Actin filaments mediate Dictyostelium myosin assembly in vitro.
Because myosin thick filaments form in the actin-rich cortex of nonmuscle cells, we have examined the role of Dictyostelium actin filaments in the assembly of Dictyostelium myosin (type II). Fluorescence energy transfer and light-scattering assembly assays indicate that self-association of Dictyostelium myosin into bipolar thick filaments is kinetically regulated by actin filament networks. Regulation is nucleotide dependent but does not require ATP hydrolysis. Myosin assembly is accelerated approximately 5-fold by actin filaments when either 1 mM ATP or 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-P[NH]P) is present. However, actin filaments together with 1 mM ADP abolish myosin assembly. Accelerated assembly appears to require transient binding of myosin molecules to actin filaments before incorporation into thick filaments. Fluorescence energy-transfer assays demonstrate that myosin associates with actin filaments at a rate that is equivalent to the accelerated myosin assembly rate, evidence that myosin to actin binding is a rate-limiting step in accelerated thick filament formation. Actin filament networks are also implicated in regulation of thick filament formation, since fragmentation of F-actin networks by severin causes immediate cessation of accelerated myosin assembly. Electron microscopic studies support a model of actin filament-mediated myosin assembly. In ADP, myosin monomers rapidly decorate F-actin, preventing extensive formation of thick filaments. In AMP-P[NH]P, myosin assembles along actin filaments, forming structures that resemble primitive stress fibers. Taken together, these data suggest a model in which site-directed assembly of thick filaments in Dictyostelium is mediated by the interaction of myosin monomers with cortical actin filament networks.
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021.
R K
RK
Mahajan
K T
KT
Vaughan
J A
JA
Johns
J D
JD
Pardee
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000227:Adenine Nucleotides / Q000494:pharmacology; D004023:Dictyostelium / Q000378:metabolism*; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism; D009218:Myosins / Q000378:metabolism* / Q000648:ultrastructure; D015800:Protozoan Proteins; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D000199:Actins; D000227:Adenine Nucleotides; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D015800:Protozoan Proteins; C036571:severin protein, Dictyostelium; D009218:Myosins