Obstacles on the microtubule reduce the processivity of Kinesin-1 in a minimal in vitro system and in cell extract.
Inside cells, a multitude of molecular motors and other microtubule-associated proteins are expected to compete for binding to a limited number of binding sites available on microtubules. Little is known about how competition for binding sites affects the processivity of molecular motors and, therefore, cargo transport, organelle positioning, and microtubule organization, processes that all depend on the activity of more or less processive motors. Very few studies have been performed in the past to address this question directly. Most studies reported only minor effects of crowding on the velocity of motors. However, a controversy appears to exist regarding the effect of crowding on motor processivity. Here, we use single-molecule imaging of mGFP-labeled minimal dimeric kinesin-1 constructs in vitro to study the effects of competition on kinesin's processivity. For competitors, we use kinesin rigor mutants as static roadblocks, minimal wild-type kinesins as motile obstacles, and a cell extract as a complex mixture of microtubule-associated proteins. We find that mGFP-labeled kinesin-1 detaches prematurely from microtubules when it encounters obstacles, leading to a strong reduction of its processivity, a behavior that is largely independent of the type of obstacle used here. Kinesin has a low probability to wait briefly when encountering roadblocks. Our data suggest, furthermore, that kinesin can occasionally pass obstacles on the protofilament track.
Telley
Ivo A
IA
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Bieling
Peter
P
Surrey
Thomas
T
D000818:Animals; D002457:Cell Extracts; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D049452:Green Fluorescent Proteins; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D009038:Motion; D009154:Mutation
D002457:Cell Extracts; D029721:Drosophila Proteins; D049452:Green Fluorescent Proteins; C515209:Khc protein, Drosophila; D016547:Kinesins