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

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 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, 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.in vivo antagonist | retrograde transport | organelle kinetics | mitotic index C ytoplasmic dynein is a microtubule-based motor protein involved in numerous essential cellular functions, including intracellular transport of membranous organelles and macromolecular complexes, mitosis, and cell migration (1). Cytoplasmic dynein consists of multiple subunits: two 532-kDa heavy chains (HCs), each containing a motor domain, and a number of accessory subunits, the intermediate, light intermediate, and light chains (ICs, LICs, and LCs, respectively). The ICs and LCs form a complex at the base of the dynein HC (residues 1-1,100) (2). The ICs have been implicated in subcellular targeting of cytoplasmic dynein through an interaction with a large accessory complex, dynactin (3-6). The LCs, in turn, form a complex with the dynein IC, but also interact with a large number of other proteins, including transcription factors, signaling molecules, RNA, and viral proteins. As such, the dynein LCs have been implicated as adaptors to link these diverse molecules to dynein for retrograde transport (1).Our recent structural and biophysical studies aimed at understanding role of the dynein LCs, however, do not support the direct involvement of these subunits in cargo recognition and transport (7). Specifically, the structure of the LCs [LC8 (Dynll1) and TcTex1 (Dynlt1)] bound to the dynein IC shows that the dynein IC occupies ...

A dynein independent role of Tctex-1 at the kinetochore

“…35 On one hand, these results support that Tctex-1 is not functionally important for dynein in mitotic progression. On the other hand, these results, along with other observations, indicate that kinetochore-associated Tctex-1 proteins may be not in rapid exchange with the free cytosolic pool, as is observed with dynein for endosomes and lysosomes.…”

Photocleavable Dimerizer for the Rapid Reversal of Molecular Trap Antagonists

“…We used native PAGE to follow the formation of the LC8 and FKBP-LC8 molecular trap (LC8 TRAP ) complex induced by the addition of PhAP (6). Upon the addition of PhAP to an equimolar mixture of LC8 and LC8 TRAP , we observed a new band of PhAP-LC8-LC8 TRAP that migrated the same distance as the band produced by the addition of AP (AP-LC8-LC8 TRAP ) ( Fig.…”

“…We demonstrated that the expression and subsequent dimerization of a dynein light chain LC8 or TcTex1 molecular trap immediately affects dynein-associated processes (e.g. endosome, lysosome, and Golgi dispersion) (6). The ability to reverse this perturbation would provide additional, powerful insight to molecular processes that is not available with current technologies (e.g.…”

“…Immunostaining and Microscopy-For immunostaining, transiently transfected COS1 cells were fixed with 3.7% formaldehyde at room temperature for 10 min, and subsequent immunostaining was performed as described previously (6). Briefly, COS 1 cells grown on 25-mm coverslips were washed three times with PBS, treated with 3.7% formaldehyde (Sigma) in PBS for fixation, and permeabilized in 0.5% Triton X-100 (Sigma) in PBS at room temperature for 10 min.…”

“…For EGFP-tagged FKBP-LC8 TRAP , the construct was cloned as a C-terminal fusion of enhanced green fluorescence protein (EGFP) mammalian expression vector (pEGFPC1; Clontech) as described previously (6).…”