Actin filaments mediate Dictyostelium myosin assembly in vitro

• Published in: Proceedings of the National Academy of Sciences - PNAS 1989-08, Vol.86 (16), p.6161-6165
• Main Authors: Mahajan, R.K. (Cornell University Medical College, New York, NY), Vaughan, K.T, Johns, J.A, Pardee, J.D
• Format: Article
• Language: English
• Subjects: Actins; Actins - metabolism; Actins - ultrastructure; Adenine Nucleotides - pharmacology; Analytical, structural and metabolic biochemistry; ANATOMIA DE LA PLANTA; ANATOMIE VEGETALE; Biological and medical sciences; Cell biology; CHAMPIGNON; Contractile proteins; Dictyostelium; Dictyostelium - metabolism; ENERGY METABOLISM; EXPERIMENTACION IN VITRO; EXPERIMENTATION IN VITRO; Fluorescence; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Fungal Proteins - metabolism; FUNGI; GLOBULINAS; GLOBULINE; GLOBULINS; Holoproteins; IN VITRO EXPERIMENTATION; Kinetics; Light; Light scattering; Macromolecular Substances; METABOLISME ENERGETIQUE; METABOLISMO ENERGETICO; Microfilament Proteins - metabolism; Microfilaments; MICROSCOPIA; MICROSCOPIE; MICROSCOPY; MOHO; MOISISSURE; Molecules; Monomers; MOULDS; MUSCLE; MUSCLES; MUSCULOS; myosin; Myosins - metabolism; Myosins - ultrastructure; Nucleotides; Physiological regulation; PLANT ANATOMY; PROTEINAS; PROTEINE; PROTEINS; Protozoan Proteins; Scattering, Radiation; Spectrometry, Fluorescence; ULTRAESTRUCTURA; ULTRASTRUCTURE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.16.6161

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 ≈ 5-fold by actin filaments when either 1 mM ATP or 1 mM adenosine 5′-[β ,γ -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.