|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
Journal of Cell Science, Vol 108, Issue 11 3451-3461, Copyright © 1995 by Company of Biologists
JOURNAL ARTICLES |
M Goldberg, H Jenkins, T Allen, WG Whitfield and CJ Hutchison
CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
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.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  E. R. Rafikova, K. Melikov, C. Ramos, L. Dye, and L. V. Chernomordik Transmembrane Protein-free Membranes Fuse into Xenopus Nuclear Envelope and Promote Assembly of Functional Pores J. Biol. Chem., October 23, 2009; 284(43): 29847 - 29859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. K. Shumaker, L. Solimando, K. Sengupta, T. Shimi, S. A. Adam, A. Grunwald, S. V. Strelkov, U. Aebi, M. C. Cardoso, and R. D. Goldman The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication J. Cell Biol., April 21, 2008; 181(2): 269 - 280. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H. Fischer, P. Taysavang, and S. M. Jhiang Nuclear Envelope Irregularity Is Induced by RET/PTC During Interphase Am. J. Pathol., September 1, 2003; 163(3): 1091 - 1100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kiseleva, S. Rutherford, L. M. Cotter, T. D. Allen, and M. W. Goldberg Steps of nuclear pore complex disassembly and reassembly during mitosis in early Drosophila embryos J. Cell Sci., March 12, 2002; 114(20): 3607 - 3618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Dimitrova, T. A. Prokhorova, J. J. Blow, I. T. Todorov, and D. M. Gilbert Mammalian nuclei become licensed for DNA replication during late telophase J. Cell Sci., January 1, 2002; 115(1): 51 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Moir, T. P. Spann, H. Herrmann, and R. D. Goldman Disruption of Nuclear Lamin Organization Blocks the Elongation Phase of DNA Replication J. Cell Biol., June 12, 2000; 149(6): 1179 - 1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T Tanaka and N Shimizu Induced detachment of acentric chromatin from mitotic chromosomes leads to their cytoplasmic localization at G(1) and the micronucleation by lamin reorganization at S phase J. Cell Sci., January 2, 2000; 113(4): 697 - 707. [Abstract] [PDF]
|
|
|
|
|
|
J. Ortega and M. DePamphilis Nucleoskeleton and initiation of DNA replication in metazoan cells J. Cell Sci., June 14, 1999; 111(24): 3663 - 3673. [Abstract] [PDF]
|
|
|
|
|
|
D Lourim and G Krohne Chromatin binding and polymerization of the endogenous Xenopus egg lamins: the opposing effects of glycogen and ATP J. Cell Sci., June 14, 1999; 111(24): 3675 - 3686. [Abstract] [PDF]
|
|
|
|
|
|
A Gajewski and G Krohne Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs J. Cell Sci., January 8, 1999; 112(15): 2583 - 2596. [Abstract] [PDF]
|
|
|
|
|
|
C Lang, M Paulin-Levasseur, A Gajewski, M Alsheimer, R Benavente, and G Krohne Molecular characterization and developmentally regulated expression of Xenopus lamina-associated polypeptide 2 (XLAP2) J. Cell Sci., January 3, 1999; 112(5): 749 - 759. [Abstract] [PDF]
|
|
|
|
|
|
J.-M. Lemaitre, G. Geraud, and M. Mechali Dynamics of the Genome during Early Xenopus laevis Development: Karyomeres As Independent Units of Replication J. Cell Biol., September 7, 1998; 142(5): 1159 - 1166. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Alsheimer, E Fecher, and R Benavente Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins J. Cell Sci., January 8, 1998; 111(15): 2227 - 2234. [Abstract] [PDF]
|
|
|
|
|
|
L. Yang, T. Guan, and L. Gerace Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase J. Cell Biol., December 1, 1997; 139(5): 1077 - 1087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Lenz-Bohme, J. Wismar, S. Fuchs, R. Reifegerste, E. Buchner, H. Betz, and B. Schmitt Insertional Mutation of the Drosophila Nuclear Lamin Dm0 Gene Results in Defective Nuclear Envelopes, Clustering of Nuclear Pore Complexes, and Accumulation of Annulate Lamellae J. Cell Biol., June 2, 1997; 137(5): 1001 - 1016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. P. Spann, R. D. Moir, A. E. Goldman, R. Stick, and R. D. Goldman Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis J. Cell Biol., March 24, 1997; 136(6): 1201 - 1212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Nicolas, C Zhang, M Hughes, M Goldberg, S Watton, and P Clarke Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts J. Cell Sci., January 12, 1997; 110(24): 3019 - 3030. [Abstract] [PDF]
|
|
|
|
|
|
Z. Lu, D. Sittman, D. Brown, R Munshi, and G. Leno Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract J. Cell Sci., January 11, 1997; 110(21): 2745 - 2758. [Abstract] [PDF]
|
|
|
|
|
|
D. Ellis, H Jenkins, W. Whitfield, and C. Hutchison GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication J. Cell Sci., January 10, 1997; 110(20): 2507 - 2518. [Abstract] [PDF]
|
|
|
|
|
|
M. Goldberg, C Wiese, T. Allen, and K. Wilson Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly J. Cell Sci., January 2, 1997; 110(4): 409 - 420. [Abstract] [PDF]
|
|
|
|
|
|
C Zhang, H Jenkins, M. Goldberg, T. Allen, and C. Hutchison Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract J. Cell Sci., January 9, 1996; 109(9): 2275 - 2286. [Abstract] [PDF]
|
|
|
|
|
|
D Lourim, A Kempf, and G Krohne Characterization and quantitation of three B-type lamins in Xenopus oocytes and eggs: increase of lamin LI protein synthesis during meiotic maturation J. Cell Sci., January 7, 1996; 109(7): 1775 - 1785. [Abstract] [PDF]
|
|
