Microtubule-dependent effect of phorbol ester on the contractility of cytoskeleton of cultured fibroblasts.

Abstract

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) upon the contractility of permeabilized cell models (cytoskeletons) of mouse fibroblasts was examined. Contraction was induced by incubation of cell models in a solution containing ATP and was assessed quantitatively by measuring alterations of the area of cell model projection on the substrate. Immunofluorescence microscopy was used to assess alterations ofcytoskeleton morphology in the course of permeabilization and contraction. It was found that contractility of cell models from PMA-treated fibroblasts was considerably diminished as compared with the models from control fibroblasts. ATP induced only local contraction of certain zones of actin cortex in models from PMA-treated fibroblasts; it did not induce general contraction, characteristic of control models. Normal high contractility was characteristic of the models from the cells preincubated with PMA in combination with Colcemid. PMA is a specific activator of protein kinase C, one of the key enzymes of the membrane signal-transduction pathway. It is suggested that protein kinase C regulates contractility of actin cortex and that the pathway of this regulation has a microtubule-dependent stage blocked by Colcemid.The tumor promoter phorbol 12-myristate 13-acetate (PMA), a phorbol ester that is a specific activator of protein kinase C (1), has been reported to induce various types of alterations of morphology, proliferation, and differentiation in cultured cells (2-6). In particular, PMA induces a special type of morphological alteration in certain lines of cultured fibroblasts: these cells are reversibly segregated into motile lamella-forming zones and nonmotile long "tail" processes (7). Experimental analysis of this effect gave us reason to suggest that shape changes are due to PMA-induced alterations of interrelationships between the microtubular system and actin cortex. Danowski and Harris (8,28) have shown that when cultured fibroblasts are spread on an elastic substrate, PMA decreases their ability to wrinkle this substrate; these data suggest that PMA can regulate the contractility of actin cortex.To study further functional alterations of actin cortex in PMA-treated fibroblasts we examined the effect of ATP on permeabilized preparations of cells (also called cell models or cytoskeletons) from differently treated cultured fibroblasts. These "cell models" have their cytoskeletal structures largely exposed to the external environment. When models are incubated with ATP in the appropriate buffer solution they contract; this contraction is probably due to the actinmyosin interactions within their cortices (9-12).We have found that ATP contractility of models of PMAtreated cells was much weaker than that of the models of control cells. This inhibition of contractility was prevented by Colcemid: cytoskeletons of cells treated by PMA after incubation with Colcemid contracted in ATP solution as readily as did the cytoskeletons of controls.These experiments indicate that protein ...

Disruption of the Golgi apparatus by brefeldin A blocks cell polarization and inhibits directed cell migration.

“…(Fig. 5 B and C), similar to the effect of pretreatment with Colcemid (19 (Fig. 6A), with ruffles containing a dense actin network (Fig.…”

Reversible structural alterations of undifferentiated and differentiated human neuroblastoma cells induced by phorbol ester.

“…Spectacular enhancement of these transformations can be induced in fibroblasts by the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), an agent that activates protein kinase C (PKC) (2). Analysis of PMA-induced segregation suggests that activation of PKC may alter the interrelationships among the actin microfilament, microtubule, and intermediate filament networks (3)(4)(5)(6).…”

“…Interestingly, their results leave open the possibility that actin polymerization in PMA-sensitive cells could occur through a direct effect of PMA on a membrane-associated actin nucleation factor rather than through a PKC-dependent pathway. After completion of lamellar extension, the formation of cylindrical processes involves the disassembly of lamellar actin networks and loss of lamellar contractility (3,4). Synergistic interactions among microtubules, intermediate filaments, and actin filaments may be essential for these reorganizations (14) and phosphorylation of cytoskeletal proteins, such as myosin II (15) Actinoplast-tubuloplast segregation may certainly depend on the interactions of several regulatory pathways.…”

Vasopressin-induced disruption of actin cytoskeletal organization and canalicular function in isolated rat hepatocyte couplets: Possible involvement of protein kinase C

“…41,[44][45][46] Furthermore, in addition to the effect on actin conformation, PKC activation was reported to induce changes in other major cytoskeletal elements such as intermediate filament proteins [47][48][49] and tubulin. 42 Finally, PKC was reported to have a role in hepatocyte apoptosis induced by a number of compounds such as the bile salt, glycochenodeoxycholate, 50 [2][3][4] Dissipation of osmotic gradients induced by an increase of ''leakiness'' of the paracellular barrier appears to play a key role in this phenomenon. Indeed, VP as well as a number of agents affecting Ca 2ϩ mobilization such as the Ca 2ϩ -ionophore, A23187, and the inhibitor of microsomal Ca 2ϩ sequestration, t-butyl-benzohydroquinone, were all shown to increase tightjunctional permeability in both isolated perfused rat liver 4,[7][8][9] and in isolated rat hepatocyte couplets.…”

“…One likely candidate is PKC. Indeed, PKC activation has been shown to induce extensive actin filament reorganization in a variety of cell lines, [40][41][42] presumably by the phosphorylation of actin 43,44 as well as other actin-associated proteins such as vinculin, filamin, ␣-actinin, and calspectin. 41,[44][45][46] Furthermore, in addition to the effect on actin conformation, PKC activation was reported to induce changes in other major cytoskeletal elements such as intermediate filament proteins [47][48][49] and tubulin.…”