Probing Intracellular Motor Protein Activity Using an Inducible Cargo Trafficking Assay

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utili...

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Bibliographic Details
Published in:Biophysical journal 2010-10, Vol.99 (7), p.2143-2152
Main Authors: Kapitein, Lukas C., Schlager, Max A., van der Zwan, Wouter A., Wulf, Phebe S., Keijzer, Nanda, Hoogenraad, Casper C.
Format: Article
Language:English
Subjects:
Animals
Assaying
Bioassays
Biological Assay - methods
Biological Transport
Biophysics
Cell Line
Cell Polarity
Cells
Cytoskeleton
Dynamical systems
Dynamics
Enzyme kinetics
Humans
Imaging
Intracellular Space - metabolism
Kymography
Molecular Motor Proteins - metabolism
Motors
Muscle, Motility, and Motor Proteins
Peroxisomes - metabolism
Proteins
Recruitment
Surgical implants
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Summary:Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2010.07.055