Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth

Abstract

Phalloidin, the toxic drug from the mushroom Amanita phalloides, was injected into the cytoplasm of tissue culture cells and the changes in intracellular actin distribution were followed by immunofluorescence microscopy with actin antibody. At low concentrations, phalloidin recruits the nonor less highly polymerized forms of cytoplasmic actin into stable "islands" of aggregated actin polymers and does not interfere with the preexisting thick bundles of microfilaments (stress fibers). Differential focusing shows that these islands of phalloidin-induced actin polymers occur at a level in the cytoplasm that is above the submembranous bundles of microfilaments present on the adhesive side of the cells. The pattern of cytoplasmic microtubules remains unaffected by the injection of phalloidin; however, filamin, a protein usually associated with actin in the cytoplasm, is also recruited into the islands. At higher phalloidin concentrations, contraction of the cell is observed. These results are discussed in the light of previous biochemical studies by Wieland and Faulstich and their coworkers [for a review see Wieland, T. (1977) Naturwissenschaften 64, 303-3091 on the in vitro interaction of phalloidin with muscle actin, which have documented that phalloidin reacts stoichiometrically with actin, promotes actin polymerization, and stabilizes actin polymers.In addition, we show that microinjection of phalloidin interferes in a concentration-dependent manner with cell locomotion and cell growth. These results indicate that a well-balanced controlled reversible equilibrium between different polymerization states of actin may be a necessary requirement for cell locomotion and may also influence other cellular functions such as growth.Actin is the major structural protein of the cytoplasm of eukaryotic cells. The structural organization and polymerization of cytoplasmic actin is still poorly understood (for a review see ref. 1). Although in muscle cells actin is exclusively organized in nearly crystalline ordered arrays of thin filaments, the actin organization in nonmuscle cells can be rather diverse. Actin is the major protein of the thick bundles of microfilaments (stress fibers) typical for a variety of fibroblastic, epithelial, and other cells (2-4), but less ordered actin polymerization occurs in the ruffling edge of these cells, and many other cell types show only thin microfilamentous structures (2, 3, 5, 6). In addition, the mechanisms that govern the polymerization and depolymerization of these various types of cellular actin structures are still unclear and it is currently difficult to assess how much of the cellular actin is present in an unpolymerized form (G actin) and how much is present in different types of polymers (F actin), including the individual microfilaments and the stress fibers.Recently phalloidin, a cyclic peptide of the mushroom Amanita phalloides, was introduced as an actin-specific drug in vitro. Wieland, Faulstich, and their coworkers have carefully documented in several studies tha...

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

“…The prior observation that jasplakinolide decreases the cellular pool of identifiable G-actin (25,26,29) correlates with the expected drop in both free and sequestered G-actin caused by jasplakinolide in vitro. Phalloidin, without a large effect on the rate constant for nucleation (17), has been reported to induce similar changes in stress fibers when loaded directly into cells at high concentrations (30).…”

Versatile fluorescent probes for actin filaments based on the actin‐binding domain of utrophin

“…Further, because only a fraction of actin is in polymer form at any given time [Korn, 1982], imaging with fluorescent G-actin entails high background signal, unless specialized techniques such as speckling are employed [Waterman-Storer et al, 1998]. Phalloidin must be microinjected, and stabilizes actin filaments [Dancker et al, 1975], and thus changes the balance of actin assembly/ disassembly in vivo [Planques et al, 1991;Dancker et al, 1975;Wehland et al, 1977]. Moreover, fluorescent phalloidin poorly labels dynamic actin in vivo .…”

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.

“…Because phallotoxins are not membrane permeable for many cell types (32, 35,36,38), the effects of these drugs can be tested reliably only when the drugs are introduced directly into the cytoplasm. We have used the perfusion technique of Tazawa et al (33) to introduce drugs into the interior of Chara cells.…”

“…Unlike CB, these bi-cyclic peptides are generally not membrane permeable (32,35, 36,38). However, upon microinjection, phalloidin inhibits cytoplasmic streaming and causes ultrastructural disruption in Amoeba proteus and Physarum polycephalum (32, 36) and interferes with locomotion of fibroblastic cells (35). A recent report suggests that phalloidin applied in the extracellular medium stops streaming in Allium, Chara, and Nitella (28), while another report finds no inhibition ofstreaming in Vaucheria (8) .…”