Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes

Dileep Varma, Amrita Dawn, Anindya Ghosh-Roy, Sarah J. Weil, Kassandra M. Ori-McKenney, Yanqiu Zhao, James Keen, Richard B. Vallee, John C. Williams

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate thespecificityandefficacyof these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dyneinmotor complex directly affects some, but not all, dyneinmediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.

Original languageEnglish (US)
Pages (from-to)3493-3498
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number8
DOIs
StatePublished - Feb 23 2010

Keywords

  • In vivo antagonist
  • Mitotic index
  • Organelle kinetics
  • Retrograde transport

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes'. Together they form a unique fingerprint.

Cite this