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Lamin-binding fragment of LAP2 inhibits increase in nuclear volume during the cell cycle and progression into S phase.

Yang L, Guan T, Gerace L - J. Cell Biol. (1997)

Bottom Line: Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization.We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1-398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase.Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization. We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1-398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase. This effect appears to be specifically due to lamin binding, because it also is caused by microinjection of the minimal lamin-binding region of LAP2 (residues 298-373) but not by the chromatin-binding domain (residues 1-88). Injection of the lamin-binding region of rat LAP2 into early G1 phase HeLa cells also strongly affects nuclear growth; it almost completely prevents the threefold nuclear volume increase that normally occurs during the ensuing 10 h. Moreover, injection of the fragment during early G1 phase strongly inhibits entry of cells into S phase, whereas injection during S phase has no apparent effect on ongoing DNA replication. Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth. These data also suggest that lamina dynamics are required for growth of the NE and for nuclear volume increase during the cell cycle, and that progression into S phase is dependent on the acquisition of a certain nuclear volume.

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Effect of microinjected LAP2 fragments on nuclear reformation and lamin assembly at the end of mitosis. Metaphase  HeLa cells were injected with a variety of proteins and incubated  for 2 h at 37°C before fixation and immunofluorescent staining.  All injectates contained rabbit IgG to mark the injected cells. In  addition, the injectates contained: A and E, LAP2 (1–398); B and  F, rabbit IgG only; C and G, MBP-LAP2 (298–373); D and H,  MBP-LAP2 (1–88). Shown are phase contrast images (left column), or epifluorescence images staining with anti–rabbit IgG  (middle column), and with antilamins (right column; A–D, antilamins A/C; E–H, antilamin B). Bar, 20 μm.
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Figure 2: Effect of microinjected LAP2 fragments on nuclear reformation and lamin assembly at the end of mitosis. Metaphase HeLa cells were injected with a variety of proteins and incubated for 2 h at 37°C before fixation and immunofluorescent staining. All injectates contained rabbit IgG to mark the injected cells. In addition, the injectates contained: A and E, LAP2 (1–398); B and F, rabbit IgG only; C and G, MBP-LAP2 (298–373); D and H, MBP-LAP2 (1–88). Shown are phase contrast images (left column), or epifluorescence images staining with anti–rabbit IgG (middle column), and with antilamins (right column; A–D, antilamins A/C; E–H, antilamin B). Bar, 20 μm.

Mentions: In initial experiments, we injected the mitotic cells with a recombinant protein comprising nearly the entire nucleoplasmic domain of LAP2 (residues 1–398; Furukawa et al., 1995) (Fig. 1, lane 3) or with various control proteins, and examined the cells 2 h later. Under these conditions, the cells that had been injected with control proteins (IgG or GST), completed mitosis, and by phase contrast microscopy appeared as pairs of daughter cells with well-defined G1 nuclei containing decondensed chromatin (Fig. 2, B and F; and data not shown). Interestingly, the cells that had been injected with LAP2 (1–398) (Fig. 2, A and E) completed mitosis like the control cells, but the nuclei in these cells had a markedly reduced size, even though they appeared to contain decondensed chromatin and nucleoluslike phase dense bodies. Immunofluorescent staining showed that despite the reduction in nuclear size, lamins A/C and B assembled in the nuclei of the LAP2 injected cells, similar to the control cells (Fig. 2, compare A and E to B and F). Lamin B appeared in a distinct nuclear rim, whereas lamins A/C appeared both in a nuclear rim and in intranuclear foci. These results on lamins A/C localization are in accord with previous findings showing that a fraction of lamin A appears in intranuclear foci as well as the NE in some cultured mammalian cells in early G1 phase, although all detectable lamin A becomes localized at the NE later in G1 phase (Bridger et al., 1993; Gerace, L., unpublished results).


Lamin-binding fragment of LAP2 inhibits increase in nuclear volume during the cell cycle and progression into S phase.

Yang L, Guan T, Gerace L - J. Cell Biol. (1997)

Effect of microinjected LAP2 fragments on nuclear reformation and lamin assembly at the end of mitosis. Metaphase  HeLa cells were injected with a variety of proteins and incubated  for 2 h at 37°C before fixation and immunofluorescent staining.  All injectates contained rabbit IgG to mark the injected cells. In  addition, the injectates contained: A and E, LAP2 (1–398); B and  F, rabbit IgG only; C and G, MBP-LAP2 (298–373); D and H,  MBP-LAP2 (1–88). Shown are phase contrast images (left column), or epifluorescence images staining with anti–rabbit IgG  (middle column), and with antilamins (right column; A–D, antilamins A/C; E–H, antilamin B). Bar, 20 μm.
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Figure 2: Effect of microinjected LAP2 fragments on nuclear reformation and lamin assembly at the end of mitosis. Metaphase HeLa cells were injected with a variety of proteins and incubated for 2 h at 37°C before fixation and immunofluorescent staining. All injectates contained rabbit IgG to mark the injected cells. In addition, the injectates contained: A and E, LAP2 (1–398); B and F, rabbit IgG only; C and G, MBP-LAP2 (298–373); D and H, MBP-LAP2 (1–88). Shown are phase contrast images (left column), or epifluorescence images staining with anti–rabbit IgG (middle column), and with antilamins (right column; A–D, antilamins A/C; E–H, antilamin B). Bar, 20 μm.
Mentions: In initial experiments, we injected the mitotic cells with a recombinant protein comprising nearly the entire nucleoplasmic domain of LAP2 (residues 1–398; Furukawa et al., 1995) (Fig. 1, lane 3) or with various control proteins, and examined the cells 2 h later. Under these conditions, the cells that had been injected with control proteins (IgG or GST), completed mitosis, and by phase contrast microscopy appeared as pairs of daughter cells with well-defined G1 nuclei containing decondensed chromatin (Fig. 2, B and F; and data not shown). Interestingly, the cells that had been injected with LAP2 (1–398) (Fig. 2, A and E) completed mitosis like the control cells, but the nuclei in these cells had a markedly reduced size, even though they appeared to contain decondensed chromatin and nucleoluslike phase dense bodies. Immunofluorescent staining showed that despite the reduction in nuclear size, lamins A/C and B assembled in the nuclei of the LAP2 injected cells, similar to the control cells (Fig. 2, compare A and E to B and F). Lamin B appeared in a distinct nuclear rim, whereas lamins A/C appeared both in a nuclear rim and in intranuclear foci. These results on lamins A/C localization are in accord with previous findings showing that a fraction of lamin A appears in intranuclear foci as well as the NE in some cultured mammalian cells in early G1 phase, although all detectable lamin A becomes localized at the NE later in G1 phase (Bridger et al., 1993; Gerace, L., unpublished results).

Bottom Line: Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization.We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1-398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase.Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization. We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1-398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase. This effect appears to be specifically due to lamin binding, because it also is caused by microinjection of the minimal lamin-binding region of LAP2 (residues 298-373) but not by the chromatin-binding domain (residues 1-88). Injection of the lamin-binding region of rat LAP2 into early G1 phase HeLa cells also strongly affects nuclear growth; it almost completely prevents the threefold nuclear volume increase that normally occurs during the ensuing 10 h. Moreover, injection of the fragment during early G1 phase strongly inhibits entry of cells into S phase, whereas injection during S phase has no apparent effect on ongoing DNA replication. Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth. These data also suggest that lamina dynamics are required for growth of the NE and for nuclear volume increase during the cell cycle, and that progression into S phase is dependent on the acquisition of a certain nuclear volume.

Show MeSH
Related in: MedlinePlus