Regulation of cytoplasmic dynein ATPase by Lis1

Abstract

Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport.

Figure 1.

Proteins used in the assays. A, A 45 kDa Lis1 band is observed in the purified recombinant Lis1 preparation by silver staining (lane 1), by Western blotting with a Lis1 antibody (lane 2), or by Coomassie staining (L in F). B, Western blots of 1 μg of recombinant Lis1 probed for the dynactin subunit p150glued, the dynein intermediate chain, α-tubulin, and Ndel1; these proteins are undetectable in the Lis1 preparation. Adult mouse brain extract was used as a positive control (br). C, Histidine-tagged Lis1 (H-Lis1) immobilized on Ni-NTA resin binds specifically to a known interacting protein, the α2 subunit of PAFAH1b (α2), indicating that the recombinant Lis1 produced in insect cells is correctly folded. D, Multiple bands that correlate by size to known dynein subunits are visualized in our dynein preparation by Coomassie staining (lane 1). HC, Heavy chain; IC, intermediate chain; LIC, light intermediate chain. HC and IC are labeled with antibodies specific to those subunits (lanes 2 and 3, respectively). E, Dynein (dyn; 1 μg) did not contain detectable levels of α-tubulin (top), nor did it contain Lis1, p150glued (p150), or the Lis1 binding protein Ndel1 (bottom) as detected by Western blotting. F, A fragment of Lis1 encompassing the WD domain (WD) was visualized by Coomassie staining along with the 4L longest splice variant of tau (t). Protein molecular weights used throughout were 1.2 MDa for dynein, 90 kDa for Lis1 homodimers, 40 kDa for WDLis1, and 65 kDa for monomeric tau. G, Microtubules were not affected by the presence of Lis1. Microtubule bundling was apparent in both mixtures, because individual structures varied in width. The thinnest structures, probably representing individual microtubules, aligned frequently to form thicker structures, probably bundles of two to six microtubules.

Figure 2.

Recombinant Lis1 specifically and directly increases the enzymatic activity of bovine brain dynein. A, Equimolar Lis1 and dynein were used in an in vitro ATPase assay. ATPase hydrolysis was analyzed using TLC to separate radiolabeled ADP from ATP. Spots on TLC plates were excised, and total counts per spot were determined by scintillation counting. Data are the mean (±SE) of three separate experiments. Con, Control; M, microtubules; D, dynein; L, Lis1; DL, dynein plus Lis1; DM, dynein plus microtubules; DML, dynein plus microtubules plus Lis1. The mean of DML is significantly different from the mean of DM (*p < 0.001 as determined by ANOVA statistical analysis). B, Dose–response data for Lis1 indicate our assays are in or near the linear range for Lis1 activity. The data represent the mean (±SE) of four data sets. C, Microtubule copelleting assay: Western blots of soluble (S) and pelleted (P) fractions, ± MTs were probed for Lis1 or DIC. The amount of DIC in the pellet relative to the soluble fraction is not increased by the presence of Lis1 (two lanes on the right). D, Additional control ATPase assays were performed with the WD repeat domain of Lis1 (WD) and with tau, an axon-enriched MAP. All reactions contained dynein and microtubules. The data are presented as the percentage of Lis1 stimulation and are the mean ± SE of three separate experiments. Neither WD nor tau stimulated dynein to the same extent as full-length Lis1 (L). In fact, tau reduced the microtubule-stimulated dynein activity. WDLis1 blocked stimulation of dynein by full-length Lis1 (L+WD).

Figure 3.

Lis1 binds to a subset of cytoplasmic dynein motors in vitro. A, Fifteen picomoles of Lis1, 5 pmol of dynein, or a mixture of the two were loaded onto separate 5–25% sucrose gradients, as indicated (left). Fractions were probed with Lis1, DIC, or DHC antibodies as indicated (right). In the combined sample, ∼1.7 pmol of Lis1 had shifted to higher fractions, where it cofractionated with DIC and DHC. No Lis1 was found in denser fractions in the absence of dynein, even when the blot was exposed overnight (long exp.). B, One picomole of dynein coimmunoprecipitated ∼0.37 pmol of Lis1. L, Lis1; D, dynein; DL, Lis1 and dynein; No Ab, no antibody control; 5% input, 5% of DL. C, No difference was observed when 74.1 IPs were performed in the presence of excess ATP or ADP. D, The amount of DHC and DIC coprecipitating with Lis1 (L) when dynein (D) is present at a twofold molar excess is visualized by Coomassie staining. IgG is an antibody band. E, Left, Purified Lis1 and dynein (dyn) probed with Lis1 and dynein antibodies. Right, 2.5 pmol of Lis1 was incubated with 2, 10, and 20 pmol of purified dynein and then precipitated with Lis1 antibodies (Lis1 IP). Western blots were probed for Lis1 and DIC. The ratio of Lis1 to dynein in each IP was 0.37, 1.2, and 1.3, indicating that dynein binding had saturated when 10 pmol was present. All experiments were repeated with similar results. Representative blots or gels are shown.

Figure 4.

Lis1 associates with a subset of dynein complexes in tissue extracts. A, Dynein was immunoprecipitated from 10 mg of adult rat extract prepared from the indicated tissues using the 74.1 DIC antibody. Br, Brain; Li, liver; T, testes. Precipitated proteins were visualized by Western blotting using Lis1 and DIC antibodies. DYN, Purified dynein; Lis1, recombinant Lis1. B, The data in A were quantified by densitometry, comparing precipitated bands to known amounts of each protein. The molar amounts were calculated using the Lis1 dimer molecular weight of 90 kDa and dynein molecular weight of 1.2 MDa. C, The amount of Lis1 and dynein in soluble extracts and insoluble pellets was compared for each tissue (see Materials and Methods). All experiments were repeated with similar results. Representative blots or gels are shown.

Figure 5.

Partial colocalization of Lis1 and dynein. A, Punctate Lis1 immunoreactivity (red) decorates microtubules (green). Bottom, An enlargement of the region indicated by the arrow. B, Left, This pattern was also seen with Lis1 (red) and dynein (74.1-green), but the overlap was not extensive. The bottom left panel shows an enlargement of the area in the white square. Right, To more easily quantify overlap (yellow), deconvolved volumes were inverted and the hues adjusted so that overlapping puncta were colorized blue (arrows). The bottom middle panel shows an enlargement of the area in the black square. A bar graph shows the percentage of dynein puncta in control (CON) and nocodazole-treated (NOC.) cells that were also positive for Lis1 (12 cells ±SD). C, Lis1 immunoreactivity (red) and actin staining (green) in E17 hippocampal neurons. Top, A stage 2 neuron with undifferentiated neurites. Bottom, A stage 3 neuron with a newly differentiated axonal growth cone enriched in Lis1 (arrow). D, Top, Dynein (red) and Lis1 (green) immunofluorescence in a stage 4 neuron. Bottom panels show enlargements of the growth cone (inset) and the axonal region indicated by the arrow. Two distinct regions in the growth cone are further enlarged (1 and 2). Scale bars: A, B, D, 10 μm; C, 5 μm.