Nuclear envelope assembly

In these experiments, chromosome decondensation and nuclear envelope assembly (late mitotic events) coincided with decreases in the cyclin B level and MPF activity. To determine whether degradation of cyclin B is required for exit from mitosis, researchers added a mutant mRNA encoding a nondegradable cyclin B to a mixture of RNase-treated Xenopus egg extract and sperm nuclei. As shown in Figure 2 l-9d, MPF activity Increased in parallel with the level of the mutant cyclin B, triggering condensation of the sperm chromatin and nuclear envelope disassembly (early mitotic events). However, the mutant cyclin B produced in this reaction never was degraded. As a consequence, MPF activity remained elevated, and the late mitotic events of chromosome decondensation and nuclear envelope formation were both blocked. This experiment demonstrates that the fall in MPF activity and exit from mitosis depend on degradation of cyclin B. 

Lohka. M. J., and Masui, Y. (1984). Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell free preparations from amphibian eggs. J. Cell Biol. 9si 1222-1230. 

The homogenate described above should be used immediately for the nuclear envelope assembly reaction. Dilute the homogenate twofold with KHM and incubate at 33°C. Over a period of about 2 hr chromosomes and chromosome... 

Boman, A. L., Delannoy M. R.. and Wilson, K. L. (1992). GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. J. Cell Biol. 116, 281-294. 

Analysis of Nuclear Envelope Assembly Using Extracts of Xenopus Eggs... 

E. Examination of Nuclear Envelope Assembly Immunofluorescence Staining of Newly A.ssembled Nuclei and Assembly Intermediates... 

We have used both types of buffers to prepare extracts capable of nuclear envelope assembly, and have detected no differences in extracts prepared with GB and those prepared with EB. For the procedures described below the two buffers give the same results, except where noted. However, once a particular buffer is used to prepare an extract, the same buffer is used for all subsequent steps of fractionation, etc. 

Nuclear envelope assembly can be followed as described below. Usually NE assembly can be detected within 30 to 45 min of incubation. 

If the bacterially expressed cyclins are not available, metaphase extracts that retain high activity of cyclin B/p34 complexes can be prepared from unfertilized Xenopus eggs, which are naturally arrested in metaphase of meiosis II. The procedure for the preparation of these extracts is similar to that used to prepare extracts capable of nuclear envelope assembly except that the buffers are different. In contrast to GB and EB, which are used to prepare extracts capable of NE assembly, the buffer given below preserves cyclin B/p34 activity, and probably also the activity of other mitotic protein kinases (Lohka and Masui, 1984 Lohka and Mailer, 1985 Lohka et al., 1988). 

I thank Dec Ann Warren and Min Yu for their helpful comments on this manuscript, and M. Carpenter for the protocol for freezing egg extracts. Research on nuclear envelope assembly is supported in the author s laboratory by the Natural Sciences and Engineering Council of Canada. 

Newport, J. W., Wilson, K. L., and Dunphy, W. G. (1990). A lamin-independent pathway for nuclear envelope assembly. J. Cell Biol. Ill, 2247-2259. 

Ulitzur, N.. and Gruenbaum, Y. (1989). Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos. FEBS Lett. 259,113-116. 

Some degree of controversy exists as to whether nuclear envelope formation precedes, parallels, or follows the assembly of a nuclear lamina (see Georgatos et ai, 1994 Lourim and Krohne, 1994). Formation of the nuclear envelope in Drosophila embryo extracts is lamin dependent (Ulitzur et ai, 1992). In contrast, lamin-independent nuclear envelope assembly in vitro has been reported in Xenopus (Newport et ai, 1990 Meier et ai, 1991) and sea urchin (Collas et ai, 1995). The latter studies corroborate immunofluorescence observations of nuclear reconstitution after mitosis in somatic mammalian cells in vivo (Chaudhary and Courvalin, 1993). Assembly of a nuclear lamina in vitro can be monitored by immunofluorescence and immunoblotting using anti-lamin antibodies. 

Sea Urchin. Ripe males (Marinus, Inc.) are blotted with paper towels to remove surface sea water, injected with 0.5 M KCl intracoelomically (--0.3 ml for L. pictus and 2 ml for S. purpuratus), and placed inside a 15°C incubator. Sperm are shed in a plastic container (weighing boat) and collected dry with a disposable transfer pipet into a 1.5-ml centrifuge tube placed on ice. Sperm can usually be stored as such however, if seawater dilutes the sample, sperm may be centrifuged for 15 sec at 1000 g in a microcentrifuge to concentrate them. Sperm can be stored without additives at 4°C for up to 5 days. We have observed low proportions of chromatin decondensation and incomplete nuclear envelope assembly with sperm stored for longer periods. 

A cytoplasmic extract consists of a crude egg lysate containing cytosol, membrane vesicles (MVs), ribosomes, and occasional mitochondria, but is free of pronuclei, essentially all yolk granules, and other large cytoplasmic inclusions. Unfertilized egg extracts support only partial chromatin decondensation (Cameron and Poccia, 1994), whereas extracts from fertilized eggs support full chromatin decondensation and nuclear envelope assembly (Zhang and Ruderman, 1993 Cameron and Poccia, 1994 Collas and Poccia, 1995a,b). 

A cytosolic extract essentially free of MVs can be prepared from the S o extract (Collas and Poccia, 1995a). Cytosolic extracts support sperm chromatin decondensation, histone phosphorylation (Green et ai, 1995), and sperm lamin removal, but not nuclear envelope assembly (Collas et ai, 1995). To our knowledge, no such extracts have been investigated for the surf clam. 

Egg cytoplasmic MVs forming the bulk of the sea urchin male pronudear envelope in vivo are derived from the endoplasmic reticulum (ER) (Congo and Anderson, 1968). Egg homogenate MVs, also largely derived from the ER, are required for nuclear envelope assembly in vitro (Collas and Poccia, 1995a, 1996b). MVs can be easily isolated from the cytoplasmic S o extract. 

Surf Clam. Lamin assembly in clam male pronuclei occurs almost invariably in extracts made from 65-min-activated eggs (Longo et ai, 1994). For chromatin decondensation and nuclear envelope assembly, no exogenous nucleotides are required. These are presumably supplied by the cytoplasmic extract. Approximately half of the decondensed sperm nuclei in unactivated or 15-min-activated extracts assemble variable deposits of lamin. 

Numerous processes occurring during pronuclear formation in vitro, such as vesicle binding, vesicle fusion, and lamina assembly, rely on the function of specific membrane-associated proteins (Wilson and Newport, 1988 Collas and Poccia, 1996a,b for reviews, see Wiese and Wilson, 1993 Gerace and Foisner, 1994). This section describes procedures to prepare nuclear envelopes from in vitro sea urchin male pronuclei and to identify proteins in MVs suspected of playing a role in nuclear envelope assembly, as well as procedures for extracting peripheral membrane proteins. 

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