Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes.

The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching have yielded the following results: (a) In the presence of both fusogenic and nonfusogenic PEG membranes are brought together at closely apposed contact regions. (b) Fluorescent lipid probes quickly spread from the membranes of erythrocytes to cultured cells in the presence of both fusogenic and nonfusogenic PEG. (c) Proteins of the erythrocyte membranes were never observed to diffuse into the cultured cell membrane. (d) Water-soluble proteins did not diffuse from the erythrocyte interior into the target cell cytoplasm until the PEG was removed. These data suggest that the coordinate action of two distinct components is necessary for fusion as mediated by PEG. Presumably, the polymer itself promotes close apposition of the adjacent cell membranes but the fusion stimulus is provided by the additives contained in commercial PEG.

Insertion of lipid domains into plasma membranes by fusion with erythrocytes.

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.

Peroxidation-induced perturbations of erythrocyte lipid organization.

Peroxidation of erythrocyte membrane lipids by hydrogen peroxide perturbs the lipid bilayer and increases phagocytosis by macrophages. This study addresses the underlying mechanism of these processes, and in particular the role of malondialdehyde, a major byproduct of lipid peroxidation. When erythrocytes were treated with hydrogen peroxide or ascorbate/iron to generate malondialdehyde, or with malondialdehyde itself, only those cells treated with hydrogen peroxide showed increased phospholipid spacing and enhanced phagocytosis. This result indicates that the alterations observed are unique to hydrogen peroxide treatment, and that malondialdehyde does not play a role in inducing these changes in surface properties. Comparison of adherence to human umbilical vein endothelial cells and phagocytosis showed that increased phagocytosis was not mirrored by enhanced adherence. This result suggests that two different signals may mediate recognition of erythrocytes by macrophages and by endothelial cells.

Characterization of protein kinases in mitotic and meiotic cell extracts.

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.

Altered lipid packing identifies apoptotic thymocytes.

To test whether apoptotic thymocytes can be identified by altered packing of lipids in their plasma membranes, thymocytes were isolated from mice injected with hydrocortisone and stained with merocyanine 540 (MC540), a fluorescent probe sensitive to lipid packing. At 9-10 h after injection, a subpopulation of H2-Klo cells with increased MC540 staining was clearly discernable. When DNA degradation was assessed by staining fixed cells with propidium iodide (PI), the fraction of cells with increased MC540 staining corresponded to the fraction with reduced PI staining. In addition, enriching for the former by fluorescence-activated cell sorting enriched for the latter. These results indicate that living apoptotic thymocytes can be identified and separated on the basis of altered lipid packing and increased staining with MC540.

Single cell analysis of factors increasing the survival of resealed erythrocytes in the circulation of mice.

Erythrocytes labeled with fluorescent membrane markers were reinfused into mice. Blood samples removed periodically were analyzed by flow cytometry for survival of labeled cells. Normal erythrocytes labeled with the markers persisted in the circulation for 45 days. A fraction of lysed and resealed erythrocytes labeled with marker were rapidly cleared from the circulation; the remainder were removed at approximately the same rate as normal cells in the same animal labeled with marker of another color. Lysis in a minimal volume of buffer and addition of ATP during lysis both increased the fraction of cells surviving initial clearance.

Injected mitotic extracts induce condensation of interphase chromatin.

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.

Cellular compartmentalization of protein kinase activity during the cell cycle.

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.