Functional dissection of LIS1 and NDEL1 towards understanding the molecular mechanisms of cytoplasmic dynein regulation

Abstract

LIS1 and NDEL1 are known to be essential for the activity of cytoplasmic dynein in living cells. We previously reported that LIS1 and NDEL1 directly regulated the motility of cytoplasmic dynein in an in vitro motility assay. LIS1 suppressed dynein motility and inhibited the translocation of microtubules (MTs), while NDEL1 dissociated dynein from MTs and restored dynein motility following suppression by LIS1. However, the molecular mechanisms and detailed interactions of dynein, LIS1, and NDEL1 remain unknown. In this study, we dissected the regulatory effects of LIS1 and NDEL1 on dynein motility using full-length or truncated recombinant fragments of LIS1 or NDEL1. The C-terminal fragment of NDEL1 dissociated dynein from MTs, whereas its N-terminal fragment restored dynein motility following suppression by LIS1, demonstrating that the two functions of NDEL1 localize to different parts of the NDEL1 molecule, and that restoration from LIS1 suppression is caused by the binding of NDEL1 to LIS1, rather than to dynein. The truncated monomeric form of LIS1 had little effect on dynein motility, but an artificial dimer of truncated LIS1 suppressed dynein motility, which was restored by the N-terminal fragment of NDEL1. This suggests that LIS1 dimerization is essential for its regulatory function. These results shed light on the molecular interactions between dynein, LIS1, and NDEL1, and the mechanisms of cytoplasmic dynein regulation.

FIGURE 1.

Effects of NDEL1 fragments on dynein function. A, schematic diagram of NDEL1 and its fragments. B, NDEL1, NN, and NC electrophoresed on a 10% polyacrylamide gel. C, MT gliding velocities of dynein in the absence (Dynein, n = 58) or presence of NDEL1 fragments (NN, n = 55). * denotes few MTs binding to the dynein-coated surface. The error bars indicate standard deviations. D, dark-field images of bound MTs in the absence (Dynein) and presence of the NC fragment (Dynein + NC). Bars, 5 μm.

FIGURE 2.

Effects of NDEL1 fragments and full-length NDEL1 on dynein-MT interactions. A, SDS-PAGE gels showing the binding of cytoplasmic dynein to MTs in the presence/absence of NC with different MT concentrations. S, supernatant; P, pellet. B, binding curves obtained from MT co-pelleting assays repeated three times. In the control condition, the dissociation constant, K_d, was 2.5 ± 0.5 μm. In the presence of the C-terminal fragment of NDEL1, the K_d was 3.4 ± 0.7 μm. C and D, amount of dynein bound to MTs in the presence (C) or absence (D) of ATP. The error bars indicate standard deviations. The p value was calculated using Student's t test (*, p < 0.005).

FIGURE 3.

Activities of NDEL1 fragments in restoring dynein motility following LIS1 suppression. A, MT gliding velocities of dynein in the presence of LIS1 and NDEL1 fragments. B, modified in vitro gliding assays. Dynein was introduced into the chamber first, followed sequentially by the other proteins. Buffer A with 1 mm ATP and 40 μm taxol was added between each protein introduction to remove excess, unbound proteins. Binding means that MTs were bound to the dynein-coated surface and did not move.

FIGURE 4.

Dimerization of LIS1 is necessary for its function. A, schematic diagram of GST-LIS1, GST-ΔN-LIS1, and ΔN-LIS1, and SDS-PAGE gel image. B, MT gliding velocities of dynein in the presence of 1 mm ATP. The error bars indicate standard deviations. The numbers of MTs measured were 55 (Dynein), 68 (Dynein + ΔN-LIS1), 66 (Dynein + GST-ΔN-LIS1), and 61 (Dynein + GST-ΔN-LIS1 + NN). The p value was calculated using Student's t test (*, p < 0.001).

FIGURE 5.

Schematic diagram of the proposed regulation of dynein motility. A, regulation by full-length LIS1 and full-length NDEL1. B, regulation by truncated LIS1 and NDEL1 fragments. The sizes of dynein, truncated LIS1 fragments, and NDEL1 fragments are not to scale.