title: Identification of Kinesin-1 Cargos Using Fluorescence Microscopy.
abstract: Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai; clement.lee@mssm.edu.
authors.forename: Clement M
authors.initials: CM
authors.lastname: Lee
mesh_terms: D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000302:isolation & purification*; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism
chemical_list: C488101:KIF5B protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D016547:Kinesins