Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Abstract

Abstract, Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin Ltti did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.

Assembly of nuclear pore complexes mediated by major vault protein

“…S5). As previously shown, such Nup62-positive structures, which are readily visualized in the light microscope, represent stacks of AL (Dabauvalle et al, 1991). This observation is consistent with the notion that MVP-associated membranes are recruited into nascent AL and hence are no longer available for NE formation when chromatin is added to the preincubated extract.…”

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The experimental system Previously, we have noted that incubation of egg extract without addition of chromatin promoted the formation of flat membrane cisternae studded with pore complexes, termed annulate lamellae (AL) (Dabauvalle et al, 1991). In a subsequent study, we found that AL formation apparently depleted a specific type of membrane vesicles from the egg extract that were required for NPC assembly from the egg extract (Ewald et al, 1997).

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“…Samples for immunofluorescence microscopy were processed as described (Dabauvalle et al, 1991). Images were recorded with a Zeiss Axiophot (Carl Zeiss, Oberkochen, Germany) equipped with epifluorescence optics and a CCD camera.…”

“…Images were recorded with a Zeiss Axiophot (Carl Zeiss, Oberkochen, Germany) equipped with epifluorescence optics and a CCD camera. EM was performed as described (Dabauvalle et al, 1991).…”

Pre-M Phase-promoting Factor Associates with Annulate Lamellae inXenopusOocytes and Egg Extracts

“…AL membranes are continuous with and embedded within the ER (this study, Figure 6, A-C;Kessel, 1992;Terasaki et al, 2001), and it is not clear why they separate from the rest of the ER during fractionation, although the high concentration of nuclear pores is likely to significantly affect their density. AL accumulate in interphase Xenopus egg extracts (Dabauvalle et al, 1991;Meier et al, 1995) and disassemble in parallel with the nuclear envelope at mitosis/meiosis (Cordes et al, 1996;Imreh and Hallberg, 2000;Terasaki et al, 2001) and thus will be maximally accumulated at the time we made our extracts, favoring their identification in our study. We were able to confirm that part of the MPF population in the undisturbed cell colocalizes with AL by two independent visualization methods applied to whole oocytes: immunofluorescence on fixed material and live imaging of GFP-CyclinB2.…”