obtained by western blotting experiments (Fig. S2).


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

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

Figure 1. 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 1: Figure 1. 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: To examine the function of ELYS/Mel28 in postmitotic NE reformation, we followed recruitment and localization of INM proteins to mitotic chromosomes using live imaging with HeLa cells stably expressing the yellow fluorescent protein (YFP) derivative Venus fused to the LBR and the super enhanced cyan fluorescent protein (SECFP) fused to emerin (Fig. 1A). We compared control cells (control: either untreated or transfected with control oligo; see also Fig. S1) with cells depleted of ELYS/Mel28 (ELYS/Mel28 kd) by RNA interference (RNAi). The position of each cell observed with live imaging was marked and checked for an efficient depletion of ELYS/Mel28 by immediate immunofluorescence (IF) staining with ELYS/Mel28 antibody after live observation (see legend of Fig. 1). Comparing the signal intensities of ELYS/Mel28 by IF staining (Fig. 1C and D) in control cells and cells depleted of ELYS/Mel28 showed an average depletion efficiency of 80%, which is in agreement with the results obtained by western blotting experiments (Fig. S2).

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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