Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein - PubMed (original) (raw)
Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein
Morgan E DeSantis et al. Cell. 2017.
Abstract
Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here-a combination of biochemistry, single-molecule assays, and cryoelectron microscopy-led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein's AAA3 domain. The low-affinity state requires Lis1 to also bind to dynein at a novel conserved site, mutation of which disrupts Lis1's function in vivo. We propose a new model for the regulation of dynein by Lis1.
Keywords: AAA+; Lis1; cryo-EM; cryoelectron microscopy; dynein; lissencephaly; microtubule; molecular motor; single-molecule; transport.
Copyright © 2017 Elsevier Inc. All rights reserved.
Figures
Figure 1. Lis1 has two modes of regulating dynein
(A) Schematic of the dynein construct used in this study. The semi-transparent ovals represent the nucleotide-binding sites in AAA1-4. The second protomer is faded out for clarity. The β-propellers in the Lis1 dimer (orange) are represented as rings. (B) AAA3 variants used in this study: Dynwt (wild type AAA3), DynWA (Walker A mutation in AAA3), and DynWB (Walker B mutation in AAA3). (C) Average velocities of dynein variants (n>103 events per data point). See Figure S1B for representative kymographs. (D) Normalized average velocities of dynein variants in the absence (solid bars) and presence (hatched bars) of 300 nM Lis1 (n>103 events per data point). Velocities were normalized by setting those in the absence of Lis1 [from (C)] to 100%. (E) Binding densities of dynein variants in the presence of ATP (top) or ATP-Vi (bottom) (n>8 fields of view per data point). See also Table S1. (F) Normalized binding densities of dynein variants alone (solid bars), or in the presence of 300 nM Lis1 (hatched bars), or 300 nM Lis15A (cross-hatched bars) in the presence of ATP. Binding densities were normalized by setting those in the absence of Lis1 [from (E), top] to 100% (n=12 fields of view per data point). (G–I) Normalized binding densities of dynein variants alone (solid bars), or in the presence of 300 nM Lis1 (hatched bars) in the presence of 1 mM ATP-Vi (G), 1 mM ADP (H) and 2.5 units/mL apyrase (“Apo”) (I) (n=8 fields of view per data point). Normalized binding densities in the absence of Lis1 shown in (G) are those in (E, bottom) without normalization. Statistical significance was calculated using an unpaired t-test with Welch’s correction for both velocity (C and D) and binding density (E–I). P-values: ns, not significant; *, <0.05; **, <0.01; ***, <0.001, ****, <0.0001. Data are shown as mean and standard error of mean.
Figure 2. Structural basis for the tight microtubule binding state of dynein induced by Lis1
(A) Additional structural features of the schematic shown in Figure 1A. The inset on the right shows the architecture of the AAA ring, with each AAA domain composed of both large (AAAL) and small (AAAS) subdomains (exemplified for AAA3). Large and small subdomains are arranged in two separate planes. Semi-transparent ovals represent nucleotide-binding sites. The linker domain forms a third layer, above the AAA ring. Insets on the left illustrate how the buttress couples the conformation of dynein’s ring to dynein’s affinity for MTs by changing the register between the two helices (CC1 and CC2) in the stalk’s coiled coil. (B) Cryo-EM structure of the Dynwt-M:Lis1 complex, solved in the presence of ATP. The cryo-EM map was filtered using local resolution and is shown as a semi-transparent surface, with the atomic model shown as a ribbon diagram. The cartoon (bottom left) indicates the portion of dynein observed in our cryo-EM map. (C) The structure viewed from the stalk, with the Dynwt-M-Lis1 interface indicated by the dashed rectangle. (D) Close-up view of the Dynwt-M-Lis1 interface, seen in a direction perpendicular to the dashed rectangle. Side chains shown on AAA4 are residues that prevent Lis1 binding when mutated (KDEE) (Huang et al., 2012). AAA domains that contribute motifs to the interface are labeled. (E) Ring architecture of human Dynein-2wt-M (ATP-Vi) (Schmidt et al., 2014) (PDB: 4RH7), yeast Dynwt-M (ATP) (Bhabha et al., 2014) (EMDB 6054), and our map of Dynwt-M:Lis1 (ATP). Colored dots and rod highlight equivalent positions in AAA4 (yellow), AAA5 (orange) and AAA6 (red). Colored arrows indicate the N-terminus of the linker (purple), the boundary between AAA5 and AAA6L (orange/red), and the boundary between AAA6L and AAA6S (red/dark red). The structure of human dynein-2 was converted into an EM-like density and both it and our Dynwt-M:Lis1 map were filtered to 20Å. The structure of Dynwt-M (ATP) (EMDB: 6054) has a resolution of 17Å. (F–I) Ring conformations of the Dynwt-M:Lis1 structure (F); the low-affinity, wild-type human Dynein-2wt-M solved in the presence of ATP-Vi (PDB: 4RH7) (G); and the high-affinity, wild-type S. cerevisiae dynein solved in the absence of nucleotide (PDB: 4AKI) (H). We removed the linker and, when present, Lis1 for clarity. (I) Maps of pairwise alpha carbon interatomic distances between the Dynwt-M:Lis1 structure and the low-affinity, wild-type human Dynein-2wt-M (left), and high-affinity, wild-type S. cerevisiae Dynwt-M (right). Structures were aligned using their AAA4L domains. The length and thickness of the vectors are proportional to the calculated interatomic distances. See also Figure S2.
Figure 3. Two Lis1 β-propellers are bound to dynein in the weak microtubule binding state
(A) Cryo-EM structure of the DynWB-M:Lis1 complex, solved in the presence of ATP-Vi. The cryo-EM map is shown as a semi-transparent surface, with the atomic model generated with Rosetta shown as a ribbon diagram. The cartoon (bottom left) indicates the portion of dynein observed in our cryo-EM map. (B) Structure viewed from the stalk, with the two DynWB-M-Lis1 interfaces indicated by the dashed rectangles. (C) Close-up views of the two DynWB-M-Lis1 interfaces, located on dynein’s ring (SiteRing) and stalk (SiteStalk), viewed perpendicular to the dashed rectangles. AAA domains that contribute motifs to the interfaces are labeled. (D–E) Ring conformations of the DynWB-M:Lis1 structure (D) and the low-affinity, wild-type human dynein-2 solved in the presence of ATP-Vi (PDB: 4RH7) (E). We removed the linker and, when present, Lis1 for clarity. (F) Map of pairwise alpha carbon interatomic distances between the DynWB-M:Lis1 structure and the low-affinity, wild-type human dynein-2. Structures were aligned using their AAA3L domains. The length and thickness of the vectors are proportional to the calculated interatomic distances. See also Figure S3.
Figure 4. Lis1’s opposite modes of dynein regulation are associated with rigid body motion conformational changes in dynein’s ring
(A) Map of pairwise alpha carbon interatomic distances between the Dynwt-M:Lis1 and DynWB-M:Lis1 structures. We removed the linker and Lis1 for clarity. Structures were aligned using their AAA3L domains. The length and thickness of the vectors are proportional to the calculated interatomic distances. (B) 1D plot of the interatomic distances shown in panel (A). Large and small AAA subdomains are indicated below the plot, along with amino acid numbers at their boundaries. The positions of the arginine fingers of domains AAA2-5, which act on domains AAA1-4, are labeled (“R”). (C) Superposition of the Dynwt-M:Lis1 and DynWB-M:Lis1 structures, aligned using their AAA4S domains. The Dynwt-M:Lis1 structure is shown in lighter colors and the Lis1 bound to SiteStalk in DynWB-M:Lis1 was faded for clarity. The square highlights SiteRing, and is the area represented in panels (D) and (E). (D) Close-up of the SiteRing in dynein, with Lis1 faded for clarity. The bi-tone yellow arrow indicates good alignment for the base of the stalks. The tri-color multi-headed arrows point to the AAA3, AAA4 and AAA5 elements in SiteRing in both Dynwt-M:Lis1 and DynWB-M:Lis1. (E) Positions of the SiteRing-bound Lis1 in the Dynwt-M:Lis1 and DynWB-M:Lis1 structures. Same view as in (D) but with dynein faded for clarity. Light and dark orange arrows point to the equivalent positions in Lis1 in the Dynwt-M:Lis1 and DynWB-M:Lis1 structures, respectively. (F) Modeling of a second Lis1 into the Dynwt-M:Lis1 structure interacting either with SiteStalk (left) or with the Lis1 bound at SiteRing (right). Grey arrows point to gaps present in the models. (G) For comparison, we show the same view of the experimentally observed DynWB-M:Lis1 structure.
Figure 5. The second Lis1 binding site is required for the Lis1-induced weak microtubule binding state of dynein
(A) Sequence conservation around the putative SiteStalk. Sequence in the region of dynein’s stalk identified as the putative second binding site for Lis1, extracted from a full sequence alignment of dynein heavy chain genes. Residues with 70% conservation or higher are shaded grey. The three residues we mutated—E, Q, N, shaded in yellow—are conserved in model organisms that have Lis1 orthologs in their genomes (Saccharomyces cerevisiae; Homo sapiens; Mus musculus; Canis familiaris; Dictyostelium discoideum; Drosophila melanogaster; Danio rerio; Aspergillus nidulans, shaded in orange), but not in a group of fission yeasts (Schizosaccharomyces pombe; Schizosaccharomyces octosporus; Schizosaccharomyces cryophilus) that do not appear to have a Lis1 ortholog in their genome. (B) Atomic model of the DynWB-M:Lis1 complex and close-up view of SiteStalk. The conserved EQN triad is shown in stick representation and nearby dynein motifs are labeled. DynEQN is a construct that carries an EQN to AAA mutation but has a wild-type AAA3. (C) Average velocities of Dynwt (grey) and DynEQN (yellow) in the absence (solid bars) or presence (hatched bars) of 300 nM Lis1 (n>154 events per data point). See Figure S5B for representative kymographs. (D) Binding densities of Dynwt (semi-transparent grey; data reproduced from Figure 1 for comparison) and DynEQN (yellow) in the presence of ATP (left) or ATP-Vi (right). See also Table S1. (E) Normalized binding densities of Dynwt (semi-transparent grey) and DynEQN (yellow) in the absence (solid bars) or presence (hatched bars) of 300 nM Lis1 (n=12 fields of view per data point), in the presence of ATP (left) or ATP-Vi (right). Binding densities were normalized by setting those in the absence of Lis1 to 100%. Data for Dynwt are reproduced from Figure 1 for comparison). Statistical significance was calculated using unpaired t-test with Welch’s correction for both velocity (C) and binding density (D–E). P-values: ns, not significant; **, <0.01; ***, <0.001, ****, <0.0001. Data are shown as mean and standard error of mean.
Figure 6. The second binding site for Lis1 is required for dynein’s microtubule plus end localization in vivo and in vitro
(A) Schematic of dynein and Lis1 function in spindle positioning in S. cerevisiae. Dynein is localized to the SPB (1), transported to the MT plus end by a kinesin in a process that also requires Lis1 (2), maintained at the MT plus end by Lis1 (3), and “off-loaded” to the cell cortex (4), where it pulls on SPB-attached MTs to position the mitotic spindle. (B) Dynein localization in dividing S. cerevisiae. First column: Representative brightfield images. Second column: maximum projections of 3xGFP-labeled dynein (Dyneinwt or DyneinEQN). Third column: maximum projections of tdTomato-labeled SPC110, a SPB marker. Fourth column: merged 3xGFP-dynein and tdTomato-SPB images. White arrowheads: co-localized dynein and SPB signals. Strains imaged: Dyneinwt (top row); Dyneinwt in a ΔLis1 background (middle row); DyneinEQN (bottom row). Both dynein-3xGFP and SPC110-tdTomato are expressed under their endogenous promoters. (C, D) Quantification of the data presented in (B). (C) Average number of dynein foci per cell colocalized with SPBs; and (D) Average number of dynein foci per cell not colocalized with SPBs for Dyneinwt (grey), Dyneinwt/ΔLis1 (hatched grey) and DyneinEQN (yellow) strains (n>50 cells per data point). (E) Schematic representation of our in vitro reconstitution of kinesin-mediated dynein transport to the MT plus end. Brightly-labeled, GMPCPP-stabilized MT seeds are attached to the coverslip via biotin-streptavidin interactions. A dimly-labeled MT extension grows faster at the plus end of the seed, allowing MT polarity to be determined. Addition of dynein (labeled with TMR), Lis1, Bik1, Kip2 and Bim1 results in plus-end-directed transport of dynein by kinesin. Known interactions are shown with double-headed arrows color-coded according to the proteins involved. MT plus and minus ends are labeled. (F, G) Representative kymographs from the assay outlined in (E), with MT (488) and dynein (TMR) channels shown in black and white, and the merged image in pseudocolor, for Dynwt (F) and DynEQN (G). Plus (+) and minus (−) indicate MT polarity. White arrowheads point to the start of plus-end-directed runs. (H) Quantification of the percentage of plus- and minus-end-directed runs for Dynwt (Grey) and DynEQN (Yellow). (I) Quantification of the percentage of non-motile runs. (n = 4 technical replicates). Statistical significance was calculated using Mann-Whitney test for both average number of foci per cell (C, D) and percentage of runs (H, I). P-values: ns, not significant; *, < 0.05; **, <0.01; ****, <0.0001. Data are shown as mean and standard error of mean. See also Figure S6.
Figure 7. Model for the opposing modes of regulation of dynein by Lis1)
(A, B) The nucleotide state of AAA3 determines which regulatory mode is used by Lis1. The figure illustrates how the ATP hydrolysis cycle at AAA3 affects the affinity of dynein for MTs (A) and how Lis1 acts on these different states (B). We show dynein with ATP/ADP-Pi bound at its AAA1 site to reflect that our data suggest this is the state where Lis1 regulation is apparent. Dynein alone has high affinity for MTs when its AAA3 is either empty (“apo”) (A, state 1), or bound to ATP/ADP-Pi (A, state 2). Phosphate release from AAA3 leads to the AAA3:ADP-bound, low-affinity state of the motor (A, state 3), which is expected to be the predominant state when dynein walks along MTs. Lis1 acts in opposite ways on states 1/3 versus 2: one β-propeller binds when AAA3 is either empty or contains ADP, leading to tight MT binding (B, states 4 and 6); while 2 β-propellers bind when AAA3 has ATP/ADP-Pi, leading to weak MT binding (B, state 5). (C) Proposed biological roles of the Lis1-mediated weak and tight MT-binding states of dynein. In S. cerevisiae, dynein is transported towards the plus-end of MTs by Kip2, a process that requires Lis1. Binding of two Lis1 β-propellers (B, state 5) keeps the motor in a weak affinity state and promotes the formation of the kinesin transport complex. Once at the plus-end of the MT, dynein cycles to a AAA3:ADP or AAA3:apo state (by a process not currently understood); binding of one Lis1 β-propeller (B, states 4 and 6) keeps the motor in a tightly bound to the MT in preparation for cargo loading.
References
- Burgess SA, Walker ML, Sakakibara H, Knight PJ, Oiwa K. Dynein structure and power stroke. Nature. 2003;421:715–718. - PubMed
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