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.
Mahajan
R K
RK
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021.
Vaughan
K T
KT
Johns
J A
JA
Pardee
J D
JD
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