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

Abstract

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin . To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB . Use of two fluorescent actin probes, fluorescein isothiocyanateheavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phalIacid in (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB . Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment . NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions .The highly organized cytoplasmic streaming exhibited by the giant internodal cells of characean algae has long made these cells an attractive system for the study of cell motility (4) . Accumulated evidence has suggested that motility in a variety of eukaryotic cells is actomyosin-dependent (21) . Measurements of streaming-velocity profiles in characean cells have suggested that the motive force is generated primarily at the interface between the stationary ectoplasm and motile endoplasm (17) . Early ultrastructural studies have revealed the presence of microfilament bundles attached to the chloroplasts at this interface (26) . Subsequent electron and, more recently, fluorescence microscope studies using labeling by heavy meromyosin (20, 27, 29) or antiactin antibodies (45) have demonstrated the presence of actin in these subcortical microfilament bundles and established the unidirectional orientation of the actin filaments (19) . Other light microscope studies have suggested the presence of endoplasmic filaments that appeared to branch from the subcortical actin bundles and participate in the generation of the motive force (1, 2) .The demonstration of actin in characean cells has encouraged workers to search for evidence of myosin in these cells as well . Using a perfusion technique that permitted manipulation of internal pH and ion and ATP concentrations, Williamson observed the ATP-dependent moti...

Fluorescent phallotoxins as probes for filamentous actin

“…Using rhodamine-labelled phalloidin and an image intensification system Yanagida et al (1984) showed that single actin filaments could be readily visualized, in solution, by fluorescence microscopy. The flexural rigidity of actin was apparently unchanged by phalloidin binding and this parameter could be investigated with respect to the presence or absence of regulating proteins (troponin and tro- Euteneuer Kordell et al, 1987Schatten et al, 1986aMaekawa et al, 1987;Yonemura & Mabuchi, 1987;Schatten & Schatten, 1987 Henson & Schatten, 1983u & Kinoshita, 1986Tamm & Tamm, 1987Warn & Magrath, 1983Warn et al, 1984;Gutzeit, 1986Bertin et al, 1987Strome, 1986Shimizu, 1986Priess & Hirsch, 1986Parthasarathy, 1985Heslop-Harrison et al, 1986Quader et al, 1987Parke et al, 1986Tiwari et al, 1984Pierson et al, 1986;Palevitz et al, 1987;Seagull et al, 1987;Traas et al, 1987Nothnagel et al, 1984Bassi & Donini, 1984Adams & Pringle, 1984Kilmartin & Adams, 1984;Marks & Hyams, 1985Hoch & Staples, 1983Runeberg et al, 1986;Heath, 1987Naib-Majani, 1983Ishigami, 1986Ishigami et al, 1987Uyeda & Furuya, 1986Rubino et al, 1984Roos ...…”

Analysis of the role of microtubules and actin in erythrophore intracellular motility.

“…The pigment granules continue to move normally after NEM-HMM injection, presumably providing ample opportunity for the injected actomyosin inhibitor to locate its binding site on actin. These probes for actin and actomyosin have been shown to inhibit a diverse set of motility events including cytokinesis (7,22), some forms of axoplasmic transport (11,14), and cytoplasmic streaming (25,39,43). It seems improbable to us that none of them would be successful in affecting pigment transport if it were actin-or actomyosin-based.…”

“…Because actin is involved in a wide variety of nonmuscle motile events (18,25,28,40) it represents a prime candidate for participation in pigment granule transport. Actin microfilaments have been identified in melanophores by heavy meromyosin decoration (27,37), but their role in pigment migration remains controversial.…”

F-actin in conifer roots

“…Our current understanding is based on extrapolations from studies of Characean algal cells, which have shown that microfilament bundles are composed of F-actin and play an essential role in the generation of cytoplasmic streaming (11)(12)(13)(14). This idea seems relevant to higher plants, based on studies showing that (i) microfilament bundles are present in streaming cells (5,15); (ii) the direction of streaming is parallel to the longitudinal axis of the microfilament bundles; and (iii) streaming is inhibited by cytochalasin B (5,(16)(17)(18), a drug known to affect microfilament bundles (19), by the ionophore A23187 (20), and by the chelating agent EGTA (5), chemicals known to affect the distribution of ions important to the generation of streaming in F-actin-containing systems (12).…”