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Updated: Aug 10, 2026
Microinjection of Xenopus Laevis Oocytes
Published on: February 23, 2009
Morphogenesis of endoplasmic reticulum in Xenopus oocytes after microinjection of rat liver smooth microsomes
J Paiement1, J M Dominguez, J McLeese
1Département d'Anatomie, Université de Montréal, Québec, Canada.
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.
Insights
Rat-liver smooth microsomes rapidly self-assemble into endoplasmic reticulum-like networks in Xenopus oocytes. This microtubule-independent process demonstrates inherent molecular information for membrane structure formation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Endoplasmic reticulum (ER) structure is crucial for cellular function.
- Smooth microsomes (SM) are vesicles derived from the ER.
- Understanding ER self-assembly is key to cell biology.
Purpose of the Study:
- To investigate the kinetics of ER reconstitution from rat-liver SM in Xenopus oocytes.
- To determine if SM self-assembly into ER-like structures is microtubule-dependent.
- To establish a novel assay for membrane subfraction self-assembly.
Main Methods:
- Microinjection of rat-liver SM into Xenopus oocytes.
- Electron microscopy (thin-section and thick-section).
- Cytochemistry and 3D reconstruction.
- Drug treatments (vinblastine, nocodazole).
Main Results:
- SM transformed into anastomosing tubules, forming an ER-like network within 80 minutes.
- The reconstituted network closely resembled native ER in rat hepatocytes.
- Network formation and maintenance were independent of microtubules.
- Pre- and post-injection drug treatments did not inhibit network assembly or organization.
Conclusions:
- Smooth microsomes possess intrinsic molecular information for self-assembly into ER structures.
- ER assembly and maintenance in oocyte cytoplasm are microtubule-independent processes.
- This study provides a valuable in vivo assay for studying membrane subfraction self-assembly.
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