Xenopus 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. 

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

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). 

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. 

Extracts prepared from unfertilized Xenopus eggs thus contain all the materials required for multiple cell cycles, including the enzymes and precursors needed for DNA replication, the histones and other chromatin proteins Involved in assembling the replicated DNA into chromosomes, and the proteins and phospholipids required in formation of the nuclear envelope. These egg extracts also synthesize proteins encoded by mRNAs in the extract, including cyclln B. 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

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