Chemical-genetic inhibition of sensitized mutant unconventional myosins.
The construction of a sensitized mutant unconventional myosin is an excellent method for determining the function of the individual myosin against a background of related myosins with partially overlapping functions. In this chapter, we outline the steps involved in sensitizing myosin by mutation and screening them against panels of nucleotide analogs, including transfection, microinjection, and actin co-sedimentation in vitro. We also describe conditions and considerations involved in designing functional experiments after a mutant and cognate analog have been identified. The powerful strategy of chemical genetics, when correctly applied to unconventional myosins, enables both the specific and selectable inhibition of the target motor with outstanding internal controls.
Karcher
Ryan L
RL
McLaughlin Research Institute, Great Falls, MT, USA.
Provance
D William
DW
Gillespie
Peter G
PG
Mercer
John A
JA
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D024461:Myosin Type I / Q000737:chemistry*; D024701:Myosin Type V / Q000737:chemistry*; D009218:Myosins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D011817:Rabbits
D000255:Adenosine Triphosphate; D024461:Myosin Type I; D024701:Myosin Type V; C460367:MYO1C protein, human; D009218:Myosins