Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes.
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 the specificity and efficacy of 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 dynein motor complex directly affects some, but not all, dynein-mediated 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.
Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.
Dileep
D
Varma
Amrita
A
Dawn
Anindya
A
Ghosh-Roy
Sarah J
SJ
Weil
Kassandra M
KM
Ori-McKenney
Yanqiu
Y
Zhao
James
J
Keen
Richard B
RB
Vallee
John C
JC
Williams
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D056445:Cytoplasmic Dyneins / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism; D055503:Protein Multimerization; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism
C533388:Dynlt1 protein, rat; D008024:Ligands; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C486069:Dynll1 protein, rat; D056445:Cytoplasmic Dyneins; D004398:Dyneins; D022021:Tacrolimus Binding Proteins