Cargo loading onto kinesin powered molecular shuttles

Yolaine Jeune-Smith, Ashutosh Agarwal, Henry Hess

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport. These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available. Building on the classic inverted motility assay, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface. The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA, quantum dots or a wide variety of antigens via biotinylated antibodies.

Original languageEnglish (US)
Article numbere2006
JournalJournal of Visualized Experiments
Issue number45
DOIs
StatePublished - Nov 2010
Externally publishedYes

Fingerprint

Kinesin
Microtubules
Nanospheres
Streptavidin
Adenosinetriphosphate
Tubulin
Proteins
Adenosine Triphosphate
Casein
Microtubule Proteins
Antigens
Rhodamines
Antibodies
Semiconductor quantum dots
Quantum Dots
Machinery
Biotin
Caseins
Assays
Fluorescein

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Cargo loading onto kinesin powered molecular shuttles. / Jeune-Smith, Yolaine; Agarwal, Ashutosh; Hess, Henry.

In: Journal of Visualized Experiments, No. 45, e2006, 11.2010.

Research output: Contribution to journalArticle

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