Tail chimeras ofDictyosteliummyosin II support cytokinesis and other myosin II activities but not full development

Abstract

IntroductionClass II myosins consist of two identical heavy chains and two pairs of light chains (Sellers, 1999). Because the cellular slime mold Dictyostelium discoideum contains only a single myosin II heavy chain gene (De Lozanne et al., 1985;Warrick et al., 1986), Dictyostelium myosin II heavy chain-null cells provide an excellent system for determining the biological roles of myosin II and the abilities of mutant myosin II heavy chains to substitute for wild-type heavy chain. In this way, it has been shown that Dictyostelium myosin II (DdMII) is required for cytokinesis and growth of vegetative amoeboid cells in suspension culture, for development and differentiation beyond the mound stage, the first developmental stage, (Knecht and Loomis, 1987;De Lozanne and Spudich, 1987;Manstein et al., 1989) and for capping of concanavalin A (ConA) receptors [(Pasternak et al., 1989b;Fukui et al., 1990) but see AguadoVelasco and Bretscher, for a contrary view (Aguado-Velasco and Bretscher, 1997)]. DdMII heavy chain-null cells are motile and capable of cAMP-induced chemotaxis but both motility and chemotaxis in a spatial gradient of cAMP are impaired compared to wild-type cells (De Lozanne and Spudich, 1987;Wessels et al., 1988;Peters et al., 1988). Null cells can phagocytose bacteria but at the time the experiments described in this paper were carried out phagocytosis by null cells had not been quantified.Cytokinesis in suspension culture, capping of ConA receptors and full development of Dictyostelium to fruiting bodies depend on both the actin-dependent MgATPase activity of DdMII and its ability to form filaments. For example, myosins with mutations in the motor domain of the heavy chain that eliminate ATPase activity but have no effect on polymerization competence (Yumura and Uyeda, 1997a) and polymerization-competent myosin rods devoid of the motor domain (Zang and Spudich, 1998) localize properly to the cleavage furrow but neither protein supports cytokinesis. On the other hand, myosin with mutations in the tail domain that prevent polymerization but have no effect on catalytic activity (Egelhoff et al., 1993) and catalytically active but polymerization incompetent heavy meromyosin (De Lozanne and Spudich, 1987;Fukui et al., 1990) Recently, we showed that the actin-activated MgATPase activity of myosin chimeras in which the tail domain of Dictyostelium myosin II heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle myosin II is unregulated and about 20 times higher than wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium myosin and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different...

Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

“…Cells were washed and resuspended in starvation buffer, 20 mM morpholinoethanesulfonic acid, pH 6.8, allowed to attach to a coverslip and then incubated for 15 min with 50 l of tetramethyl rhodamine isothiocyanate-conjugated Con A (Molecular Probes), 30 g͞ml (8). For myosin II redistribution after capping, cells were incubated with Con A for 3 min to allow capping, incubated with or without blebbistatin for 25 min, fixed and permeabilized in acetone͞1% formalin at Ϫ20°C for 15 min, and mounted directly to visualize the Con A caps or reacted with anti-Dictyostelium myosin II antibody (8) for colocalization of myosin II by indirect immunofluorescence.…”

“…For cell streaming, cells were harvested, resuspended at 5 ϫ 10 6 ͞ml, and 1.5 ϫ 10 7 cells were plated on 60-mm Petri dishes and allowed to adhere for 30 min (8). The medium was removed by aspiration and 2 ml of starvation buffer, containing 100 M blebbistatin or an equal volume of DMSO, was carefully applied and the cells placed in the dark for 6-8 h. Development was assessed 48 h after spotting small aliquots of amoebae on black filters (Millipore HABP4700) saturated with starvation buffer with and without 100 M blebbistatin.…”

“…A suspension of heat-killed Klebsiella aerogenes containing fewer than 100 Dictyostelium cells was seeded onto black filters saturated with HL5 medium in 60-mm Petri dishes (8). Either blebbistatin, 150 M, or an equivalent volume of DMSO was added to both the mixture and the HL5 mediumsaturated pads.…”

“…For phagocytosis (8), cells were collected, washed in Sorenson's buffer (16 mM KH 2 PO 4 , 2 mM Na 2 HPO 4 , pH 6.1), diluted to 5 ϫ 10 6 cells per ml and incubated for 1 h at room temperature with 150 M blebbistatin or an equivalent volume of DMSO on a rotary shaker at 100 rpm. Washed Fluoresbrite yellow orange carboxylate-conjugated polystyrene-latex beads (1 m in diameter; Polysciences) were added at a ratio of 300 beads per cell.…”

“…After confirming that blebbistatin inhibits the basal and actin-activated ATPase activities of purified Dictyostelium myosin II, we evaluated the effects of blebbistatin on three cellular activities known to require myosin II [growth in suspension culture, capping of Con A receptors, and development to fruiting bodies (3-7)]; on five processes that do not require myosin II (8) [growth on plates, cell streaming, macropinocytosis, which involves class-I myosins (9, 10), contractile vacuole function, which involves class-V myoJ (11), and phagocytosis in suspension, which is myosin VII-dependent (12, 13)]; and on the organization of the actomyosin cytoskeleton. In all of the experiments, we compared the effects of blebbistatin on myosin II heavy chainnull (HS1) cells, HS1 cells expressing wild-type myosin II heavy chain (WT), and HS1 cells expressing an enzymatically inactive myosin II heavy chain mutant (E476K) (14).…”