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Journal of Cell Science, Vol 84, Issue 1 105-127, Copyright © 1986 by Company of Biologists

JOURNAL ARTICLES

Nuclear matrix generation during reactivation of avian erythrocyte nuclei: an analysis of the protein traffic in cybrids

CL Woodcock and H Woodcock

It has previously been shown that an internal nuclear matrix is generated during the reactivation of the chick erythrocyte nucleus in mouse L-cell cytoplasts. This experimental system has now been used to identify the major polypeptides that migrate into the nucleus during the reactivation process. Mouse L-cells were prelabelled with [35S]methionine, enucleated using cytochalasin B, and fused with 14- to 17-day embryonic chick erythrocytes. Sixteen hours post-fusion, the redistribution of the labelled proteins was examined by electron microscopic autoradiography, and two-dimensional polyacrylamide gel fluorography of the isolated nuclei was used to identify the major imported species. After allowing for cytoplasmic contamination, 15 nucleus-associated polypeptides were identified, two of which also matched with counterparts in the L-cell nuclear preparation. Five of the nucleus-associated polypeptides were tentatively identified (on the basis of one-dimensional gel matches) as nuclear matrix proteins; these five included the two that had counterparts in L-cell nuclei. The autoradiographic results showed that 16 h post-fusion, the specific activity (silver grains/unit area) of the reactivated nucleus was about half that of the cytoplasm, with no evidence for an accumulation of labelled protein at the nuclear periphery. When well-reactivated nuclei were distinguished from poorly reactivated nuclei on the basis of the extent of chromatin decondensation, it was found that their specific activities were quite similar, but because of the difference in size, the well-reactivated nuclei contained about twice as much labelled protein. Estimates of the protein traffic based upon the autoradiographic data indicated that the nuclei had increased in mass by 10-20% during the 16 h reactivation period.




© The Company of Biologists Ltd 1986