Immunocytochemical detection of the intranuclear variations of phosphatidylinositol 4,5-bisphosphate amount associated with changes of activity and amount of phospholipase C beta 1 in cells exposed to mitogenic or differentiating agonists.
The intracellular localizations of phosphatidylinositol 4,5-bisphosphate (PIP2) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the beta 1 isoform) have been evaluated by electron microscope immunocytochemistry in cells exposed to mitogenic or differentiating agents. These cells have been previously demonstrated to present a signal transduction system based on the polyphosphoinositide hydrolysis localized at the nuclear level, which can be specifically modulated by agonists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mitogenically stimulated by insulin-like growth factor I (IGF-I), a rapid and transient decrease of the PIP2 detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activation of the PLC beta 1 isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF-I, when the PLC beta 1 activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumulation of PIP2 in the nucleus, accompanied by a decrease of the intranuclear amount of PLC beta 1 isozyme, have been observed in mouse erythroleukemia Friend cells, induced to erythroid differentiation by dimethylsulfoxide (DMSO). These results indicate that quantitative immunocytochemistry represents an increment in the available methodologies to investigate the complex regulation of nuclear PI-signalling.
Maraldi
N M
NM
Institute of Citomorfologia Normale e Patologica, CNR, Bologna, Italy.
Zini
N
N
Ognibene
A
A
Martelli
A M
AM
Barbieri
M
M
Mazzotti
G
G
Manzoli
F A
FA
D016475:3T3 Cells; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D007942:Leukemia, Experimental / Q000473:pathology; D051379:Mice; D008854:Microscopy, Electron; D008934:Mitogens / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D054799:Phospholipase C beta; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D008934:Mitogens; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D007334:Insulin-Like Growth Factor I; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C518034:Plcb1 protein, mouse; D004121:Dimethyl Sulfoxide