Evidence for a Unique Profile Synthesis in Late Mitotic Cells of Phosphatidylcholine

Abstract

Evidence is presented that the structural rearrangements in late mitosis are accompanied by an alteration in membrane lipid synthesis. This evidence was derived from analyzing phospholipid classes after rapid-labeling, as well as from determining the intracellular site of incorporation of choline by HeLa $3 cells as they progressed from metaphase into early interphase (G1). Compared with postmitotic cell data, the recent mitotic cell data indicate a specific twoto threefold increase in the net synthesis of phosphatidylcholine (PC) species, which appeared to contain the more saturated fatty acids. Since this was observed with glycerol, choline, and orthophosphate labelings, and not with methyl labeling, it appears that the CDP-choline plus diacylglycerol pathway rather than the phosphatidylethanolamine to PC pathway was augmented. Electron microscope autoradiography of anaphase, telophase, and early G1 cells demonstrated that the reformed nuclear envelope was the incorporation site of a significant proportion of the newly synthesized PC. This incorporation occurred by early telophase prior to chromosome decondensation. The potential significance of PC metabolism with regard to membrane rearrangements, such as nuclear envelope reformation, is discussed. The late mitotic cell undergoes a series of dynamic ultrastructural changes (11, 19) resulting in progeny cells in which macromolecular synthesis (16, 22, 23) has been re-established. Among the ultrastructural events are several that involve the membranous components of the cell, such as reforming the nuclear envelope (NE), rearranging the endoplasmic reticulum (ER), and breaking and resealing the cytoplasmic membrane. Although ultrastructural changes in mitosis have long been recognized, the biochemical basis of most of these events, except for those involving the spindle apparatus (for a review see reference 18), is largely unknown. For example, this is the case for NE reformation, which is an early event in telophase. It is thought that membrane fragments for this reformation are, at least in part, carried through cell division at the telomeres of chromsomes (10). Recently, it has been argued that peripheral dense lamina polypeptides (putatively, NE components) play a role in this process (14). These polypeptides, which have been designated lamins A, B, and C, apparently become less phosphorylated during nuclear reformation. What role, if any, newly synthesized membrane lipids play in either nuclear or cytoplasmic membranous events cannot be evaluated at the present time because there is no information that can be associated unequivocally with cells in late stages of mitosis. The available data, on the other hand, have been obtained from partially synchronized cells and indicate that the incorporation of membrane lipids into the NE occurs only after the cell has entered interphase (G~) (8) and, thus, suggest that newly synthesized lipids are not involved in this process. In this paper, however, data are presented that suggest a specific role for a particular class of membrane lipids in late mitotic events. We have determined the amount and the profile of phospholipid synthesized in tightly synchronized populations, and the intracellular site of incorporation of one class of these membrane components. Specifically, aspects of phospholipid biosynthesis were explored in the human derived cell line, HeLa $3, by following the incorporation of radioactive orthophosphate, glycerol, choline, and methionine under conditions of "rapid-labeling." For this analysis, metaphase cells were collected and then, by incubation at 37"C, were permitted to traverse through the final stages of mitosis. In addition, the individual stages of late mitosis were analyzed for the intracellular distribution of choline incorporation by electron microscope autoradiography (EMAR). Our data indicate that late mitotic cells have an augmented phosphatidylcholine (PC) biosynthetic activity, in particular, and that a substantial portion of this newly synthesized PC can be visualized in association with the NE by early telophase before chromosome decondensation. MATERIALS AND METHODS Cell Culture and Synchronization: HeLa S3 cells were maintained at 37°C in suspension culture in Eagle's minimal essential medium (gibco Laboratories, Grand Island, NY) supplemented with 7% calf serum and 2 mM

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Cite this paper

@inproceedings{Henry2002EvidenceFA, title={Evidence for a Unique Profile Synthesis in Late Mitotic Cells of Phosphatidylcholine}, author={Scott M Henry and Lashanda Hodge}, year={2002} }