Cytoskeletal reorganizations responsible for the phorbol ester-induced formation of cytoplasmic processes: possible involvement of intermediate filaments.

Abstract

The tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes of cell shape in certain fibroblastic lines: motile lamellas are transformed into noncontractile narrow processes; simultaneously, the actin microframent network of lamellas is locally disorganized. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentincontaining intermediate filaments (IF) in the development of PMA-induced reorganizations. PMA readily induced formation of noncontractile processes in Taxol-treated fibroblasts; these cells had a profoundly altered microtubular system but noncollapsed IF. A short (1 hr) exposure to PMA induced formation of processes in control cells but not in the Colcemidtreated cells, which had depolymerized microtubules and IF that collapsed around the nucleus. Longer (3-4 hr) exposure of the Colcemid-treated cells to PMA induced partial reversal of the IF collapse; those parts of the peripheral lamellas that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction ofIF with the actin system is an essential step in the formation of processes from lamellas. The microtubular system controls distribution of IF in the cytoplasm and thus plays an indirect role in the reorganization of the actin cortex.Alterations of the degree of cell segregation into two types of cytoplasmic domains, motile lamellas and nonmotile narrow processes, are characteristic of many morphological differentiations (1). Reversible enhancement of this segregation accompanied by the pronounced formation of nonmotile long processes from motile lamellas is induced in certain fibroblastic and epithelial cultures by phorbol 12-myristate 13-acetate (PMA). This phenomenon is convenient for the experimental analysis of mechanisms controlling segregation (1-5, t). PMA-induced transformation of lamellas into stable processes was found to be accompanied by profound reorganization of the cytoskeleton, especially by the disappearance of the network of actin microfilaments filling lamellas and by the loss ofcontractility ofthe actin cortex (3-5). These alterations of the microfilament system were shown to be microtubule dependent (4, t): their development after shortterm PMA treatment was prevented by preincubation of cells with Colcemid, which caused complete depolymerization of microtubules and was accompanied by perinuclear collapse of vimentin-containing intermediate filaments (IF). These data suggest that the cell has a special microtubule-dependent mechanism controlling the actin microfilament system (4).Experiments presented in this paper show that when the time of exposure of Colcemid-treated cells to PMA is increased to 3-4 hr, microtubules remain depolymerized, but IF collapse is reversed; simultaneously, formation of narrow processes from lamellas takes place. The main cytoskeletal elements pres...

Post-translational modification of microtubules is a component of synergic alterations of cytoskeleton leading to formation of cytoplasmic processes in fibroblasts.

“…What is the nature of these possible interactions? The presence of microtubules is not essential for disorganization of actin accompanying lamella-to-stalk transformation: these transformations can be induced by PMA in the cells with microtubules depolymerized by Colcemid (31). Intermediate filaments are the only type of cytoskeletal element that is always found in cytoplasmic stalklike processes induced by PMA in all types of experiments; as already indicated, intermediate filaments are usually absent from lamellas before their transformation into these processes.…”

“…Besides microtubule modification, lamella-to-stalk transformation also involves the profound reorganization of another cytoskeletal system, actin microfilaments; the network of microfilaments present in the lamellas is dismantled and actin cortex loses its contractility (3,4,30,31).…”

Cytoplasmic Linker Proteins Regulate Neuronal Polarization through Microtubule and Growth Cone Dynamics

“…In fact, fibroblast studies showed that microtubules and actin filaments mutually influence each other. For example, increased microtubule growth in fibroblasts after the washout of the microtubule-depolymerizing drugs nocodazole or colcemid increases actin polymerization and dynamics of lamellipodia (Bershadsky et al, 1990;WatermanStorer et al, 1999) by activating the Rho GTPase Rac1 (Waterman-Storer et al, 1999). Consistent with such interpretation, we found that modest nocodazole concentrations caused neurons with splayed microtubules at the tips, enlarged growth cones, and impaired growth cone dynamics and axon growth, recapitulating the phenotype of DN-CLIP-expressing neurons (data not shown).…”

Actin-dependent Lamellipodia Formation and Microtubule-dependent Tail Retraction Control-directed Cell Migration

“…It was therefore interesting to investigate whether this disruption also would inhibit stimulation-induced ruffling and lamellipodium formation. Because PMA has been shown to induce the reorganization of the actin cytoskeleton Bershadsky et al, 1990;Downey et al, 1992), we used this agent to study ruffle and lamellipodium formation in the presence or absence of microtubules. Upon PMA treatment of GFP-actin-transfected cells, it was possible to induce extensive ruffling and lamellipodium formation ( Figure 3A).…”