Experimental evidence for the contractile activities of Acanthamoeba myosins IA and IB.

Abstract. Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin-IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.

Section: Relevance Of Myosin-i Kinetics To In Vivo Functionmentioning

confidence: 99%

Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.

Ostap

Pollard

1996

“…Furthermore, immunofluorescence microscopy reveals that myosin I is concentrated in the lamellopodial and pseudopodial projections at the front of locomoting Dictyostelium ameba (Fukui et al, 1989). This finding, together with the fact that actomyosin I is known to be capable of producing movement in vitro (Albanesi et al, 1985;Fujisaki et al, 1985;Adams and Pollard, 1986), suggests that actomyosin I contributes to the forces that cause extension at the leading edge of motile cells. If so, then myosin I almost certainly plays a key role (perhaps an essential role) in the extension of pseudopods and lamellopods, cell locomotion, and chemo~ taxis.…”

mentioning

confidence: 92%

Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform.

Jung

Hammer

1990

131

118

Abstract. Motile activities such as chemotaxis and phagocytosis, which occur in Dictyostelium cells lacking myosin II, may be dependent upon myosin I. To begin to explore this possibility, we have engineered a disruption of the Dictyostelium myosin I heavy chain (DMIHC) gene described recently (Jung, G., C. L. Saxe III, A. R. Kimmel, and J. A. Hammer III. 1989. Proc. Natl. Acad. Sci. . The double-crossover, gene disruption event that occurred resulted in replacement of the middle approximate one-third of the gene with the neomycin resistance marker. The resulting cells are devoid of both the 3.6-kb DMIHC gene transcript and the 124-kD DMIHC polypeptide. DMIHC-cells are capable of chemotactic streaming and aggregation, but these processes are delayed. Furthermore, the rate of phagocytosis by DMIHC-cells is reduced, as assessed by growth rate on lawns of heat-killed bacteria and on the initial rate of uptake of FITC-labeled bacteria. Therefore, this Dictyostelium myosin I isoform appears to play a role in supporting chemotaxis and phagocytosis, but it is clearly not required for these processes to occur. Using a portion of the DMIHC gene as a probe, we have cloned three additional Dictyostelium small myosin heavy chain genes. Comparison of these four genes with three genes described recently by Titus et al. (Titus, M. A., H. M. Warrick, and J. A. Spudich. 1989. Cell Reg. 1:55-63) indicates that there are at least five small myosin heavy chain genes in Dictyostelium. The probability that there is considerable overlap of function between these small myosin isoforms indicates that multiple gene disruptions within a single cell may be necessary to generate a more striking myosin Iphenotype.

“…However, the heavy chains of myosins IA and IB contain a second actin-binding site on the nonhelical, COOH-terminal domain (29). The presence of two actinbinding sites in the heavy chain allows myosins IA and IB to cross-link actin filaments (5,16) and, when phosphorylated, support superprecipitation in vitro (16). Phosphorylated myosins I can also support the translocation of latex beads along Nitella actin cables (6).…”

mentioning

confidence: 99%

Plasma membrane association of Acanthamoeba myosin I.

Miyata

Bowers

Korn

1989

Self Cite

117

Abstract. Myosin I accounted for "~2 96 of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F-actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI-extracted plasma membranes in a saturable manner with maximum binding four-to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40/~M in 0.1 M KCI plus 2 mM MgATP). Thus, neither the MgATP-sensitive actinbinding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP-sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actinbinding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block stericaUy its interaction with actin.ANTHAMOEBA castellanii contains approximately equal concentrations of two classes of myosins: myosin II (31) and myosin I (33, 36, for review see 26). Myosin II is a conventional myosin comprising two heavy chains and two pairs of light chains and forming typical bipolar filaments with actin-activated Mg2÷-ATPase activity (when the three phosphorylation sites at the tip of the tail of each heavy chain are unphosphorylated). Acanthamoeba myosins IA and IB are the first, and best characterized, examples of a group of monomolecular, nonfilamentous myosins that is known to include a third isozyme from Acanthamoeba, a myosin I from Dictyostelium discoideum (15), and the 110-kD protein-calmodulin complex from intestinal brush border (10,(12)(13)(14).The comparatively short heavy chains of myosins IA and IB (140 and 127 kD, respectively [33]) each comprise a single globular head (with striking similarities of amino acid sequence [23] and functional sites [4,9,29,30] to the corresponding domain of myosins II) and a short, nonhelical COOH-terminal domain (that is rich in glycine, proline, and alanine [23]). Phosphorylation of a single amino acid (21) situated between the...