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Nuclear envelope remnants: fluid membranes enriched in sterols and polyphosphoinositides.

Garnier-Lhomme M, Byrne RD, Hobday TM, Gschmeissner S, Woscholski R, Poccia DL, Dufourc EJ, Larijani B - PLoS ONE (2009)

Bottom Line: Although most of the sperm nuclear envelope disassembles, remnants of the envelope at the acrosomal and centriolar fossae do not and are subsequently incorporated into the newly forming male pronuclear envelope.Using lipid mass spectrometry and a new application of deuterium solid-state NMR spectroscopy we have characterized the lipid composition and membrane dynamics of the sperm nuclear envelope remnants in isolated sperm nuclei.The localization of the polybasic effector domain of MARCKS illustrates the non-nuclear aspect of the polyphosphoinositides.

View Article: PubMed Central - PubMed

Affiliation: Cell Biophysics Laboratory, Lincoln's Inn Fields Laboratories, Cancer Research UK, London, United Kingdom.

ABSTRACT

Background: The cytoplasm of eukaryotic cells is a highly dynamic compartment where membranes readily undergo fission and fusion to reorganize the cytoplasmic architecture, and to import, export and transport various cargos within the cell. The double membrane of the nuclear envelope that surrounds the nucleus, segregates the chromosomes from cytoplasm and regulates nucleocytoplasmic transport through pores. Many details of its formation are still unclear. At fertilization the sperm devoid of nuclear envelope pores enters the egg. Although most of the sperm nuclear envelope disassembles, remnants of the envelope at the acrosomal and centriolar fossae do not and are subsequently incorporated into the newly forming male pronuclear envelope. Remnants are conserved from annelid to mammalian sperm.

Methodology/principal findings: Using lipid mass spectrometry and a new application of deuterium solid-state NMR spectroscopy we have characterized the lipid composition and membrane dynamics of the sperm nuclear envelope remnants in isolated sperm nuclei.

Conclusions/significance: We report nuclear envelope remnants are relatively fluid membranes rich in sterols, devoid of sphingomyelin, and highly enriched in polyphosphoinositides and polyunsaturated phospholipids. The localization of the polybasic effector domain of MARCKS illustrates the non-nuclear aspect of the polyphosphoinositides. Based on their atypical biophysical characteristics and phospholipid composition, we suggest a possible role for nuclear envelope remnants in membrane fusion leading to nuclear envelope assembly.

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Cholesterol removal from nuclear envelope remnants inhibits membrane fusion events of nuclear envelope formation.(A) 0.1% L. pictus nuclei were treated with 10mM MβCD or untreated (control). Nuclei were filipin stained, normalized to an equal nuclei concentration and excited at 360nm. The fluorescence intensity of the emission peak at 479/480nm was measured, and data were normalized to the control value. Data shown are mean±SEM of four experiments conducted in duplicate. (B) 0.1% L. pictus nuclei were treated with 10mM MβCD (white) or untreated (black). Nuclear envelope precursor MVs were subsequently bound to nuclei (ATP), and in parallel reactions their fusion was triggered with GTP to complete envelope formation. At least 20 nuclei were scored for the presence of a fully-formed nuclear envelope in 3 independent experiments. Data shown are mean±SEM.
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pone-0004255-g006: Cholesterol removal from nuclear envelope remnants inhibits membrane fusion events of nuclear envelope formation.(A) 0.1% L. pictus nuclei were treated with 10mM MβCD or untreated (control). Nuclei were filipin stained, normalized to an equal nuclei concentration and excited at 360nm. The fluorescence intensity of the emission peak at 479/480nm was measured, and data were normalized to the control value. Data shown are mean±SEM of four experiments conducted in duplicate. (B) 0.1% L. pictus nuclei were treated with 10mM MβCD (white) or untreated (black). Nuclear envelope precursor MVs were subsequently bound to nuclei (ATP), and in parallel reactions their fusion was triggered with GTP to complete envelope formation. At least 20 nuclei were scored for the presence of a fully-formed nuclear envelope in 3 independent experiments. Data shown are mean±SEM.

Mentions: The data presented describe nuclear envelope remnants as double membrane structures with a unique lipid composition that are relatively fluid in nature. The 42 mole% sterol content of nuclear envelope remnants is closest to that of cortical vesicles (57%) [45]. Given the role of cholesterol in facilitating cortical vesicle fusion to the plasma membrane [20] we hypothesized that cholesterol in the nuclear envelope remnants may play a similar role in promoting fusion events of nuclear envelope assembly. To test this, we treated 0.1% nuclei with 10mM MβCD, a sterol depleting agent [51], [52]. Detection of sterols with filipin after MβCD treatment of nuclei indicated that there was considerably less sterols present, as indicated by a decrease of over 30% in filipin fluorescence intensity, compared to untreated nuclei (Figure 6A). Moreover, the same MβCD treated nuclei were less able to support nuclear envelope assembly in a cell free system (Figure 6B). In the presence of a fertilized egg cytoplasmic extract and ATP nuclei decondense to a spherical mass and bind egg MVs which fuse in the presence of GTP to form a nuclear envelope. Binding of MVs to MβCD treated nuclei was not affected but the ability of the MVs to subsequently fuse was inhibited by 35% (Figure 6B). Together, these indicate a functional role for nuclear envelope remnant sterols in the fusion events of nuclear envelope formation.


Nuclear envelope remnants: fluid membranes enriched in sterols and polyphosphoinositides.

Garnier-Lhomme M, Byrne RD, Hobday TM, Gschmeissner S, Woscholski R, Poccia DL, Dufourc EJ, Larijani B - PLoS ONE (2009)

Cholesterol removal from nuclear envelope remnants inhibits membrane fusion events of nuclear envelope formation.(A) 0.1% L. pictus nuclei were treated with 10mM MβCD or untreated (control). Nuclei were filipin stained, normalized to an equal nuclei concentration and excited at 360nm. The fluorescence intensity of the emission peak at 479/480nm was measured, and data were normalized to the control value. Data shown are mean±SEM of four experiments conducted in duplicate. (B) 0.1% L. pictus nuclei were treated with 10mM MβCD (white) or untreated (black). Nuclear envelope precursor MVs were subsequently bound to nuclei (ATP), and in parallel reactions their fusion was triggered with GTP to complete envelope formation. At least 20 nuclei were scored for the presence of a fully-formed nuclear envelope in 3 independent experiments. Data shown are mean±SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626249&req=5

pone-0004255-g006: Cholesterol removal from nuclear envelope remnants inhibits membrane fusion events of nuclear envelope formation.(A) 0.1% L. pictus nuclei were treated with 10mM MβCD or untreated (control). Nuclei were filipin stained, normalized to an equal nuclei concentration and excited at 360nm. The fluorescence intensity of the emission peak at 479/480nm was measured, and data were normalized to the control value. Data shown are mean±SEM of four experiments conducted in duplicate. (B) 0.1% L. pictus nuclei were treated with 10mM MβCD (white) or untreated (black). Nuclear envelope precursor MVs were subsequently bound to nuclei (ATP), and in parallel reactions their fusion was triggered with GTP to complete envelope formation. At least 20 nuclei were scored for the presence of a fully-formed nuclear envelope in 3 independent experiments. Data shown are mean±SEM.
Mentions: The data presented describe nuclear envelope remnants as double membrane structures with a unique lipid composition that are relatively fluid in nature. The 42 mole% sterol content of nuclear envelope remnants is closest to that of cortical vesicles (57%) [45]. Given the role of cholesterol in facilitating cortical vesicle fusion to the plasma membrane [20] we hypothesized that cholesterol in the nuclear envelope remnants may play a similar role in promoting fusion events of nuclear envelope assembly. To test this, we treated 0.1% nuclei with 10mM MβCD, a sterol depleting agent [51], [52]. Detection of sterols with filipin after MβCD treatment of nuclei indicated that there was considerably less sterols present, as indicated by a decrease of over 30% in filipin fluorescence intensity, compared to untreated nuclei (Figure 6A). Moreover, the same MβCD treated nuclei were less able to support nuclear envelope assembly in a cell free system (Figure 6B). In the presence of a fertilized egg cytoplasmic extract and ATP nuclei decondense to a spherical mass and bind egg MVs which fuse in the presence of GTP to form a nuclear envelope. Binding of MVs to MβCD treated nuclei was not affected but the ability of the MVs to subsequently fuse was inhibited by 35% (Figure 6B). Together, these indicate a functional role for nuclear envelope remnant sterols in the fusion events of nuclear envelope formation.

Bottom Line: Although most of the sperm nuclear envelope disassembles, remnants of the envelope at the acrosomal and centriolar fossae do not and are subsequently incorporated into the newly forming male pronuclear envelope.Using lipid mass spectrometry and a new application of deuterium solid-state NMR spectroscopy we have characterized the lipid composition and membrane dynamics of the sperm nuclear envelope remnants in isolated sperm nuclei.The localization of the polybasic effector domain of MARCKS illustrates the non-nuclear aspect of the polyphosphoinositides.

View Article: PubMed Central - PubMed

Affiliation: Cell Biophysics Laboratory, Lincoln's Inn Fields Laboratories, Cancer Research UK, London, United Kingdom.

ABSTRACT

Background: The cytoplasm of eukaryotic cells is a highly dynamic compartment where membranes readily undergo fission and fusion to reorganize the cytoplasmic architecture, and to import, export and transport various cargos within the cell. The double membrane of the nuclear envelope that surrounds the nucleus, segregates the chromosomes from cytoplasm and regulates nucleocytoplasmic transport through pores. Many details of its formation are still unclear. At fertilization the sperm devoid of nuclear envelope pores enters the egg. Although most of the sperm nuclear envelope disassembles, remnants of the envelope at the acrosomal and centriolar fossae do not and are subsequently incorporated into the newly forming male pronuclear envelope. Remnants are conserved from annelid to mammalian sperm.

Methodology/principal findings: Using lipid mass spectrometry and a new application of deuterium solid-state NMR spectroscopy we have characterized the lipid composition and membrane dynamics of the sperm nuclear envelope remnants in isolated sperm nuclei.

Conclusions/significance: We report nuclear envelope remnants are relatively fluid membranes rich in sterols, devoid of sphingomyelin, and highly enriched in polyphosphoinositides and polyunsaturated phospholipids. The localization of the polybasic effector domain of MARCKS illustrates the non-nuclear aspect of the polyphosphoinositides. Based on their atypical biophysical characteristics and phospholipid composition, we suggest a possible role for nuclear envelope remnants in membrane fusion leading to nuclear envelope assembly.

Show MeSH