Vesicle urchin extracts

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). 

Fusion of chromatin-bound vesicles in vitro is promoted by GTP hydrolysis in sea urchin egg extracts (Collas and Poccia, 1995a), as shown previously in Xenopus (Boman et al., 19%). MVs are bound to chromatin in the presence of an ATP-generating system as described above. To induce fusion, GTT (Type II, Sigma G8752) is added to the extract from a frozen 5 mM stock in egg lysis buffer to a final concentration of 100 pM (Cameron and Poccia, 1994 Collas and Poccia, 1995a). Fusion usually occurs within 15-20 min of incubation at room temperature, but we routinely allow fusion to proceed for up to 40 min to ensure completion of the reaction. MV fusion can be assessed directly by... 

Sea urchin male pronuclei formed in vitro under the conditions described in this chapter remain small ( 4 /tm in diameter) and do not contain a lamina. Pronuclear swelling is promoted only if extra ATP is added to the egg extract. Nuclear swelling has been shown to be associated with, and dependent on, assembly of a nuclear lamina from a cytosolic pool of soluble lamins (Collas et al., 1995). More recent unpublished observations have shown that B-type lamins are also associated with a minor fraction of MV2i3 vesicles. The contribution of these vesicle-associated lamins to the nuclear lamina is under investigation. In the surf clam, sperm chromatin decondensation and pronuclear expansion are continuous processes that occur in parallel with nuclear envelope and lamina assembly in 65-min-activated egg extracts (Longo et al., 1994). Manipulations of this in vitro system to control each of these processes have not been reported. 

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. 

Fig. 7 Extractability of lamin B from membrane vesicles. Sea urchin egg MVs were extracted for 30 min at 4 C in TN buffer (Cont.), or TN supplemented with 1 M NaCI, 2 M urea, 0.1 M NajCO.i (pH 11.5), or 250 mAf MgClj. Vesicles were pelleted at 150,000 g for 10 min and dissolved in SDS sample buffer. Extracted proteins were TCA precipitated and dissolved in SDS sample buffer. Pelleted vesicles (P) and supernatants (S) were run on 10% SDS-PAGE and immunoblotted using the ECM-20 antibody. Note that lamin B of MVs migrates with an apparent M, of 68,000. Lamin B is recovered exclusively from the supernatants following salt, urea, and alkaline extractions. MgCb partially extracts lamin B from vesicles.

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