Dictyostelium myosin bipolar thick filament formation: importance of charge and specific domains of the myosin rod.
Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) "head" is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines.
Hostetter
Daniel
D
Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA.
Rice
Sarah
S
Dean
Sara
S
Altman
David
D
McMahon
Peggy M
PM
Sutton
Shirley
S
Tripathy
Ashutosh
A
Spudich
James A
JA
D008841:Actin Cytoskeleton / Q000737:chemistry; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002942:Circular Dichroism; D003599:Cytoskeleton / Q000737:chemistry; D004023:Dictyostelium / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000737:chemistry*; D024462:Myosin Type II / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000737:chemistry; D014461:Ultracentrifugation
D015879:Myosin Subfragments; D049452:Green Fluorescent Proteins; D013912:Threonine; D024462:Myosin Type II; D009218:Myosins