Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

Abstract

The presence of two ras-related proteins (22 and 23 kDa) was demonstrated in Xenopus embryonic extracts by selective immunoprecipitation using anti-ras monoclonal antibodies 142-24E05 and Y13-259. We further describe the cytological effects of the microinjection of anti-ras monoclonal antibody Y13-259 into early cleavage blastomeres of Xenopus embryos. Injection of the antibody into a blastomere at the two-, four-, or eight-cell stage caused cleavage arrest in the descendants of the injected blastomere. Light microscopy (LM) of cleavage-arrested cells revealed extensive deformation of the cells as well as heterogeneity of distribution of yolk platelets and pigment granules. LM analysis of serial sections of cleavage-arrested cells revealed the presence of multiple nuclei. Although the nuclei expressed similar morphological properties, indicating that they were probably in the same stage of the nuclear cycle, they revealed highly variable chromatin densities. Electron microscope (EM) analysis of the cytoplasm of cleavage-arrested cells revealed the accumulation of vesicles and large membranous elements coincident with cleavage arrest. Furthermore, endoplasmic reticulum (ER) existed in two forms, as closed, circular profiles and as long, linear arrays. Mitochondria were characteristically aligned in single file on both sides of the two types of ER cisternae. EM analysis of nuclei confirmed variations in chromatin organization and suggested the occurrence of unique nuclear envelope fusion among micronuclei in cleavage-arrested cells. Cleavage arrest and changes in cytological features were not observed in the cytoplasm of cells microinjected with normal rat IgG. Thus the immunochemical data and microinjection experiments suggest that ras-like or ras antigenicity exists within rapidly replicating Xenopus blastomeres and may be involved in the organization of a number of its cytoplasmic elements.

Expression of ras‐like proteins in embryonic and adult cells of Xenopus laevis

“…Finally, PA1 antibody has been used to immunoprecipitate ras-like proteins from various Xenopus cell types. The immunoprecipitated peptides have a Mr -22,500, in agreement with the recently described data on Xenopus ras proteins: Mr -23,000 (Andeol et al, 1990) and Mr -22,000-23,000 (Miron et al, 1990:. Except for a brief report on the presence of ras sequences in the Xenopus genome by Mechali and co-workers (Moreau et al, 1989) and works on Ki type cDNA sequences (Andeol et al, 1990;Baum and Bebernitz, 19901, we have no data about the number and nature of ras genes in Xenopus. From what is known for other species (yeast, Aplysia, and Drosophila) (Barbacid, 1987), we inferred that there is more than one ras gene type in Xenopus also.…”

“…We previously described inhibition of Axolotl egg cleavage following injection of a commercial antibody raised against a c-Ha-ras p21 (Baltus et al, 1988). Miron et al (1990) obtained the same result onXenopus egg, using another anti-ras antibody. By electron microscope analysis, these authors observed in arrested embryos a number of abnormalities related to membrane structure.…”

Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres.

“…However, there are other studies which also implicate the inositol cascade in the cell cycle. The most relevant such study reported that microinjection of an antibody against a p2 ' H-ras onco-protein inhibited cell division in axolotl embryos (2) as well as inXenopus embryos (43). This ras gene protein is found predominantly on the cytoplasmic side of the plasma membrane and is a GTP-binding protein that activates various target enzymes, including PLC which hydrolyzes PIP2 (22,39,45,55) .…”