Neurodegeneration mutations in dynactin impair dynein-dependent nuclear migration

Abstract

Neurodegenerative disease in humans and mice can be caused by mutations affecting the microtubule motor dynein or its biochemical regulator, dynactin, a multiprotein complex required for dynein function (1–4). A single amino acid change, G59S, in the conserved cytoskeletal-associated protein glycine-rich (CAP-Gly) domain of the p150^glued subunit of dynactin can cause motor neuron degeneration in humans and mice, which resembles ALS (2, 5–8). The molecular mechanism by which G59S impairs the function of dynein is not understood. Also, the relevance of the CAP-Gly domain for dynein motility has not been demonstrated in vivo. Here, we generate a mutant that is analogous to G59S in budding yeast, and show that this mutation produces a highly specific phenotype related to dynein function. The effect of the point mutation is identical to that of complete loss of the CAP-Gly domain. Our results demonstrate that the CAP-Gly domain has a critical role in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus, but it is otherwise dispensable for dynein-based movement. The need for this function appears to be context-dependent, and we speculate that CAP-Gly activity may only be necessary when dynein needs to overcome high force thresholds to produce movement.

The cytoplasmic dynein motor is an ancient ATPase that powers directional motility along microtubule tracks. Cells use dynein to organize the cytosol by manipulating the position of various cargoes with respect to the microtubule cytoskeleton, and the mitotic spindle and the microtubule organizing centers (MTOCs) with respect to regions of the cell cortex. The latter function is particularly important during asymmetric cell divisions and cell migration.

Cytoplasmic dynein is a large multisubunit complex, and every known function of cytoplasmic dynein requires a second multisubunit complex, dynactin (9, 10). Dynactin includes a short actin-like filament composed of the actin-related protein, Arp1, overlaid by a shoulder-sidearm complex composed of the p24, dynamitin/p50, and p150^glued subunits (11). The amino terminus of the p150^glued subunit contains a cytoskeletal-associated protein glycine-rich (CAP-Gly) domain, which can bind to microtubules and the microtubule-binding proteins, CLIP-170 and EB1 (12–14). In vitro, antibodies that bind to the amino-terminal region of p150^glued diminish the run length of dynein–dynactin in motility assays; however, the manner in which this domain contributes to the function of dynein–dynactin remains unclear (15, 16). In fact, recent work has shown that loss of the CAP-Gly domain of p150^glued does not affect the transport of several cargoes along microtubules in Drosophila S2 cells (17) or the organization of Golgi in HeLa cells (18), suggesting that the CAP-Gly domain may not be required for all dynein–dynactin functions.

A point mutation in the CAP-Gly domain of human p150^glued, G59S, has been linked to a slowly progressive form of lower motor neuron disease that resembles ALS (2). Although neurons use the dynein motor for several distinct tasks, the connection between dynein dysfunction and neuronal degeneration is not well understood. The specific nature of the G59S mutation provides an opportunity to identify functions that are linked to disease. In mouse models, a homozygous G59S mutation results in embryonic lethality, similar to complete loss of p150^glued (6). Heterozygous G59S-mutant mice are viable, and adults exhibit cellular abnormalities in lower motor neurons that are reminiscent of changes seen with human diseases, including ALS (6–8). The G59S phenotype appears to be a dominant-negative effect rather than the result of haploinsufficiency, because animals bearing a single wild-type gene for p150^glued do not display these phenotypes (6). The motor neurons in adult G59S-mutant mice accumulate large inclusions that contain p150^glued, raising the possibility that the disease pathology could either be due to the toxic accumulation of mis-folded p150^glued, altered dynein function, or a combination of these effects (7, 8). In vitro, the G59S mutation disrupts the interaction of p150^glued with microtubules and EB1; however, fibroblasts from G59S patients exhibit only a mild dynein-like phenotype in maintaining Golgi distribution (5).

The key questions at this point are what is the role of the CAP-Gly domain in dynein motility, and how does the G59S mutation impair that function? Here, we demonstrate that the CAP-Gly domain of Nip100, the budding yeast homologue of p150^glued, makes a specific contribution to dynein motility. The function of dynein in yeast is to draw the mitotic spindle and nucleus into the bud neck by pulling on cytoplasmic microtubules from sites at the cell cortex (19–21). The CAP-Gly domain of Nip100 is well-conserved, and we show that a mutation analogous to G59S abolishes the function of this domain. Mutations that disrupt the predicted binding interface or that completely remove the CAP-Gly domain have phenotypes identical to those of G59S. The CAP-Gly domain appears to be involved specifically in the initial movement of the spindle and nucleus into the bud neck. The specificity of the phenotype is remarkable in that these CAP-Gly mutations have no effect in several other assays of dynein function in vivo. Most notably, the CAP-Gly domain is completely dispensable for the dynein-dependent sliding of free microtubules along the cell cortex. These data support a model in which the CAP-Gly domain of dynactin is important for dynein function in scenarios that require maximal force production by the motor.

Results

Residue G59 of human p150^glued is conserved in CAP-Gly domains, and it corresponds to G45 of yeast Nip100 (Fig. 1A). Based on crystal structures of human p150^glued, G59 is embedded in a β sheet, and does not appear to participate in interactions with binding partners (Fig. 1B; see refs. 13, 14). We created homology models for the WT and G45S forms of Nip100 using Rosetta (22). These structures were similar to the CAP-Gly domain of human p150^glued, with G/S45 similarly positioned within the second strand of the β sheet. We used molecular dynamics simulations to investigate the effect of changing Gly 45 to Ser (SI Materials and Methods). The hydroxyl group of the serine allowed the residue to make hydrogen bonding interactions, disrupting the first 2 strands of the β sheet, in particular the strands from K31 to E35 and from I42 to S45. This loss of secondary structure elicited conformational changes throughout the β sheet, and altered the dynamics of the regions between the strands (Fig. 1C). These regions of p150^glued are known to be involved in protein–protein interactions, from structural studies (14), so we hypothesized that the mutation might impair the functional interactions of the CAP-Gly domain in cells.

Fig. 1.

The G59S mutation of human p150^glued constructed in yeast. (A) Sequence alignment of the CAP-Gly domains of human p150^glued, yeast Nip100, human CLIP170, and yeast Bik1. The conserved glycine at position 59 of p150^glued is shown in black. Dashed lines indicate residues of human p150^glued that are involved in interactions with binding partners, as determined by Weisbrich et al. (14). (B) Predicted structures of residues 25–83 for wild-type Nip100 and the Nip100-G45S mutant. The sequence of Nip100 was threaded onto the structure of p150^glued (13) by using Rosetta, and both structures were analyzed by molecular dynamics simulations (see SI Materials and Methods). Regions that exhibited significantly altered dynamics in the G45S mutant are indicated in blue (G53-N55) and green (S58-G62). (C) Root-mean-square fluctuation plot for each residue of Nip100 (black) and Nip100-G45S (red), based on molecular dynamics simulations. The blue and green regions here correspond to those in B. (D) Levels of soluble Nip100 in wild-type and mutant strains. Mutations were introduced at the endogenous NIP100 locus, and each allele was tagged with the 13myc epitope. Endogenous Cdc3 is shown as a loading control. Strain numbers: WT, yJC6056; nip100-G45S, yJC6300; nip100ΔCAP-Gly, yJC5811; nip100-K54E,N55D, yJC6301; nip100-K54A,N55A, yJC6302; nip100-K54A, yJC6307; nip100-N55A, yJC6303. (E) Anaphase spindle position assay for dynein function. Percentage of cells with abnormal spindle position is plotted for the mutant strains, with comparison to WT, and nip100 and dyn1 null strains. Values are the mean of 5 sets of at least 50 cells, and error bars are the SE of the mean. Strain numbers: WT, 5919; nip100Δ, yJC6047; dyn1Δ, yJC5603; nip100-G45S, yJC6295; nip100ΔCAP-Gly, yJC5795; nip100-K54E,N55D, yJC6296; nip100-K54A,N55A, yJC6297; nip100-K54A, yJC6298; nip100-N55A, yJC6299.

To test the consequences of G45S in cells, we introduced the mutation into haploid yeast at the chromosomal NIP100 locus such that the mutant allele provided the only source of Nip100 (Table S1). For comparison, we mutated residues K54 and N55, which correspond to K68 and N69 in mammalian p150^glued, and have been shown to be important for the CAP-Gly domain to interact with the EEY/F motifs found in α-tubulin, EB1, and CLIP170 (13, 14, 23). Also, we excised codons 2-104 from the chromosomal NIP100 locus to remove the entire CAP-Gly region of the protein. In this truncated protein, the first predicted coiled-coil is located at the amino terminus (Fig. S1). Mammalian p150^glued contains a region of basic residues between the CAP-Gly and coiled-coil domains, which has been shown to bind microtubules and enhance the processivity of dynein in vitro (24); however, Nip100 does not appear to contain an analogous region. For each Nip100 mutant, expression was tested, and the level of soluble Nip100 was not decreased in any of the mutants, including the ΔCAP-Gly truncate, for which the level was actually increased by 2-fold (Fig. 1D).

To determine whether and how the CAP-Gly domain might be important for dynein–dynactin activity, we tested each mutant for several phenotypes indicative of loss of dynein function. First, we used a simple assay, observing anaphase spindle position at a single time-point in a population of asynchronous cells. The CAP-Gly mutants were indistinguishable from wild-type cells and did not exhibit accumulation of spindles within the mother cell, a trait characteristic of dynein or dynactin null mutants (Fig. 1E). In contrast, a larger truncation that removed the first coiled-coil region of Nip100 along with the CAP-Gly domain was defective in this assay, at a level comparable with that of nip100 and dyn1 null mutants (Fig. S1). In vertebrates, this coiled-coil region is required for interaction with dynein (15).

Next, we scored the position of preanaphase spindles in the absence of Kar9, which helps position the spindle via an independent mechanism (25, 26). Loss of the CAP-Gly domain of Nip100 decreased the number of kar9Δ cells in which the preanaphase spindle was found in the bud (Fig. S1C), suggesting that the CAP-Gly domain is important for the translocation of short spindles through the bud neck. Later in anaphase, the frequency of mis-positioned spindles was normal, indicating that this defect resolves over time, as the cell cycle progresses (Fig. S1D).

To examine dynein–dynactin function more carefully in the CAP-Gly mutants, we assayed the kinetics of spindle movement in synchronized cells expressing GFP-tubulin. After release of cells from G1 arrest, we recorded time-lapse movies and measured the time until the movement of 1 spindle pole into the daughter cell. Cells expressing either nip100-G45S or nip100ΔCAP-Gly required more time to move the spindle across the bud neck than did wild-type cells (Fig. 2A). Therefore, mutations in the CAP-Gly domain appear to impair the efficiency of dynein function during spindle translocation.

Fig. 2.

The CAP-Gly region of p150^Glued/Nip100 contributes to dynein-dependent spindle movements. (A) Time elapsed between bud emergence and 1 spindle pole crossing the bud neck. Cells were arrested in G1 with 0.6 μM αfactor, released into new media, and mounted on agarose pads for microscopy. Confocal images of GFP-tubulin were captured at 45-s intervals through a Z series of 7 planes separated by 0.8 μm. Asterisks indicate statistical significance (P < 0.05; compared with WT) as determined by t test. Strains: WT, yJC5920; nip100-G45S, yJC6446; nip100ΔCAP-Gly, yJC5798; dyn1Δ, yJC5603. (B) Percentage of HU-arrested kar9Δ mutant cells in which the preanaphase spindle crosses the bud neck over the course of a 10-min timelapse movie. Neck crossing events were defined as at least 1 pole crossing the plane of the bud neck within a unidirectional movement. At least 90 cells were scored for each strain. Error bars are the SE of proportion. Asterisks indicate statistical significance (P < 0.05; compared with WT) as determined by Fisher's exact test. Strain numbers: WT, yJC5802; nip100-G45S, yJC6308 and 6309; nip100ΔCAP-Gly, yJC5803; nip100-K54E,N55D, yJC6310; nip100-K54A,N55A, yJC6311; nip100-K54A, yJC6329; nip100-N55A, yJC6312. (C) Timelapse images of GFP-labeled microtubules in kar9Δ mutants arrested with HU. Each image is a composite of 9 planes separated by 0.5 μm. Stacks were captured at 15-s intervals on a confocal microscope. The arrow marks the spindle pole that is tracked in D. Strains: WT, yJC5802; nip100ΔCAP-Gly, yJC5803. (D) The distance between one end of the spindle and the bud neck was plotted with respect to the long axis of division. The position of the spindle poles indicated in C was determined for each time point, and distances were calculated by using ImageJ.

To explore this hypothesis further, we developed a live-cell assay to monitor the movement of preanaphase spindles labeled with GFP-tubulin. To eliminate the possibility that spindle pole movement could be influenced by spindle elongation, we arrested cells in S phase with short spindles by treatment with hydroxyurea (HU). In wild-type cells, we observed the spindle to move in both directions through the bud neck; these movements coincided with lateral sliding of a cytoplasmic microtubule along the cell cortex (Movie S1). These events require dynein–dynactin function; nip100Δ null mutants displayed frequent contacts between microtubule ends and the cell cortex, but no spindle movements or microtubule sliding events occurred (Table S2 and Movie S2). A kar9Δ mutation dramatically enhanced dynein-dependent spindle movements in HU-arrested cells, consistent with previous findings (27). In this background, short preanaphase spindles oscillated across the bud neck, moving completely between the mother and bud (Table S2 and Movie S3).

To examine the contribution of the CAP-Gly domain of Nip100 in this setting, we collected timelapse movies of GFP-labeled microtubules for each CAP-Gly mutation combined with kar9Δ. The number of cells, in which the spindle moved from the mother cell compartment through the neck, was significantly decreased in each CAP-Gly mutant (Fig. 2B; Movie S4). However, in CAP-Gly mutant cells where the spindle did move through the neck, the frequency of transits of the spindle, back-and-forth through the neck, was similar to that of wild-type cells (Fig. S2).

To quantitate spindle movement, we tracked the position of spindle poles over time (Fig. 2 C and D; for histograms of complete datasets from these analyses, see SI Materials and Methods). Compared with wild-type Nip100, CAP-Gly mutants tended to produce shorter displacements (Table 1). In these abbreviated movements, spindles often approached the neck, but did not pass through it, or they made brief excursions into the neck. The instantaneous velocities observed between individual frames for CAP-Gly mutants were similar to those observed for wild-type Nip100 (mean = 2.6 ± 0.1 μm/min; see Table 1).

Table 1.

Spindle movements and microtubule-cortex interactions in CAP-Gly mutants

Strain	Spindle movements			Microtubule-cortex interactions
	Neck crosses, min−1	Displacement, μm	Velocity, μm·min−1	Sliding events, min−1	Unproductive contacts, min−1	Sliding ratio
NIP100 kar9Δ	0.23 ± 0.01 (203)	3.4 ± 0.2 (25)	2.6 ± 0.1	0.30 ± 0.02 (44)	0.44 ± 0.03	0.41 ± 0.02
nip100-G45S kar9Δ	0.09 ± 0.01 (210)	1.8 ± 0.1 (19)	2.4 ± 0.1	0.14 ± 0.03 (20)	1.02 ± 0.08	0.13 ± 0.02
nip100ΔCAP-Gly kar9Δ	0.10 ± 0.01 (301)	1.9 ± 0.2 (28)	2.4 ± 0.1	0.11 ± 0.01 (44)	0.79 ± 0.04	0.13 ± 0.02
nip100-K54E,N55D kar9Δ	0.11 ± 0.01 (170)	1.9 ± 0.2 (20)	2.3 ± 0.1	0.15 ± 0.03 (20)	1.05 ± 0.11	0.14 ± 0.03
nip100-K54A,N55A kar9Δ	0.09 ± 0.01 (141)	2.2 ± 0.2 (20)	2.5 ± 0.1	0.13 ± 0.02 (20)	0.81 ± 0.07	0.15 ± 0.03
nip100-K54A kar9Δ	0.11 ± 0.01 (133)	3 ± 0.2 (19)	2.8 ± 0.1	0.11 ± 0.03 (20)	0.78 ± 0.07	0.12 ± 0.03
nip100-N55A kar9Δ	0.08 ± 0.01 (127)	2.0 ± 0.2 (20)	2.2 ± 0.1	0.12 ± 0.03 (20)	0.73 ± 0.08	0.15 ± 0.04
NIP100 bim1Δ kar9Δ	0.05 ± 0.01 (148)	1.8 ± 0.3 (24)	2.1 ± 0.1	0.08 ± 0.02 (20)	0.36 ± 0.04	0.12 ± 0.38
nip100ΔCAP-Gly bim1Δ kar9Δ	0.06 ± 0.01 (169)	2.0 ± 0.3 (18)	2.6 ± 0.1	0.06 ± 0.02 (20)	0.44 ± 0.04	0.17 ± 0.45

Data were collected from 10-min movies of cells arrested in HU (see Materials and Methods). Mean values ± SEM are shown. Boldface indicates statistical significance (P < 0.05) compared with NIP100 kar9Δ, determined by t test. Numbers in parentheses are the number of cells scored. Strain numbers: WT, yJC5802; nip100-G45S, yJC6308 and 6309; nip100Δ CAP-Gly, yJC5803; nip100-K54E,N55D, yJC6310; nip100-K54A,N55A, yJC6311; nip100-K54A, yJC6329; nip100-N55A, yJC6312; nip100Δ CAP-Gly bim1Δ, yJC6596 and 6597; bim1Δ, yJC6599.

Dynein-dependent spindle movements depend on microtubule sliding along the cell cortex; therefore, we examined microtubule-cortex interactions in the timelapse movies. The CAP-Gly mutants all exhibited a decrease in the frequency of microtubule sliding events (Table 1). Conversely, the frequency of unproductive microtubule-cortex interactions, in which the microtubule plus end contacted the cortex, but did not transition into sliding, was increased (Table 1). Together, these data indicate that the CAP-Gly domain of Nip100 promotes the initiation and persistence of the dynein-dependent microtubule sliding events that power spindle translocation through the bud neck.

Mitosis in yeast is closed, meaning that the nuclear envelope does not disassemble. Electron micrographs reveal that the diameter of the nucleus within the mother cell is far greater than the diameter of the bud neck, and that the nuclear space is greatly constricted during passage through the neck (28). To investigate how constriction of the nucleus might influence spindle movement, we labeled the nuclear pore complex with Nup133-RFP, and captured 2-color timelapse movies of HU-arrested kar9Δ cells that also expressed GFP-tubulin. In these movies, the initial movement of the nucleus into the bud neck coincided with a bud-directed microtubule-sliding event that brought the spindle to the proximal edge of the nucleus (Fig. 3A). The nuclear membrane appeared to squeeze through the neck and, then, to expand as it entered the bud. After this point, the nucleus adopted a bilobed shape and remained lodged across the bud neck. In cells where the spindle translocated back-and-forth across the neck, the outline of the nuclear envelope changed very little, displaying only transient disfigurations when one end of the spindle collided with it (Fig. 3B; Movie S5 and Movie S6). Thus, the deformation of the nuclear envelope and volume is coupled with the initial movement of the nucleus and spindle into the bud neck, and not subsequent spindle oscillations. In HU-arrested Nip100ΔCAP-Gly cells in which the spindle failed to enter the bud neck, we observed that the entire nucleus remained within the mother compartment (37/40 cells; Movie S7).

Fig. 3.

Nuclear envelope position during spindle movements. (A) The nuclear envelope enters the neck with the spindle. (B) The outline of the nuclear envelope remains in the neck and moves rather little, whereas the spindle moves back-and-forth across the neck. Timelapse images of GFP-labeled microtubules and RFP-labeled Nup133 in kar9Δ mutants arrested with HU. Images are composites of Z projections from 9 confocal sections at 0.5-μm increments captured every 20 s. Strain: yJC6288. (Scale bar, 1 μm.) (C) Timelapse images of GFP-labeled microtubules and RFP-labeled Nup133 in bni1Δkar9Δ mutants arrested with HU. Images were collected as described in A and B. Strain: yJC6289. (D) Mean instantaneous velocity during initial spindle movement into the neck and subsequent spindle oscillations in kar9Δ cells, and initial spindle movement in bni1Δkar9Δ cells. Initial movements began with the entire nucleus located within the mother cell and proceeded to bring at least 1 spindle pole across the bud neck. At least 25 cells were analyzed for each category. Error bars are the SE of the mean.

To compare these movements, we measured the speed of mitotic spindle translocation during initial nuclear movement into the neck and subsequent spindle oscillations in these movies. The spindle entered the neck with a mean instantaneous speed of 1.7 ± 0.1 μm/min, which was significantly less than the speed of spindle movements that occurred after the spindle was positioned across the neck (3.0 ± 0.1 μm/min; P < 0.01; see Fig. 3D). To test whether the constriction of the nucleus retards spindle movement into the neck, we examined spindle movement in cells with wide necks; bni1Δ mutants have wide necks, thought to arise from defective septin organization (29). In bni1Δ cells, the nuclear envelope was wider at the neck, and spindles moved into the neck at greater speeds (2.2 ± 0.1 μm/min; P < 0.01; see Fig. 3 C and D). This result supports the idea that constriction of the nucleus antagonizes dynein-induced movement of the spindle into the neck.

To test the role of the CAP-Gly domain in dynein-dependent motility further, we examined cases where movement of the cytoplasmic microtubule was uncoupled from movement of the nucleus. In wild-type cells, we occasionally observed cytoplasmic microtubules pull out of the spindle pole body (SPB) spontaneously as the preanaphase spindle began to move (Fig. S3 and Movie S8). When a microtubule pulled out, the spindle snapped back into the mother, and the detached microtubule slid along the bud cortex at high speed (Fig. S3).

To allow for a quantitative analysis of free microtubule sliding, we enhanced the frequency of cytoplasmic microtubule detachment from the SPB by introducing a cnm67Δ mutation, which destabilizes the cytosolic face of the SPB (30). In this background, the number of free cytoplasmic microtubules in mitotic cells was >10-fold higher than in CNM67 cells, whether cells expressed full-length or ΔCAP-Gly Nip100 (Table S3). As expected, free microtubules in dyn1Δ cnm67Δ or nip100Δ cnm67Δ double mutants exhibited end-on contacts with the cell cortex, but did not slide. In these double mutants, the number of detached microtubules was decreased compared with cnm67Δ single mutants, indicating that the force generated by dynein contributes to detaching the microtubule from the SPB.

To measure the movement of free sliding microtubules, we first collected 2-color movies of cnm67Δ cells expressing GFP-tubulin and RFP-Spc97, a component of the γ-tubulin complex (31). The persistent association of Spc97 at 1 end of the free sliding microtubule confirmed the minus end-directed polarity of all sliding events, and demonstrated that the minus end was stabilized by the γ-tubulin complex (Fig. S4A, Movie S9, and Movie S10). We measured the movement of free microtubules by tracking the position of their minus ends. During individual sliding events, we observed changes in speed and occasional pauses, but no reversals of direction (Fig. S4B, Movie S11, and Movie S12). The distribution of instantaneous velocities was similar for cells expressing full-length Nip100 (6.4 ± 0.2 μm/min) and the ΔCAP-Gly truncate (6.2 ± 0.2 μm/min) (Fig. S4C; P = 0.56). These speeds are similar to the microtubule gliding speeds observed in vitro for purified dynein (5.4 ± 1.6 μm/min; see ref. 32). Also, the duration of individual sliding events was not affected by loss of the CAP-Gly domain (Fig. S4D; P = 0.16).

To examine the character of these movements more closely, we separated each sliding event into segments in which the speed did not change. We found that segments of longer duration were associated with slower speeds, whereas the speeds of shorter segments were greater and more varied (Fig. S4E). The results for this analysis were similar for full-length Nip100 and the ΔCAP-Gly mutant. Overall, the CAP-Gly domain of Nip100 does not appear to influence the sliding of free microtubules along the cell cortex, which suggests that the role of the CAP-Gly domain in dynein motility is context-dependent.

We evaluated several molecular mechanisms by which the CAP-Gly domain of p150^glued/Nip100 might enhance the dynein motility. Dynactin is required for the localization of dynein to the cell cortex, which is presumably necessary for microtubule sliding (33). To test whether the CAP-Gly domain contributes to this function, we examined the localization of dynein heavy chain (Dyn1–3GFP, expressed from the endogenous locus) in asynchronous and HU-arrested cells, expressing full-length Nip100 or the ΔCAP-Gly truncate. Loss of the CAP-Gly domain did not decrease the number of cortical dynein foci in either case (Fig. S5). In fact, the number of cortical dynein foci was slightly increased in Nip100ΔCAP-Gly cells, by a statistically significant amount (P < 0.01). We also quantitated the fluorescence intensity of individual foci, and found that the amount of dynein per cortical focus was not altered (Fig. S5; P = 0.84). Therefore, the diminution of dynein function in CAP-Gly mutants was not due to defective anchoring of dynein to the cortex.

As an alternative, we considered whether decreased nuclear movement could be due to poor dynein–dynactin function at the SPB. Dynein–dynactin has been shown to organize microtubule minus-ends at the MTOC in other organisms (17, 34, 35). If the CAP-Gly domain were required for anchoring cytoplasmic microtubules at the yeast SPB, then an increased frequency of microtubule detachment in CAP-Gly mutants could account for the failure to pull the nucleus through the neck. To the contrary, we found that the frequency of microtubule detachment during spindle movements was less in CAP-Gly mutant cells (Table S3). Also, loss of the CAP-Gly domain did not alter the number or length of cytoplasmic microtubules at any phase of the cell cycle (Table S4). Although these data argue that the CAP-Gly domain is dispensable for microtubule organization at the SPB, one cannot completely exclude a role for the CAP-Gly domain at this site. Detachment of cytoplasmic microtubules during spindle translocation appears to be caused by dynein pulling from the cell cortex (Movies S1–S8). Thus, it is possible that diminished activity of cortical dynein in the CAP-Gly mutants may mask a defect at the SPB.

Last, we asked whether the CAP-Gly domain was necessary for the association of Nip100 with microtubules in the cell. First, we examined Nip100 localization in cells with a 3X-GFP tandem fusion at the C terminus of Nip100, expressed from the chromosomal locus. In haploid cells expressing Nip100-ΔCAP-Gly-3GFP, the fluorescence distribution along cytoplasmic microtubules was qualitatively similar to that of full-length Nip100–3GFP (Fig. S6). Quantitation of the fluorescence intensity at microtubule plus ends revealed similar values for the truncated and full-length versions of Nip100, 117 ± 16 and 85 ± 18 (mean ± SEM), respectively, in cells with short bipolar spindles. This result is consistent with our previous finding that the localization of Nip100 to microtubule ends is mediated by dynein (33). Next, we examined the microtubule-pelleting behavior of Nip100 and Nip100ΔCAP-Gly from yeast cell lysates. Truncated Nip100 pelleted with microtubules similar to full-length Nip100 (Fig. S6). The CAP-Gly domain alone exhibited little to no microtubule pelleting, indicating that the microtubule-pelleting ability of Nip100 from the cell extract probably depends on other interactions with binding partners. Thus, the CAP-Gly domain of Nip100 is not the primary link between dynactin and microtubules.

We considered the possibility that the CAP-Gly domain of Nip100 may indirectly link dynein–dynactin to microtubules by interaction with CLIP-170 or EB1. The CLIP-170 homologue, Bik1, participates in targeting dynein–dynactin to microtubule plus ends, and disruption of BIK1 leads to a complete loss of dynein function (Fig. S1; see refs. 33 and 36). This phenotype precludes the analysis of bik1Δ null mutants in our spindle movement assay. The EB1 homologue, Bim1, is not necessary for dynein function. Therefore, we examined the movement of GFP-labeled spindles in bim1Δ kar9Δ double mutant cells during HU arrest. Spindle movements were rare in the presence of the bim1Δ mutation (Table 1). This effect may be partly due to suppression of microtubule dynamics, and subsequent reduction in the frequency of total microtubule contacts with the cortex (Table 1; see ref. 19). However, we found that the ratio of microtubule-cortex interactions that transitioned into sliding events was low in these cells, similar to the level observed for the CAP-Gly mutants (Table 1). When sliding events were initiated in bim1Δ kar9Δ mutants, they typically produced short displacements of the spindle (Table 1). To determine whether the CAP-Gly domain was still contributing to dynein function in bim1Δ mutants, we analyzed triple mutants expressing Nip100ΔCAP-Gly in the bim1Δ kar9Δ background. These cells exhibited phenotypes that were nearly identical to those observed for bim1Δ kar9Δ (Table 1). This result indicates that the loss of Bim1 is epistatic to the disruption of the CAP-Gly domain of Nip100, consistent with the hypothesis that the CAP-Gly domain binds to Bim1.

Discussion

In this study, we report that a mutation in the CAP-Gly domain of the dynactin complex, which can cause motor neuron degeneration in humans, abolishes the dynein-based function of this domain in yeast. Although mutation of the CAP-Gly domain did not abrogate dynein–dynactin motility, we found that these mutants displayed defects in specific settings. CAP-Gly function was important for the initiation and persistence of dynein-dependent microtubule sliding, which is directly coupled to the initial movement of the mitotic spindle and nucleus into the bud neck. Once the nucleus was positioned across the neck, loss of CAP-Gly function had only a mild effect on spindle movement. Also, the sliding of free microtubules, ones that had detached from the spindle, along the cortex was not affected by loss of the CAP-Gly domain.

How might the movement of the spindle and nucleus into the bud neck be distinct from dynein motility in other contexts? Several lines of evidence indicate that this movement introduces a load burden that antagonizes dynein motility. First, we observed that the velocity of dynein-dependent microtubule sliding was significantly reduced when coupled with movement of the nucleus (Fig. 3D). Second, by monitoring the morphology of the nuclear envelope during spindle movements, we found that when the spindle was initially pulled into the neck, the nuclear envelope deformed, causing a substantial constriction of the nuclear volume as it extended into the bud (Fig. 3A). Once the nucleus was positioned across the neck, subsequent microtubule sliding events produced spindle movements that did not require a change in nuclear shape. Thus, nuclear constriction only occurs during the initial movement into the bud. Third, we found that lessening the constriction of the nucleus, by enlarging the bud neck, increased the velocity of spindle and nuclear movement into the neck. This velocity dependence is reminiscent of results from in vitro assays of dynein motility, in which the rate at which the dynein motor moves along a microtubule is inversely proportional to the magnitude of load burden applied to the motor by optical tweezers (37, 38). Thus, our results in cells suggest that attempting to move the nucleus into the neck imposes a greater load burden on dynein–dynactin than do subsequent spindle oscillations or free microtubule sliding.

The force required to move the nucleus may antagonize the initiation and persistence of microtubule sliding events by disrupting either the interaction between dynein–dynactin and its cortical receptor, or the processive association of dynein–dynactin with the microtubule substrate. In our experiments, the CAP-Gly domain was not necessary for the association of dynein with the cortex (Fig. S5). It is more likely that the CAP-Gly domain mediates association with microtubules. Although our results provide no evidence of a direct interaction between the Nip100 CAP-Gly domain and tubulin, we found that loss of EB1/Bim1 elicited defects in microtubule sliding that were similar to CAP-Gly mutants. Also, double mutants that lacked both the CAP-Gly domain and EB1/Bim1 were nearly identical to the EB1/Bim1 null mutation alone. These data are consistent with the notion that EB1/Bim1 is necessary for the function of the CAP-Gly domain. It will important to further test the roles of EB1/Bim1 and CLIP-170/Bik1 in this mechanism by generating mutations that specifically disrupt the EEY/F motifs in each protein.

We hypothesize that the role of the CAP-Gly domain of dynactin is to enhance the ability of dynein to produce movement under load by helping the dynein–dynactin complex resist detachment from the microtubule and maintain directional motility. Our model is consistent with the observation that dynactin increases the run length of dynein in in vitro motility assays, and informed by results from optical trap experiments, where purified dynein has been shown to detach or drift backwards on microtubules when subjected to increasing load (15, 16, 37, 38). Based on this model, we predict that the CAP-Gly domain of dynactin provides a module to tune the dynein motor for tasks that require greater force generation in other cells, including neurons.

In the context of adult motor neurons, what might be the function of the CAP-Gly domain of p150^glued? Dynein–dynactin is required for retrograde transport of organelles through the axon, and it is important for the structure of the neuron. In Drosophila adults, dynein–dynactin stabilizes microtubule networks at neuromuscular junctions (39). During development in mammals, dynein–dynactin promotes axonal outgrowth by organizing microtubule networks in the growth cone, and it positions the nucleus in migrating neuronal precursors (40, 41). To our knowledge, it is not known whether similar roles are needed to maintain the structure of adult motor neurons. Transgenic mice homozygous for the p150^glued G59S mutation die during development, and heterozygotes slowly accumulate defects in motor neuron morphology, neurofilament organization, and neuromuscular junction structure (6–8). Axonal transport appears to be unaffected in the G59S heterozygous adult mice, indicating that not all functions of dynein–dynactin are impaired (7). Our results suggest that the G59S mutation may impair the function of dynein under load, such as during the reorganization of the microtubule cytoskeleton, and we speculate that this defect may disrupt the homeostasis of adult neurons. In the future, our system may provide a framework for rapid testing of protein-expression or small-molecule therapies aimed at restoring dynein–dynactin function in these individuals.

Materials and Methods

Chemicals and reagents were from Fisher Scientific and Sigma-Aldrich, unless stated otherwise.

Yeast Strains and Manipulation.

General yeast manipulation, media, and transformation were performed by standard methods (42). Strains are listed in Table S1. To generate mutants of Nip100 expressed from the endogenous chromosomal locus, we used a site-specific genomic mutagenesis strategy (43). Further details are provided in SI Materials and Methods.

Microscopy and Data Analysis.

Timelapse Z series images of spindle oscillations and free microtubule sliding were captured on an Olympus Bmax-60F microscope equipped with a 1.35NA 100× UPlanApo objective, spinning disc Confocal Scanner Unit (CSU10), Picarro Cyan laser (488 nm), and a Stanford Photonics XR-Mega10 ICCD camera, by using QED software (Media Cybernetics). Image analysis was preformed by using ImageJ. Two-color imaging of GFP-tubulin, and RFP-Nup133 was also carried out on this microscope, by using Picarro Cyan and Cobolt Jive (561 nm) lasers.