Sequential degradation of proteins from the nuclear envelope during apoptosis

2001 ◽  
Vol 114 (20) ◽  
pp. 3643-3653 ◽  
Author(s):  
Madeleine Kihlmark ◽  
Gabriela Imreh ◽  
Einar Hallberg
Keyword(s):  
Nuclear Envelope ◽  
Apoptotic Cell ◽  
Nuclear Periphery ◽  
Condensed Chromatin ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Nuclear Pores ◽  
Double Labeling ◽  
Lamin B ◽  
Nuclear Apoptosis

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.

Nuclear envelope disassembly in mitotic extract requires functional nuclear pores and a nuclear lamina

1998 ◽  
Vol 111 (9) ◽  
pp. 1293-1303 ◽  
Author(s):  
P. Collas
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Nuclear Membrane ◽  
Critical Role ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Lamin B ◽  
Nuclear Membranes

Using sea urchin embryonic and in-vitro-assembled nuclei incubated in sea urchin mitotic extract, I provide evidence for a requirement for functional nuclear pores and a nuclear lamina for nuclear envelope disassembly in vitro. In interphase gastrula nuclei, lamin B interacts with p56, an integral protein of inner nuclear membrane cross-reacting with antibodies to human lamin B receptor. Incubation of gastrula nuclei in mitotic cytosol containing an ATP-generating system rapidly induces hyperphosphorylation of p56 and lamin B. Subsequently, p56-lamin B interactions are weakened and the two proteins segregate into distinct nuclear envelope-derived vesicles upon disassembly of nuclear membranes and of the lamina. Nuclear disassembly is accompanied by chromatin condensation. Blocking nuclear pore function with wheat germ agglutinin or antibodies to nucleoporins prevents p56 and lamin B hyperphosphorylation, nuclear membrane breakdown and lamina solubilization. These events are not rescued by permeabilization of nuclear membranes to molecules of 150, 000 Mr with lysolecithin. In-vitro-assembled nuclei containing nuclear membranes with functional pores but no lamina do not disassemble in mitotic cytosol in spite of p56 hyperphosphorylation. Nuclear import of soluble lamin B and reformation of a lamina in interphase extract restores nuclear disassembly in mitotic cytosol. The data indicate a role for functional nuclear pores in nuclear disassembly in vitro. They show that p56 hyperphosphorylation is not sufficient for nuclear membrane disassembly in mitotic cytosol and argue that the nuclear lamina plays a critical role in nuclear disassembly at mitosis.

Nuclear Envelope Dynamics During Mitosis

1988 ◽  
Vol 46 ◽  
pp. 224-225
Author(s):  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Membrane Vesicles ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Animal Cells ◽  
Interphase Chromosome ◽  
Lamin B ◽  
Chromosome Architecture ◽  
Complex Membrane ◽  
Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

scholarly journals Induction of a Massive Endoplasmic Reticulum and Perinuclear Space Expansion by Expression of Lamin B Receptor Mutants and the Related Sterol Reductases TM7SF2 and DHCR7

2010 ◽  
Vol 21 (2) ◽  
pp. 354-368 ◽  
Author(s):  
Monika Zwerger ◽  
Thorsten Kolb ◽  
Karsten Richter ◽  
Iakowos Karakesisoglou ◽  
Harald Herrmann
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Cell Lines ◽  
Nuclear Membrane ◽  
Cultured Cells ◽  
Membrane Separation ◽  
Reductase Activity ◽  
Nuclear Pore ◽  
Lamin B ◽  
Lamin B Receptor

Lamin B receptor (LBR) is an inner nuclear membrane protein involved in tethering the nuclear lamina and the underlying chromatin to the nuclear envelope. In addition, LBR exhibits sterol reductase activity. Mutations in the LBR gene cause two different human diseases: Pelger-Huët anomaly and Greenberg skeletal dysplasia, a severe chrondrodystrophy causing embryonic death. Our study aimed at investigating the effect of five LBR disease mutants on human cultured cells. Three of the tested LBR mutants caused a massive compaction of chromatin coincidental with the formation of a large nucleus-associated vacuole (NAV) in several human cultured cell lines. Live cell imaging and electron microscopy revealed that this structure was generated by the separation of the inner and outer nuclear membrane. During NAV formation, nuclear pore complexes and components of the linker of nucleoskeleton and cytoskeleton complex were lost in areas of membrane separation. Concomitantly, a large number of smaller vacuoles formed throughout the cytoplasm. Notably, forced expression of the two structurally related sterol reductases transmembrane 7 superfamily member 2 and 7-dehydrocholesterol reductase caused, even in their wild-type form, a comparable phenotype in susceptible cell lines. Hence, LBR mutant variants and sterol reductases can severely interfere with the regular organization of the nuclear envelope and the endoplasmic reticulum.

scholarly journals Piecing together nuclear pore complex assembly during interphase

2009 ◽  
Vol 185 (3) ◽  
pp. 377-379 ◽  
Author(s):  
Michael Rexach
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Supramolecular Structures ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Assembly Process ◽  
Nuclear Pores ◽  
Complex Assembly ◽  
Cell Biol ◽  
Pore Complexes

All nucleocytoplasmic traffic of macromolecules occurs through nuclear pore complexes (NPCs), which function as stents in the nuclear envelope to keep nuclear pores open but gated. Three studies in this issue (Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Böttcher, and E. Hurt. 2009. J. Cell Biol. 185:387–395; Makio, T., L.H. Stanton, C.-C. Lin, D.S. Goldfarb, K. Weis, and R.W. Wozniak. 2009. J. Cell Biol. 185:459–491; Onishchenko, E., L.H. Stanton, A.S. Madrid, T. Kieselbach, and K. Weis. 2009. J. Cell Biol. 185:475–491) further our understanding of the NPC assembly process by reporting what happens when the supply lines of key proteins that provide a foundation for building these marvelous supramolecular structures are disrupted.

scholarly journals Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts

1995 ◽  
Vol 108 (11) ◽  
pp. 3451-3461 ◽  
Author(s):  
M. Goldberg ◽  
H. Jenkins ◽  
T. Allen ◽  
W.G. Whitfield ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Nuclear Matrix ◽  
Nuclear Pore ◽  
Major Protein ◽  
Protein Species ◽  
Nuclear Pores ◽  
Thin Sections ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of ‘depleted’ extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.

Relationships at the nuclear envelope: lamins and nuclear pore complexes in animals and plants

2010 ◽  
Vol 38 (3) ◽  
pp. 829-831 ◽  
Author(s):  
Jindriska Fiserova ◽  
Martin W. Goldberg
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nucleocytoplasmic Trafficking ◽  
Animal Cells ◽  
Cellular Processes ◽  
Associated Proteins ◽  
Pore Complexes

The nuclear envelope comprises a distinct compartment at the nuclear periphery that provides a platform for communication between the nucleus and cytoplasm. Signal transfer can proceed by multiple means. Primarily, this is by nucleocytoplasmic trafficking facilitated by NPCs (nuclear pore complexes). Recently, it has been indicated that signals can be transmitted from the cytoskeleton to the intranuclear structures via interlinking transmembrane proteins. In animal cells, the nuclear lamina tightly underlies the inner nuclear membrane and thus represents the protein structure located at the furthest boundary of the nucleus. It enables communication between the nucleus and the cytoplasm via its interactions with chromatin-binding proteins, transmembrane and membrane-associated proteins. Of particular interest is the interaction of the nuclear lamina with NPCs. As both structures fulfil essential roles in close proximity at the nuclear periphery, their interactions have a large impact on cellular processes resulting in affects on tissue differentiation and development. The present review concentrates on the structural and functional lamina–NPC relationship in animal cells and its potential implications to plants.

Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis

1999 ◽  
Vol 112 (13) ◽  
pp. 2253-2264 ◽  
Author(s):  
K. Bodoor ◽  
S. Shaikh ◽  
D. Salina ◽  
W.H. Raharjo ◽  
R. Bastos ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Time Course ◽  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Confocal Immunofluorescence Microscopy ◽  
Pore Complexes ◽  
Nuclear Membrane Protein

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. With a mass of about 125 MDa, NPCs are thought to be composed of 50 or more distinct protein subunits, each present in multiple copies. During mitosis in higher cells the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized. Using both conventional and digital confocal immunofluorescence microscopy we have been able to define a time course of post-mitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a component of the nuclear basket, associates with chromatin towards the end of anaphase, in parallel with the inner nuclear membrane protein, LAP2. However, immunogold labeling suggests that the initial Nup153 chromatin association is membrane-independent. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, 54, 45) during mitosis and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates.

scholarly journals Identification and characterization of autoantibodies against the nuclear envelope lamin B receptor from patients with primary biliary cirrhosis.

1990 ◽  
Vol 172 (3) ◽  
pp. 961-967 ◽  
Author(s):  
J C Courvalin ◽  
K Lassoued ◽  
H J Worman ◽  
G Blobel
Keyword(s):  
Primary Biliary Cirrhosis ◽  
Membrane Protein ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Biliary Cirrhosis ◽  
Lamin B ◽  
Lamin B Receptor

We have identified autoantibodies from two patients with primary biliary cirrhosis (PBC) that recognize the nuclear envelope of mammalian cells on indirect immunofluorescence microscopy. These antibodies bind to a 58-kD integral membrane protein (p58) of the turkey erythrocyte nuclear envelope, which has been previously identified as a membrane receptor for lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531). The antibodies also bind to a 61-kD integral membrane protein (p61) of the rat liver nuclear envelope. Affinity-purified antibodies eluted from turkey p58 bind to rat p61, showing that the two proteins share an epitope(s) and that p61 is likely the rat liver lamin B receptor. In human nuclear envelopes, the antigen recognized has an apparent molecular mass close to that of avian protein. These findings, along with the previous discovery of autoantibodies against an integral membrane glycoprotein (gp210) of the nuclear pore membrane in patients with PBC, suggest that antibodies against integral membrane proteins of the nuclear envelope are characteristic of a subset of patients with PBC.

scholarly journals The nuclear basket mediates perinuclear mRNA scanning in budding yeast

2015 ◽  
Vol 211 (6) ◽  
pp. 1131-1140 ◽  
Author(s):  
Mark-Albert Saroufim ◽  
Pierre Bensidoun ◽  
Pascal Raymond ◽  
Samir Rahman ◽  
Matthew R. Krause ◽  
...  
Keyword(s):  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Messenger Rnas ◽  
Nuclear Pores ◽  
Transport Channel ◽  
Mrna Binding Protein ◽  
Live Cell Microscopy ◽  
Scanning Behavior ◽  
Mrna Binding ◽  
Cell Microscopy

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.

scholarly journals Dynamic Rearrangement of Nucleoporins during Fungal “Open” Mitosis

2008 ◽  
Vol 19 (3) ◽  
pp. 1230-1240 ◽  
Author(s):  
Ulrike Theisen ◽  
Anne Straube ◽  
Gero Steinberg
Keyword(s):  
Nuclear Envelope ◽  
Protein Import ◽  
Complex Dynamics ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Pores ◽  
Basic Principles ◽  
Early Anaphase ◽  
Pore Complexes ◽  
Corn Smut

Mitosis in animals starts with the disassembly of the nuclear pore complexes and the breakdown of the nuclear envelope. In contrast to many fungi, the corn smut fungus Ustilago maydis also removes the nuclear envelope. Here, we report on the dynamic behavior of the nucleoporins Nup214, Pom152, Nup133, and Nup107 in this “open” fungal mitosis. In prophase, the nuclear pore complexes disassembled and Nup214 and Pom152 dispersed in the cytoplasm and in the endoplasmic reticulum, respectively. Nup107 and Nup133 initially spread throughout the cytoplasm, but in metaphase and early anaphase occurred on the chromosomes. In anaphase, the Nup107-subcomplex redistributed to the edge of the chromosome masses, where the new envelope was reconstituted. Subsequently, Nup214 and Pom152 are recruited to the nuclear pores and protein import starts. Recruitment of nucleoporins and protein import reached a steady state in G2 phase. Formation of the nuclear envelope and assembly of nuclear pores occurred in the absence of microtubules or F-actin, but not if both were disrupted. Thus, the basic principles of nuclear pore complex dynamics seem to be conserved in organisms displaying open mitosis.

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