Wilhelm bernhard workshop on the cell nucleus

DYNAMICS OF PML DURING HYPOTONIC SHOCK–INDUCED FORMATION OF MICRO-NUCLEI.

Kurchashova S. Yu., Kireyev I. I., *Gulak P. V., Polyakov V. Yu.

A.N. Belozersky Institute of Physico–Chemical Biology, Moscow State University and *Insitute of Biology of Gene, Moscow, Russia.
The promyelocytic leukemia nuclear bodies (PML NBs) are nuclear multiprotein structures that are tightly bound to the nuclear matrix.. Cells typically contain 10–30 PML NBs per nucleus with diameters 0.2–1 m, their number and size change during the cell cycle. Although the functions of PML and PML NBs remain largely unknown, evidence is accumulating for a role of PML in transcription regulation (Fuchsova et al., 2002). The PML protein is necessary for the proper formation of PML NBs and a defining marker for this structure. We have studied the role of individual chromosomes in postmitotic reconstruction of PML NBs. An experimental model of micronuclei formation offers several advantages for the study of nuclear domain assembly at the end of mitosis. The process of nuclear domain formation occurs more slowly than in intact cells and the micronuclei formed around individual chromosomes allow us to estimate the participation of the individual chromosomes in the formation of nuclear domains. Here, we demonstrate that PML protein is specifically associated with certain chromosomes in Hela and PK cells during micronuclei formation. The formation of micronuclei around individual chromosomes was induced by hypotonic treatment and transfer of cells to the culture medium The interaction between chromosomes and PML starts during hypotonic treatment and further persists upon subsequent transfer of cells to the culture medium. PML localization was observed as a spot associated with some chromosomes and also diffusely distributed in the cytoplasm. Immunofluorescent localization of PML protein in micronuclei revealed characteristic pattern of small discrete foci. The integrity of these foci was dependent on the association with RNA but not with DNA as shown by DNase and RNase treatment of cells. Based on these data we propose that PML specifically associates with certain chromosomes, and this formation does not require spatial association of chromosomes inside one nucleus.