A novel split kinesin assay identifies motor proteins that interact with distinct vesicle populations.
Identifying the kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess kinesin-vesicle interactions and use it to identify kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of "split kinesins," comprising an axon-selective kinesin motor domain and a series of kinesin tail domains that can attach to their native vesicles; when the split kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon. This method provided highly specific results, showing that three Kinesin-3 family members-KIF1A, KIF13A, and KIF13B-interacted with dendritic vesicle populations. This experimental paradigm allows a systematic approach to evaluate motor-vesicle interactions in living cells.
The Jungers Center for Neurosciences Research, Oregon Health and Science University, Portland, OR 97239, USA.
Brian
B
Jenkins
Helena
H
Decker
Marvin
M
Bentley
Julie
J
Luisi
Gary
G
Banker
D000818:Animals; D002478:Cells, Cultured; D003584:Cytological Techniques / Q000379:methods*; D022162:Cytoplasmic Vesicles / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism* / Q000648:ultrastructure; D011247:Pregnancy; D021381:Protein Transport / Q000235:genetics / Q000502:physiology*; D051381:Rats
C490575:Kif1a protein, rat; D020409:Molecular Motor Proteins; C504977:KIF13B protein, rat; D016547:Kinesins