Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation

Abstract

Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.