Background: Cytoplasmic dynein performs a great variety of cellular functions using a diversity of regulators. Results: NudE and dynactin compete for a common site within the dynein complex. Conclusion: This mechanism prevents dual regulation by dynactin and LIS1 and suggests a major new mode of regulatory control. Significance: This is the first insight into coordination of cytoplasmic dynein regulators.
DYNC1H1 encodes the heavy chain of cytoplasmic dynein 1, a motor protein complex implicated in retrograde axonal transport, neuronal migration, and other intracellular motility functions. Mutations in DYNC1H1 have been described in autosomal dominant Charcot-Marie-Tooth type 2 and in families with distal spinal muscular atrophy (SMA) predominantly affecting the legs (SMA-LED). Recently, defects of cytoplasmic dynein 1 were also associated with a form of mental retardation and neuronal migration disorders. Here we describe two unrelated patients presenting a combined phenotype of congenital motor neuron disease associated with focal areas of cortical malformation. In each patient we identified a novel de novo mutation in DYNC1H1: c.3581A>G (p.Gln1194Arg) in one case and c.9142G>A (p.Glu3048Lys) in the other. The mutations lie in different domains of the dynein heavy chain, and are deleterious to protein function as indicated by assays for Golgi recovery after nocodazole washout in patient fibroblasts. Our results expand the set of pathological mutations in DYNC1H1, reinforce the role of cytoplasmic dynein in disorders of neuronal migration and provide evidence for a syndrome including spinal nerve degeneration and brain developmental problems.
Dynactin is the longest known cytoplasmic dynein regulator, with roles in dynein recruitment to subcellular cargo and in stimulating processive dynein movement. The latter function was thought to involve the N-terminal microtubule binding region of the major dynactin polypeptide p150Glued, though recent results disputed this. To understand how dynactin regulates dynein we generated recombinant fragments of the N-terminal half of p150Glued. We find that the dynein-binding coiled-coil α-helical domain CC1B is sufficient to stimulate dynein processivity, which it accomplishes by increasing average dynein step size and forward step frequency, while decreasing lateral stepping and microtubule detachment. In contrast, the immediate upstream coiled-coil domain, CC1A, activates a novel diffusive dynein state. CC1A interacts physically with CC1B and interferes with its effect on dynein processivity. We also identify a role for the N-terminal portion of p150Glued in coordinating these activities. Our results reveal an unexpected form of long-range allosteric control of dynein motor function by internal p150Glued sequences, and evidence for p150Glued auto regulation.
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 ...
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