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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 5. The depletion of the LBR does not inhibit focusing of emerin and Lap2α at the core region. Live imaging was performed with cells stably expressing LBR-Venus/SECFP-emerin, like described in Figure 1. (A) Untreated cells (control). (B) Cells depleted of LBR by RNAi for 48 h (LBR kd). Arrowheads show SECFP-emerin accumulating at the core region, and arrows show initial targeting of SECFP-emerin to the chromosomes. Scale bars = 10 µm. (C) The duration time of SECFP-emerin at the core region as observed in live imaging in Figures 1A and B, 5A and B (n = 24 for the control, n = 15 for the ELYS/Mel28 kd, n = 10 for the LBR kd). (D) Endogenous Lap2α was examined by IF staining in HeLa cells stably expressing LBR-Venus/SECFP-emerin depleted of ELYS/Mel28 as in Figure 1B, or depleted of LBR as in Figure 5B. Scale bars = 10 µm. (E) Quantification results of D are shown. Control cells (n = 40), ELYS/Mel28 kd (n = 39) and LBR kd cells (n = 25).
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Figure 5: Figure 5. The depletion of the LBR does not inhibit focusing of emerin and Lap2α at the core region. Live imaging was performed with cells stably expressing LBR-Venus/SECFP-emerin, like described in Figure 1. (A) Untreated cells (control). (B) Cells depleted of LBR by RNAi for 48 h (LBR kd). Arrowheads show SECFP-emerin accumulating at the core region, and arrows show initial targeting of SECFP-emerin to the chromosomes. Scale bars = 10 µm. (C) The duration time of SECFP-emerin at the core region as observed in live imaging in Figures 1A and B, 5A and B (n = 24 for the control, n = 15 for the ELYS/Mel28 kd, n = 10 for the LBR kd). (D) Endogenous Lap2α was examined by IF staining in HeLa cells stably expressing LBR-Venus/SECFP-emerin depleted of ELYS/Mel28 as in Figure 1B, or depleted of LBR as in Figure 5B. Scale bars = 10 µm. (E) Quantification results of D are shown. Control cells (n = 40), ELYS/Mel28 kd (n = 39) and LBR kd cells (n = 25).

Mentions: The depletion of ELYS/Mel28 altered the recruitment of emerin to the chromosomal core region (Figs. 1Band5C). We next examined whether the depletion affects other INM components known to concentrate at the chromosomal core region in postmitosis. For this, we examined the localization of endogenous Lap2α (Fig. 4A; Fig. S4D), lamin A/C (Fig. 4B), and BAF (Fig. 4D; Fig. S4C and S4D) with IF staining in control cells or cells depleted of ELYS/Mel28, using two RNAi oligos as described above. In control cells, Lap2α and BAF often accumulated first at both sides of the core region next to the central spindle- and spindle pole area, and then focused at the core region next to the central spindle area (Fig. S4A and S4B). In ELYS/Mel28-depleted cells, Lap2α and BAF either bound all around the chromosomes instead of accumulating mainly at the core region, or only at the core region next to the spindle pole area, which is the opposite to what was observed in control cells (Figs. 4B and C, 5D; Fig. S4D: see arrows for localization at the spindle pole side). Lamin A/C, like Lap2α and BAF, was also affected by ELYS/Mel28 depletion, but differently, as it bound all around mitotic chromosomes in telophase and did not concentrate to a specific side or region (Fig. 4B). The protein expression levels of lamin A/C were unchanged (Fig. S2B). More than 90% of ELYS/Mel28-depleted cells displayed aberrant localization of A-type lamin and Lap2α, as described above (Fig. 4D).


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 5. The depletion of the LBR does not inhibit focusing of emerin and Lap2α at the core region. Live imaging was performed with cells stably expressing LBR-Venus/SECFP-emerin, like described in Figure 1. (A) Untreated cells (control). (B) Cells depleted of LBR by RNAi for 48 h (LBR kd). Arrowheads show SECFP-emerin accumulating at the core region, and arrows show initial targeting of SECFP-emerin to the chromosomes. Scale bars = 10 µm. (C) The duration time of SECFP-emerin at the core region as observed in live imaging in Figures 1A and B, 5A and B (n = 24 for the control, n = 15 for the ELYS/Mel28 kd, n = 10 for the LBR kd). (D) Endogenous Lap2α was examined by IF staining in HeLa cells stably expressing LBR-Venus/SECFP-emerin depleted of ELYS/Mel28 as in Figure 1B, or depleted of LBR as in Figure 5B. Scale bars = 10 µm. (E) Quantification results of D are shown. Control cells (n = 40), ELYS/Mel28 kd (n = 39) and LBR kd cells (n = 25).
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Figure 5: Figure 5. The depletion of the LBR does not inhibit focusing of emerin and Lap2α at the core region. Live imaging was performed with cells stably expressing LBR-Venus/SECFP-emerin, like described in Figure 1. (A) Untreated cells (control). (B) Cells depleted of LBR by RNAi for 48 h (LBR kd). Arrowheads show SECFP-emerin accumulating at the core region, and arrows show initial targeting of SECFP-emerin to the chromosomes. Scale bars = 10 µm. (C) The duration time of SECFP-emerin at the core region as observed in live imaging in Figures 1A and B, 5A and B (n = 24 for the control, n = 15 for the ELYS/Mel28 kd, n = 10 for the LBR kd). (D) Endogenous Lap2α was examined by IF staining in HeLa cells stably expressing LBR-Venus/SECFP-emerin depleted of ELYS/Mel28 as in Figure 1B, or depleted of LBR as in Figure 5B. Scale bars = 10 µm. (E) Quantification results of D are shown. Control cells (n = 40), ELYS/Mel28 kd (n = 39) and LBR kd cells (n = 25).
Mentions: The depletion of ELYS/Mel28 altered the recruitment of emerin to the chromosomal core region (Figs. 1Band5C). We next examined whether the depletion affects other INM components known to concentrate at the chromosomal core region in postmitosis. For this, we examined the localization of endogenous Lap2α (Fig. 4A; Fig. S4D), lamin A/C (Fig. 4B), and BAF (Fig. 4D; Fig. S4C and S4D) with IF staining in control cells or cells depleted of ELYS/Mel28, using two RNAi oligos as described above. In control cells, Lap2α and BAF often accumulated first at both sides of the core region next to the central spindle- and spindle pole area, and then focused at the core region next to the central spindle area (Fig. S4A and S4B). In ELYS/Mel28-depleted cells, Lap2α and BAF either bound all around the chromosomes instead of accumulating mainly at the core region, or only at the core region next to the spindle pole area, which is the opposite to what was observed in control cells (Figs. 4B and C, 5D; Fig. S4D: see arrows for localization at the spindle pole side). Lamin A/C, like Lap2α and BAF, was also affected by ELYS/Mel28 depletion, but differently, as it bound all around mitotic chromosomes in telophase and did not concentrate to a specific side or region (Fig. 4B). The protein expression levels of lamin A/C were unchanged (Fig. S2B). More than 90% of ELYS/Mel28-depleted cells displayed aberrant localization of A-type lamin and Lap2α, as described above (Fig. 4D).

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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