Ultrastructural localization of cPLA2 in unstimulated and EGF/A23187-stimulated fibroblasts.
The 85 kDa cytosolic phospholipase A2 is the key enzyme in the release of arachidonic acid. To gain insight into cytosolic phospholipase A2 action in mitogen-activated cells, the localization of the phospholipase was investigated in fibroblasts upon stimulation with epidermal growth factor and the calcium ionophore A23187. By the use of indirect immunofluorescence microscopy, staining of endogenous cytosolic phospholipase A2 resulted in a punctate labeling pattern randomly distributed throughout the cytoplasm of the cell. Immunogold electron microscopy revealed that this punctate labeling pattern exhibited the presence of the 85 kDa phospholipase A2 in small clusters. These clusters were found in the cytosol in the vicinity of all organellar membranes, except for the Golgi system. The enzyme showed no preference for the nuclear envelope, the endoplasmic reticulum or the plasma membrane. Stimulation of cells with epidermal growth factor or A23187 or both did not change the punctate immunofluorescence labeling pattern. Furthermore, a similar labeling pattern was observed by the artificial introduction of extremely low or high intracellular calcium concentrations. Even by electron microscopy, translocation of cytosolic phospholipase A2 to membranes was not observed after stimulation of cells with epidermal growth factor and A23187. From these results it is concluded that cytosolic phospholipase A2 is localized in clusters close to membranes in stimulated as well as unstimulated fibroblasts, without preference for a specific organellar membrane.
Bunt
G
G
Department of Molecular Cell Biology, Utrecht University, Utrecht, The Netherlands. gertrude@emsaserv.biol.ruu.nl
de Wit
J
J
van den Bosch
H
H
Verkleij
A J
AJ
Boonstra
J
J
D016475:3T3 Cells; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003599:Cytoskeleton / Q000201:enzymology / Q000648:ultrastructure; D003600:Cytosol / Q000201:enzymology* / Q000648:ultrastructure*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D010741:Phospholipases A / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D054467:Phospholipases A2
D000001:Calcimycin; D004815:Epidermal Growth Factor; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium