scholarly journals Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication

1994 ◽  
Vol 107 (12) ◽  
pp. 3259-3269 ◽  
Author(s):  
C.J. Hutchison ◽  
J.M. Bridger ◽  
L.S. Cox ◽  
I.R. Kill
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Nuclear Lamina ◽  
Proliferating Cells ◽  
Intermediate Filament Proteins ◽  
Nuclear Assembly ◽  
Residual Structure ◽  
Clear Cut ◽  
Germinal Vesicles ◽  
Nuclear Envelope Assembly

The major residual structure that remains associated with the nuclear envelope following extraction of isolated nuclei or oocyte germinal vesicles with non-ionic detergents, nucleases and high salt is the lamina (Fawcett, 1966; Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The nuclear lamina is composed of intermediate filament proteins, termed lamins (Gerace and Blobel, 1980; Shelton et al., 1980), which polymerise to form a basket-weave lattice of fibrils, which covers the entire inner surface of the nuclear envelope and interlinks nuclear pores (Aebi et al., 1986; Stewart and Whytock, 1988; Goldberg and Allen, 1992). At mitosis, the nuclear envelope and the lamina both break down to allow chromosome segregation. As a consequence, each structure has to be rebuilt during anaphase and telophase, allowing cells an opportunity to reposition chromosomes (Heslop-Harrison and Bennett, 1990) and to reorganise looped chromatin domains (Franke, 1974; Franke et al., 1981; Hochstrasser et al., 1986), which may in turn control the use of subsets of genes. Because of the position that it occupies, its dynamics during mitosis and the fact that it is an essential component of proliferating cells, the lamina has been assigned a number of putative roles both in nuclear metabolism and in nuclear envelope assembly (Burke and Gerace, 1986; Nigg, 1989). However, to date there is little clear cut evidence that satisfactorily explains the function of the lamina in relation to its structure. In this Commentary we will describe some of the recent work that addresses this problem and attempt to provide a unified model for the role of lamins in nuclear envelope assembly and for the lamina in the initiation of DNA replication.

scholarly journals A lamin-independent pathway for nuclear envelope assembly.

1990 ◽  
Vol 111 (6) ◽  
pp. 2247-2259 ◽  
Author(s):  
J W Newport ◽  
K L Wilson ◽  
W G Dunphy
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Structural Integrity ◽  
Nuclear Lamina ◽  
Limited Extent ◽  
Nuclear Pores ◽  
Nuclear Assembly ◽  
Nuclear Envelope Assembly ◽  
A Cell ◽  
Nuclear Envelope Formation

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.

scholarly journals The role of protein phosphorylation in the assembly of a replication competent nucleus: investigations in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR

1995 ◽  
Vol 108 (1) ◽  
pp. 235-244 ◽  
Author(s):  
J. Murphy ◽  
C.M. Crompton ◽  
S. Hainey ◽  
G.A. Codd ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Nuclear Lamina ◽  
Nuclear Antigen ◽  
Cyanobacterial Toxin ◽  
Nuclear Assembly ◽  
Unusual Distribution ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly

Cell-free extracts of Xenopus eggs support nuclear assembly and DNA replication in vitro. Extracts supplemented with the protein phosphatase inhibitor microcystin-LR displayed various inhibitory effects at different concentrations of the toxin. In the presence of cycloheximide, additions of microcystin did not induce histone H1-kinase activity. Nevertheless, increasing concentrations of microcystin did sequentially prevent DNA replication, nuclear lamina assembly and nuclear envelope assembly. DNA replication was prevented when microcystin was added at 250 nM. Furthermore, this effect could be reversed after the addition of the catalytic sub-unit of protein phosphatase 2A to inhibited extracts. At a concentration of 250 nM microcystin, nuclear membrane assembly, nuclear lamina assembly and nuclear transport all occurred in egg extracts. In addition single-stranded M13 DNA replication was also permitted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an unusual distribution of proliferating cell nuclear antigen (PCNA). Although PCNA was located at sites that resembled pre-replication foci, this nuclear protein was readily solubilised when nuclei were isolated and extracted sequentially with Triton, nucleases and salts. Despite this, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.

scholarly journals Characterization of the elastic properties of the nuclear envelope

2005 ◽  
Vol 2 (2) ◽  
pp. 63-69 ◽  
Author(s):  
A.C Rowat ◽  
L.J Foster ◽  
M.M Nielsen ◽  
M Weiss ◽  
J.H Ipsen
Keyword(s):  
Nuclear Envelope ◽  
Structural Stability ◽  
Large Scale ◽  
Critical Role ◽  
Coiled Coil ◽  
Nuclear Lamina ◽  
Elastic Behaviour ◽  
Nuclear Pore ◽  
Two Dimensional ◽  
Intermediate Filament Proteins

Underlying the nuclear envelope (NE) of most eukaryotic cells is the nuclear lamina, a meshwork consisting largely of coiled-coil nuclear intermediate filament proteins that play a critical role in nuclear organization and gene expression, and are vital for the structural stability of the NE/nucleus. By confocal microscopy and micromanipulation of the NE in living cells and isolated nuclei, we show that the NE undergoes deformations without large-scale rupture and maintains structural stability when exposed to mechanical stress. In conjunction with image analysis, we have developed theory for a two-dimensional elastic material to quantify NE elastic behaviour. We show that the NE is elastic and exhibits characteristics of a continuous two-dimensional solid, including connections between lamins and the embedded nuclear pore complexes. Correlating models of NE lateral organization to the experimental findings indicates a heterogeneous lateral distribution of NE components on a mesoscopic scale.

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.

scholarly journals Chromosome length and gene density contribute to micronuclear membrane stability

2021 ◽  
Author(s):  
Anna Mammel ◽  
Heather Z Huang ◽  
Amanda L Gunn ◽  
Emma Choo ◽  
Emily M Hatch
Keyword(s):  
Nuclear Envelope ◽  
Membrane Integrity ◽  
Nuclear Lamina ◽  
Chromosome Length ◽  
Gene Density ◽  
Membrane Stability ◽  
Nuclear Pore ◽  
Membrane Rupture ◽  
Lamin B1 ◽  
Nuclear Envelope Assembly

Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here we demonstrate that two characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability in human cells. Chromosome length is proportional to micronuclei size, and gene density has an additive effect with micronucleus size on membrane stability. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial nuclear pore complex recruitment during post-mitotic nuclear envelope assembly. We find that chromosome length and micronuclei size strongly correlate with lamin B1 and nuclear pore density in intact micronuclei. Unexpectedly, lamin B1 levels do not predict nuclear lamina organization and membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.

scholarly journals Chromosome length and gene density contribute to micronuclear membrane stability

2021 ◽  
Vol 5 (2) ◽  
pp. e202101210
Author(s):  
Anna E Mammel ◽  
Heather Z Huang ◽  
Amanda L Gunn ◽  
Emma Choo ◽  
Emily M Hatch
Keyword(s):  
Nuclear Envelope ◽  
Membrane Integrity ◽  
Nuclear Lamina ◽  
Chromosome Length ◽  
Gene Density ◽  
Membrane Stability ◽  
Nuclear Pore ◽  
Membrane Rupture ◽  
Lamin B1 ◽  
Nuclear Envelope Assembly

Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here, we demonstrate that two characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability and determine the timing of micronucleus rupture. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial protein recruitment during post-mitotic nuclear envelope assembly. Micronucleus size strongly correlates with lamin B1 levels and nuclear pore density in intact micronuclei, but, unexpectedly, lamin B1 levels do not completely predict nuclear lamina organization or membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.

Purification and immunological detection of pea nuclear intermediate filaments: evidence for plant nuclear lamins

1992 ◽  
Vol 103 (2) ◽  
pp. 407-414 ◽  
Author(s):  
A.K. McNulty ◽  
M.J. Saunders
Keyword(s):  
Nuclear Envelope ◽  
Intermediate Filaments ◽  
Plant Cells ◽  
Nuclear Lamina ◽  
Animal Cells ◽  
Nuclear Lamins ◽  
Antigenic Characterization ◽  
Major Structural Component ◽  
Nuclear Envelope Assembly ◽  
Type V

A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1–3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.

scholarly journals A role for nuclear lamins in nuclear envelope assembly

2001 ◽  
Vol 154 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Reynold I. Lopez-Soler ◽  
Robert D. Moir ◽  
Timothy P. Spann ◽  
Reimer Stick ◽  
Robert D. Goldman
Keyword(s):  
Nuclear Envelope ◽  
Molecular Interactions ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Chromatin Decondensation ◽  
Nuclear Lamins ◽  
Nuclear Membranes ◽  
Terminal Domain ◽  
Nuclear Envelope Assembly

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina–pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These “nonfusogenic” vesicles lack lamin B3 (LB3) and do not bind LB3T; however, “fusogenic” vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

The structure and function of the nuclear lamins

1995 ◽  
Vol 53 ◽  
pp. 762-763
Author(s):  
R.D. Goldman ◽  
A. Goldman ◽  
S. Khuon ◽  
M. Montag-Lowy ◽  
R. Moir ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Lamina ◽  
Structure And Function ◽  
Supramolecular Organization ◽  
Intermediate Filament Proteins ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Lamins ◽  
Interphase Cells ◽  
Type V ◽  
And Function

The nuclear lamins are the Type V intermediate filament proteins comprising the nuclear lamina. The lamina is located subjacent to the nucleoplasmic face of the nuclear envelope where it interfaces with chromatin. The lamins are major karyoskeletal proteins which are thought to play important roles in the formation and maintenance of nuclear shape and architecture, as well as in the supramolecular organization of chromatin. The lamins have long been thought to be stable polymeric constituents of the interphase nuclear matrix, due to their insolubility in solutions containing detergents and high salt concentrations. During mitosis, however, the nuclear lamins depolymerize during nuclear envelope breakdown. Subsequently, the lamins repolymerize around the decondensing chromosomes as the nuclear envelope reassembles at the end of mitosis. Although there is a significant amount known about the properties and potential functions of the lamins during mitosis, surprisingly little is known about their properties during interphase. In light of this, we have undertaken experiments which are aimed at determining the properties of the lamins in interphase cells.

Selective digestion of nuclear envelopes from Xenopus oocyte germinal vesicles: possible structural role for the nuclear lamina

1990 ◽  
Vol 97 (3) ◽  
pp. 571-580
Author(s):  
S. Whytock ◽  
R.D. Moir ◽  
M. Stewart
Keyword(s):  
Nuclear Envelope ◽  
Xenopus Oocyte ◽  
Structural Integrity ◽  
Marked Change ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Structural Role ◽  
Germinal Vesicles ◽  
Pore Complexes

We have used enzymic digestion as a structural probe to investigate components of the nuclear envelope of germinal vesicles from Xenopus oocytes. Previous studies have shown that these envelopes are composed of a double membrane in which nuclear pore complexes are embedded. The nuclear pore complexes are linked to a fibrous lamina that underlies the nucleoplasmic face of the envelope. The pores are also linked by pore-connecting fibrils that attach near their cytoplasmic face. Xenopus oocyte nuclear envelopes were remarkably resistant to extraction with salt solutions and, even after treatment with 1 M NaCl or 3 M MgCl2, pores, lamina and pore-connecting fibrils remained intact. However, mild proteolysis with trypsin selectively removed the lamina fibres from Triton-extracted nuclear envelopes to leave only the pore complexes and connecting fibrils. This observation confirmed that the pore-connecting fibrils were different from the lamina fibres and were probably constructed from different proteins. Trypsin digestion followed by Triton treatment resulted in the complete disintegration of the nuclear envelope, providing direct evidence for a structural role for the lamina in maintaining envelope integrity. Digestion with ribonuclease did not produce any marked change in the structure of Triton-extracted nuclear envelopes, indicating that probably neither the pore-connecting fibrils nor the cytoplasmic granules on the pore complexes contained a substantial proportion of RNA that was vital for their structural integrity.

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