Morphogenesis of endoplasmic reticulum in Xenopus oocytes after microinjection of rat liver smooth microsomes

Abstract

We have determined the kinetics of endoplasmic reticulum (ER) reconstitution following insertion of rat-liver smooth microsomes (SM) into Xenopus oocyte cytoplasm using electron microscopy as well as cytochemistry and thick-section 3-dimensional reconstruction. Oocytes were fixed 0, 10, 20, 40, 80, and 120 min after microinjection with SM and processed for thin- and thick-section electron microscopy. At 0 min postinjection, rat liver SM were observed as small vesicles and were loosely dispersed amongst oocyte organelles. At 10 min, tubules were discerned among many elongate vesicles; and these structures comprised large cytoplasmic regions delimited by mitochondria and yolk platelets. By 20 min, segregation of transplanted organelles yielded yolk-platelet-free regions composed of few vesicles but increasingly numerous, long and anastomosing tubules. By 40 min, a network with numerous tubular branches and fenestrations was observed among the few remaining vesicles. By 80 min, transformation of rat liver SM into a complex network of branching and anastomosing tubules was complete. Three-dimensional reconstruction revealed the network to be composed of interconnecting elements consisting of anastomosing tubules. The reconstituted network of anastomosing tubules in Xenopus oocytes was compared to the network of anastomosing tubules in rat liver hepatocytes and was found to be essentially identical. Network formation occurred in oocytes pretreated with either vinblastine (40 microM) or nocodazole (0.166 microM), and network organization was maintained in oocytes treated with the same drugs after microinjection and reconstitution. We conclude that SM retain sufficient molecular information for rapid self-assembly into structures resembling those in the cells from which they were derived. Both the assembly and maintenance of ER structure in oocyte cytoplasm are microtubule-independent. The formation of such structures following microinjection of SM into living cells provides a unique assay for this type of membrane subfraction.

Dynamic and reversible restructuring of the ER induced by PDMP in cultured cells

“…The available data suggest that the architecture of the organelle is determined both by intrinsic factors (proteins and/or lipids) of the ER membrane itself and by the interaction of the membranes with the microtubule cytoskeleton. Thus, although microsomal vesicles can assemble into a branching tubular network in the absence of microtubules (Dreier and Rapoport, 2000;Paiement et al, 1990) and even in the absence of any cytosolic factor (Voeltz et al, 2006) the distribution of tubules, their extension and the regulation of their branching is microtubule dependent (Dabora and Sheetz, 1988;Lee and Chen, 1988;Terasaki et al, 1986;Waterman-Storer and Salmon, 1998). The SER random tubular network is characterised by highly convoluted tubules rather than by the relatively straight ones of the polygonal meshwork and could thus represent a spontaneous arrangement of the ER -similar to the so-called 'sponge phase' of curvature theoreticians (Hyde et al, 1997) -that is attained in the absence of regulatory factors mediating interaction with microtubules.…”