Action of cytochalasin D on cytoskeletal networks.

Abstract

Extraction of BSC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body : 10-nm filaments, actin microfilaments, microtubules, and 2-to 3-nm filaments . Actin filaments and 2-to 3-nm filaments form numerous end-to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the threedimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased . Supernates of preparations treated in this way contain both short actin filaments and network fragments (i .e ., actin filaments in end-to-side contact with other actin filaments) . It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response . The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filamentfilament interactions (i .e ., disruption of end-to-side contacts) . The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci .The cytochalasins have now been used for more than a decade as tools to study motility-related phenomena in a variety of cell types (e.g., references 7, 11, 26, 27, 39; for an overview, see reference 33) . They exert a number of dramatic morphological effects on cells, including inhibition of ruffling and motility, cell retraction and arborization, zeiosis, blebbing, and enucleation (reviewed in reference 14) . Because of their profound effects on cell morphology and motility, and because of numerous electron microscope observations of marked effects on filamentous structures, it had early been assumed that their primary site of action is the cellular contractile machinery . In fact, cytochalasin-sensitivity alone has often been taken as an indication for the involvement of an actin-based contractile system in certain cellular activities, even in the absence of additional supportive morphological or physiological evidence.However, the mechanism of action of cytochalasin has begun to be understood only recently . In vitro studies using purified

Regulation of Epithelial Sodium Channels by Short Actin Filaments

“…While this interaction may serve as a means to control the spatial distribution of ion channels in the apical membrane of epithelial cells, a requirement in polarized epithelia, the possibility also existed that the colocalization of actin filaments with the apical Na ϩ channels would serve a functional role. Modification of actin filament organization with cytochalasin D, an actin filament disrupter (41)(42)(43), induced apical Na ϩ channel activity in the A6 cell (15). Because DNase I, which inhibits actin polymerization, inhibited the cytochalasin D-induced Na ϩ channel activity, the data suggested that "short" actin filaments might be involved in effect- ing ion channel activation.…”

The role of the cytoskeleton in the viscoelastic properties of human articular chondrocytes

“…Prior to testing the chondrocytes, alginate beads were incubated for 3 h in control, low, medium and high concentrations of chemical agents that disrupt the three principal cytoskeletal elements (cytochalasin D for microfilaments [44] Micropipette aspiration was used to determine the viscoelastic properties of the chondrocytes, as described previously [48]. Briefly, the solution and cells were placed in a chamber that allows for the entry of a micropipette from the side [20].…”

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…It is known that actin remodeling requires polymerization of G-actin to form F-actin. Therefore, we hypothesized that, if we inhibited the process by a pharmacological inhibitor, cytochalasin D (Cyto D), 59 we could reduce the pDNA uptake through macropinocytosis. Indeed, our experimental data revealed that pretreatment of cells with Cyto D decreased eTE in B16.F10 and HEK293 cells by 60% and 50%, respectively ( Figure 4B).…”