Motility of intracellular particles in rat fibroblasts is greatly enhanced by phorbol ester and by over-expression of normal p21N-ras.
Particle motility in cultured rat fibroblasts was studied using video-enhanced differential interference contrast microscopy. The average velocity of large bright particles (apparent diameter about 0.5-0.7 micron) was measured in control cells and in cells treated with agents which affected targets related to signal transduction pathways. A Rat-2-derived fibroblast line transfected with a construct containing multiple copies of the N-ras proto-oncogene under the control of dexamethasone-sensitive promoter was used as a main experimental model. Dexamethasone treatment was shown to induce high levels of N-ras expression in these cells. This treatment greatly increased the average particle velocity. At the same time dexamethasone did not influence the particle motility in the non-transfected parent cells and in the cells transfected with a construct which did not contain N-ras. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), also induced an approximate eightfold increase in the particle rate after several hours of incubation, while sphingosine, an inhibitor of PKC, prevented this activation. Sphingosine alone reduced the particle motility after a 20 min incubation. The particle movements were inhibited also by colcemid. These data show that the activation of N-ras and PKC produced dramatic activation of microtubule-dependent particle motility. A possible role of this activation in signal-induced alterations of cell morphology is discussed.
Alexandrova
A Y
AY
A.N. Belozersky Institute of Physico-Chemical Biology of Moscow State University, Russia.
Dugina
V B
VB
Paterson
H
H
Bershadsky
A D
AD
Vasiliev
J M
JM
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003599:Cytoskeleton / Q000502:physiology; D003703:Demecolcine / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D002479:Inclusion Bodies / Q000502:physiology* / Q000648:ultrastructure; D008870:Microtubules / Q000502:physiology; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000502:physiology; D051381:Rats; D013110:Sphingosine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D014741:Video Recording
D003907:Dexamethasone; D011493:Protein Kinase C; D016283:Proto-Oncogene Proteins p21(ras); D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D003703:Demecolcine