Dynein pulls microtubules without rotating its stalk

Abstract

Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, "cryo-positive stain" electron microscopy. The dynein molecules in the complex were shown to be active in in vitro motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP.vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).

Fig. 1.

Two possible models based on the structural changes observed with Chlamydomonas dynein c (3). (A) The stalk rotates relative to the MT to pull the MT. (B) The stalk angle is constant, but the movement of the tail with respect to the head and stalk causes relative sliding of the MTs. The resulting MT movement is expected to be smaller in B, but it seems to be enough to produce a shift of ≈8 nm. Note that a longer part of the linker is exposed in the ADP·Pi state than in no-nucleotide.

Fig. 2.

Dynein-MT complex in the no-nucleotide state. (A and B) Cryo-positive stain EM images of dynein purified from S. nudus, bound to MTs in the presence of apyrase. The B-MT minus end is at Right. Dynein molecules are regularly arranged in a single layer between 2 MTs. Individual dynein molecules may show a single ring (B, #1–3), a double ring (B, #4), or be unclear (B, #5). Stalks (arrows), the head-A-MT tethers (white arrowheads), and extra densities on the top of the tails (asterisks) are indicated. Some molecules show 2 stalks. (C and D) Interpretation of the single-ring and double-ring images. (E) Cross sections of the dynein-MT complex embedded in Epon812, with our interpretation of the images. The likely viewing direction of our cryo images is indicated. The micrographs in A, B, and Fig. 4 were Gaussian-filtered to reduce noise. (Scale bars: A and B, 20 nm; C, 50 nm.)

Fig. 3.

Orientation of dynein relative to the MT polarity. (A and B) Cryo-positive stain EM images of dynein cross-bridging anti-parallel (A) or parallel (B) MTs. (C) Proportion of MT pairs with different polarities. Although the polarity of the A-MT is not uniform, all of the dynein molecules are oriented in the same way with respect to the B-MT, with the heads and stalks to the B-MT minus-end side of the tail. The observed head/tail arrangement agrees with that suggested from the QFDE images of dynein in axonemes (11, 12), but inconsistent with the assignment in a recent report on the Chlamydomonas dynein-MT complex (18). (D and E) Averaged images of dynein cross-bridging 2 anti-parallel MTs (D; n = 30) or parallel MTs (E; n = 31), showing no detectable differences. (Scale bars: A and B, 50 nm; D and E, 10 nm.)

Fig. 4.

Structural changes of dynein with ADP·Vi. (A and B) Cryo-positive stain EM images in the ADP·Vi state, with the B-MT plus end at Left. Individual dynein images (B) show a double ring (#1–6), a single ring (#7), or an intermediate conformation (#8). Most stalks are clearly tilted toward the B-MT minus end (arrows). (C) Populations of dynein molecules in the no-nucleotide and ADP·Vi states showing a superimposed, single ring (S), a double ring (D), or, intermediate or unclear structures (I). (D) Distribution of stalk angles (θ) with respect to MTs. The averaged angles are 54.0 ± 8.7° (n = 451) and 58.6 ± 14.6° (n = 490) for the no-nucleotide and ADP·Vi states, respectively. (Scale bars: 20 nm.)

Fig. 5.

Averaged images after classification. Shown are no-nucleotide (A and C) and ADP·Vi (B and D) averages. A total of 1,062 no-nucleotide images and 988 ADP·Vi images were classified first according to the heads (A and B) and then according to the stalks (C and D). Protein is white. Two selected stalk-classes are shown below each head-class. The numbers of images in averages are: 736, 257, and 69 for #1–3 (A); 188, 144, 74, 73, 72, and 6 for #1–6 (B); between 19 and 269 (C); and 9–40 (D). (E and F) Interpretation of each class-average. Stalks (arrows), and a low-density region between the tail and head (bracket) containing linkers (white arrowheads) are indicated in some images. (Scale bar: 20 nm.)

Fig. 6.

Comparison with Chlamydomonas dynein c images. Our class-averages of sea urchin dynein (A and C) are compared with images from figure 4 of ref. (B and D). (A and B) No-nucleotide state. (C and D) ADP·Vi state. Cross correlation between the masked regions showed that the head regions of the two images agreed best when the dynein c images were rotated as shown in B and D. The hypothetical positions of the B-MT, based on our images with MTs, are indicated. (Scale bars: 10 nm.) [Reproduced with permission from ref. (Copyright 2003, Nature Publishing Group).]

Fig. 7.

Possible mechanism for dynein movement. (A) Head-class averages characteristic of each nucleotide state are selected from Fig. 5 A and B, and aligned by their A-MT attachment sites (yellow dotted line). In #2 and #3, at least one ring is shifted toward the minus end (red dotted line). (B) Stalk-class averages are superimposed on the head-class averages (red) shown in A to show both stalks and tails clearly. The stalks do not rotate but, together with their heads, shift ≈8 nm (cyan dotted line) toward the minus end in some ADP·Vi averages. (C) A model for dynein stepping (see “Mechanism of Motility” in Results and Discussion). (Scale bar: 10 nm.)