The G59S mutation disrupts the binding of p150Glued to microtubules and EB1
The G59S mutation is located within the highly conserved CAP-Gly domain of the p150Glued
polypeptide, a domain that mediates the binding of dynactin to microtubules. We compared the microtubule binding affinities of wild-type and G59S p150Glued
peptides (). The CAP-Gly domain of wild-type p150Glued
, which spans residues 1–107, bound weakly to microtubules (unpublished data). This 1–107 peptide lacks the serine-rich region of p150Glued
(111–191), which may be required for efficient microtubule binding by CAP-Gly proteins (Hoogenraad et al., 2000
). In contrast, the binding of NH2
-terminal residues 1–333 of the wild-type protein to microtubules was robust, with a Kd
of 1.1 ± 0.2 μM. The 1–333 fragment of p150Glued
carrying the G59S mutation bound to microtubules with a Kd
of 2.6 ± 0.5 μM, indicating a modest decrease in affinity. More striking, however, was the observation that even at saturating microtubule concentrations, only half of the mutant protein was able to bind to microtubules in this assay (). Similar results were observed in experiments with full-length wild-type and G59S p150Glued
Figure 1. The G59S mutation impairs the binding of p150Glued to microtubules. (A) Schematic representation of p150Glued. Glycine 59 lies in the CAP-Gly microtubule binding domain. The 1–107 fragment contains the CAP-Gly domain, and the 1–333 fragment (more ...)
We performed sequential microtubule binding experiments, in which the unbound fraction of G59S p150Glued
(1–333) protein was incubated for a second time with a saturating concentration of microtubules (25 μM), and observed that ~60% of the protein pelleted with microtubules (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200511068/DC1
). These data suggest that there may be a rapid equilibrium between two populations of the mutant polypeptide, one that can bind and one that cannot. Mixing of wild-type and G59S p150Glued
at a 1:1 ratio resulted in 60% of protein pelleting with 25 μM microtubules (Fig. S1 B). These data suggest that mutant protein does not significantly inhibit the binding of wild-type polypeptide to microtubules.
We next investigated the effects of the mutation on the binding of p150Glued
to microtubules in cells. We used transient transfection assays to compare the distribution of wild-type and G59S p150Glued
in COS7 cells as well as MN1 cells, motor neuron–like cells that extend neurites (Salazar-Grueso et al., 1991
; Brooks et al., 1998
). Although endogenous dynactin generally has a punctate cellular localization, with decoration of dynamic microtubule plus ends, overexpression of p150Glued
results in the decoration of the microtubule cytoskeleton (Waterman-Storer et al., 1995
). As shown in , 24–48 h after transfection of GFP-tagged full-length constructs of wild-type p150Glued
, there was decoration of microtubules, as assessed by colocalization with tubulin. In contrast, GFP-tagged full-length G59S p150Glued
was distributed diffusely in the cytoplasm and showed no colocalization with tubulin (). Similar results were obtained using GFP-tagged NH2
-terminal 1–333 constructs of wild-type and G59S p150Glued
, as well as untagged full-length wild-type and G59S p150Glued
constructs (unpublished data). We performed microtubule binding experiments using protein extract from COS7 cells that had been transfected with GFP-tagged, full-length p150Glued
. Almost all of the exogenous polypeptide from wild-type p150Glued
–transfected cells pelleted with taxol-stabilized microtubules. However, only approximately half of the protein from G59S p150Glued
–transfected cells pelleted with microtubules (unpublished data). This observation confirms our in vitro data that only a portion of the G59S p150Glued
protein population may be available for microtubule binding.
-terminal CAP-Gly domain of p150Glued
binds to EB1 (Ligon et al., 2003
). Crystallographic studies demonstrate that the COOH terminus of EB1 contacts p150Glued
in a hydrophobic cleft of the CAP-Gly domain (Hayashi et al., 2005
). We therefore examined the binding of G59S p150Glued
to EB1 using affinity chromatography. The wild-type peptide bound to the EB1 column and was retained until elution with high ionic strength buffer, but the G59S peptide had decreased retention on the column, indicating reduced affinity for EB1 ().
Figure 2. The G59S mutation impairs the binding of p150Glued to EB1 and to microtubule plus ends. (A) Affinity chromatography of in vitro–translated wild-type (WT) or G59S p150Glued (residues 1–333) over an EB1 column. Load (L), flow-through (F), (more ...)
Previous studies have shown that p150Glued
tracks dynamically with growing microtubule ends together with EB1 (Vaughan et al., 1999
). To investigate the effect of the G59S mutation on the localization of p150Glued
to microtubule plus ends, we transfected COS7 cells with GFP-labeled wild-type or G59S p150Glued
. We selected for cells with low levels of expression, as microtubule plus-end tracking behavior is not evident at higher expression levels because of the decoration and bundling of microtubules induced by high levels of exogenous p150Glued
. Wild-type p150Glued
tracked dynamically with growing microtubule ends ( and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200511068/DC1
), whereas the G59S construct showed no microtubule association, even at tips ( and Video 2). In cells with higher levels of expression of the G59S construct, we noted apparent aggregates of misfolded protein, but these aggregates showed no directed movement (Video 2).
The G59S mutation does not alter the structural integrity of the dynactin complex
To study the cellular effects of the G59S mutation in the p150Glued subunit of dynactin, we established fibroblast and lymphoblast cell lines from two symptomatic individuals known to be heterozygous for the G59S missense allele. Control fibroblast cell lines were obtained from two age-matched control individuals, and a control lymphoblast cell line was derived from an age-matched subject.
In these lines, we examined whether the G59S mutation alters the expression of dynein and dynactin. In both lymphoblasts and fibroblasts, quantitative RT-PCR analysis of RNA levels showed no difference in p150Glued transcript levels between cell lines heterozygous for the G59S mutation and control cell lines (). Western blots of protein extract from patient cell lines showed up-regulation of levels p150Glued, but not of dynein or other dynactin subunits, compared with control cell lines ().
Figure 3. Expression of G59S p150Glued does not alter the integrity of the dynactin complex. (A) Quantification of levels of p150Glued RNA in lymphoblast and fibroblast cell lines derived from patients carrying the G59S mutation and unaffected controls, as measured (more ...)
To determine whether the wild-type and mutant proteins are both expressed in cells cultured from patients heterozygous for the G59S mutation, we performed quantitative Western blotting using both a monoclonal antibody to the microtubule binding region of p150Glued, which binds the wild-type protein with a much higher affinity than the mutant protein, and a polyclonal antibody to p150Glued, which recognizes both forms equally well (). Analysis of patient cells indicated that the total level of p150Glued expression (as determined using the polyclonal antibody) is 147 ± 7% the level observed in control cells (). Western blots with the monoclonal antibody demonstrated that patient cells express 82 ± 4% of the wild-type p150Glued that control cells express (). Thus, we estimate that the mutant protein makes up ~44% of the total p150Glued population in patient cells.
To examine the structural integrity of the dynactin complex, we fractionated cell extracts from the patient-derived and control fibroblast cell lines by sucrose density gradient centrifugation. Intact dynactin was observed to sediment at ~19S in both the patient and control samples, consistent with the large size of the multimeric complex. No significant pool of unincorporated p150Glued subunits was observed in the lower S value fractions from either the patient or control cells (), suggesting that expression of the mutant polypeptide does not significantly disrupt dynactin structure and that the mutant polypeptide is incorporated into dynactin in these cells.
The heterozygous G59S mutation in p150Glued does not disrupt dynein/dynactin localization, Golgi morphology, microtubule organization, or spindle assembly
Incorporation of the mutant polypeptide into dynactin might be expected to disrupt dynactin localization in patient-derived cells; however, we observed no change in the cellular localization of dynactin in fibroblasts derived from patients compared with control fibroblasts ( and Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200511068/DC1
). Dynactin was present diffusely in the cytoplasm in a fine, punctate pattern, with no visible dynactin aggregates. We also noted no change in the cellular localization of cytoplasmic dynein, which was also found in a punctate cellular distribution, partially overlapping with dynactin staining in both patient and control cells (), or EB1, which was localized specifically to microtubule tips ().
Figure 4. Dynein, dynactin, and EB1 are localized normally in cells heterozygous for the G59S mutation in p150Glued. Control fibroblasts and fibroblasts from patients heterozygous for the G59S mutation were stained with antibodies to tubulin (MT; red) and dynamitin (more ...)
We examined the effects of the G59S mutation on the integrity of the Golgi and the assembly of the mitotic spindle in the patient-derived fibroblasts. Disruption of dynactin by dynamitin overexpression has been shown to disrupt the Golgi in interphase cells (Burkhardt et al., 1997
) and the mitotic spindle in dividing cells (Echeverri et al., 1996
). However, no gross morphological defects in the organization of the Golgi or the mitotic spindle were evident in patient-derived heterozygous cells under normal growth conditions ( and Fig. S2 B). In addition, no consistent defects in the growth rate were observed in the patient fibroblasts (unpublished data).
The G59S mutation in p150Glued impairs dynactin function
To test the patient fibroblasts for dynactin function, we looked at several dynein/dynactin-dependent processes. Dynactin, as well as dynein and the dynein-interacting protein LIS1, are necessary for directed fibroblast migration (Dujardin et al., 2003
). However, wounded monolayers of patient cells recovered at the same rate as control cells (unpublished data). Aggresome formation has also been shown to be dynein dependent (Johnston et al., 2002
). To test the effect of the mutation on aggresome formation, an androgen receptor containing an expanded polyglutamine repeat that induces inclusion formation (Merry et al., 1998
) was expressed in patient fibroblasts. These fibroblasts formed inclusions at a rate indistinguishable from control cells (unpublished data).
Although a single wild-type copy of the gene for p150Glued
may be sufficient to mediate dynein-dependent processes under normal conditions, conditions of cellular stress may reveal latent effects of the G59S mutation. Nocodazole, a microtubule-depolymerizing drug, causes dispersal of the Golgi. During recovery from nocodazole treatment, microtubules reassemble and the Golgi fragments coalesce near the centrosome in a dynein/dynactin-dependent manner (Corthesy-Theulaz et al., 1992
). Hafezparast et al. (2003)
have shown a slowing in the recovery of the Golgi after nocodazole treatment in fibroblasts cultured from homozygous Loa
mice. Therefore, we assayed the cytoskeletal and organelle recovery rates in heterozygous G59S and control fibroblasts after nocodazole washout. Microtubules were depolymerized and the Golgi body dispersed after 1 h of nocodazole treatment. 1 h after drug washout, microtubules had reassembled in both control and patient-derived cells; however, Golgi complex morphology was significantly different in patient cells. In control cells, 75 ± 2% of cells had an intact Golgi complex, 22 ± 3% of cells had a partially disrupted Golgi complex, and 3 ± 1% of cells had completely disrupted Golgi complex (). In contrast, in patient-derived cells only 46 ± 8% of cells had intact Golgi complexes, whereas 44 ± 5% of cells showed partial disruption and 11 ± 6% of cells showed complete disruption of the Golgi. Golgi reassembly after 24 h was essentially normal in patient-derived fibroblasts (unpublished data), indicating that expression of mutant dynactin slows but does not block the minus end–directed transport of Golgi elements toward the microtubule organizing center.
Figure 5. Cells heterozygous for the G59S mutation in p150Glued have delayed recovery after microtubule depolymerization. Nocodazole washout experiments were performed on patient and control fibroblasts. Cells were treated with nocodazole for 1 h, washed twice (more ...)
We also observed that the localization of EB1 to microtubule plus-end tips was altered in patient cells during nocodazole recovery. After microtubule depolymerization with nocodazole, EB1 demonstrated diffuse cytoplasmic staining. After 30 min of recovery in conditioned growth media, EB1 was localized specifically to the plus ends of microtubules in control cells, forming comet tails that were 1.20 ± 0.06 μm long (). In patient-derived cells, EB1 was not limited to microtubule tips but was also observed to localize along microtubules (). EB1 tail length increased significantly in patient-derived cells, often to >5 μm, although overlap of adjacent microtubules prevented exact measurements of the elongated EB1 tails. These data suggest a defect in the specific localization of EB1 to microtubule plus ends.
To compare these data to a loss of function of dynactin, we used RNA interference to knockdown p150Glued expression levels in HeLa cells by 70–90% (). This knockdown caused dispersal of the Golgi throughout the cell body (). In addition, we observed an increase in the length of EB1 comet tails from 1.08 ± 0.05 μm in mock-transfected cells to 1.28 ± 0.07 μm in cells transfected with small interfering RNA (). The lengthening of EB1 comet tails is similar to what was observed in patient fibroblasts recovering from nocodazole treatment and correlates with a loss of dynactin function.
The G59S mutation leads to aberrant aggregation of p150Glued
In the microtubule binding assays described in , we observed the binding of only half of the mutant p150Glued
polypeptide to microtubules, suggesting that some portion of the mutant protein population is unavailable for binding to microtubules. To investigate this further, we expressed differentially tagged (T7 and His) truncated forms of wild-type and G59S p150Glued
in vitro and performed immunoprecipitation with an antibody to the T7 tag. Although our constructs, which include amino acids 1–333, span part of the first coiled-coil domain of p150Glued
hypothesized to mediate dimerization (Schroer, 2004
), we observed no association of the T7- and His-tagged wild-type polypeptides (). However, we did observe coimmunoprecipitation of the differentially tagged NH2
-terminal G59S constructs. These data suggest that the G59S polypeptide, but not the wild type, has a tendency to self-associate. There was no coimmunoprecipitation after incubation of differentially tagged wild-type and G59S p150Glued
(unpublished data), indicating that the wild-type and G59S proteins do not interact under these conditions.
Figure 6. G59S p150Glued aggregates in vitro and in vivo. (A) His- and T7-tagged constructs of wild-type and G59S p150Glued were coexpressed in vitro. Immunoprecipitations were performed with anti-T7 antibody. The load (L), unbound (U), and immunoprecipitated (IP) (more ...)
We next investigated whether aberrant biochemical species of the G59S p150Glued protein could be isolated from protein extracts of cells overexpressing this protein. COS7 cells were transfected with full-length wild-type or G59S GFP-tagged p150Glued. 24 h after transfection, the extract from these cells was fractionated over a sucrose gradient and analyzed by SDS-PAGE gel electrophoresis and Western blot. In cells transfected with wild-type p150Glued, the peak concentration of dynamitin and endogenous p150Glued was at 19S (). The exogenous p150Glued protein (as determined by the increase in molecular weight that is due to the GFP tag) was present at 19S, as well as at less dense fractions. This indicates that some exogenous protein is incorporated into the dynactin complex but some remains unincorporated in lower molecular weight fractions, most likely because its expression is in excess of the other subunits of dynactin. In contrast, extracts from cells transfected with GFP-tagged G59S p150Glued demonstrated higher molecular weight species in fractions 2–4. This suggests the presence of aggregated forms of G59S p150Glued with a molecular weight well above that of endogenous dynactin (). Endogenous p150Glued and dynamitin are not present in these fractions, indicating that they do not copurify with the aggregated protein. The aggregated protein remains soluble, as we did not observe the formation of detergent-insoluble aggregates (unpublished data).
As shown in , G59S p150Glued
was cytoplasmically dispersed in COS7 cells 24–48 h after transfection, whereas wild-type p150Glued
decorated microtubules. At longer time points, however, we noted a centripetal localization of the proteins. Wild-type p150Glued
became preferentially localized along microtubules in the perinuclear region (). In contrast, G59S p150Glued
localized to inclusions surrounding the nucleus, which may correspond to the aggresomes of misfolded protein described by Johnston et al. (2002)
. These structures were also observed in very highly expressing cells at earlier time points, but their frequency increased with time after transfection (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200511068/DC1
). In some MN1 cells transfected with GFP-tagged G59S p150Glued
, single or multiple inclusions were evident most often in the cell body () and rarely in neurites. They were similar in appearance to those observed in motor neurons from the brainstem of an affected patient (Puls et al., 2005
), and their frequency increased with time after transfection (Fig. S3). Inclusions stained positive for dynein intermediate chain (DIC), the Golgi marker GM130, and the 20S proteasome but not kinesin heavy chain, microtubules, neurofilaments, vimentin, microtubule-associated protein 2, Cu/Zn superoxide dismutase (SOD1), and survival of motor neurons, (unpublished data). Thus, in both neuronal and nonneuronal cells, mutation of the glycine 59 appears to decrease microtubule binding by the p150Glued
CAP-Gly domain and leads to aggregation and inclusion formation by the mutant protein.
Inclusions of mutant protein are granular and associated with mitochondria
To look at the ultrastructure of the inclusions, transfected COS7 cells and MN1 cells were observed by EM. MN1 cells transfected with GFP-tagged full-length G59S p150Glued
and labeled with immunogold showed granular, nonfibrillar inclusions of mutant protein (). Analysis of nonimmunogold-labeled, glutaraldehyde-fixed COS7 cells demonstrated that the inclusions were not membrane bound (). These micrographs show inclusions that look remarkably like the dynein- and dynactin-containing inclusions seen in patient neurons by immunohistochemistry (Puls et al., 2005
Figure 7. The p150Glued inclusions are associated with mitochondria. (A and B) Low-magnification (A) and high-magnification (B) electron micrographs of MN1 cells that have been transfected with GFP-labeled G59S p150Glued and immunolabeled with an antibody to GFP. (more ...)
In these ultrastructural studies, mitochondria frequently surrounded or were contained within the G59S p150Glued
inclusions (). To examine the possibility that mitochondria localization was altered by the inclusions, COS7 cells were transfected with wild-type or G59S p150Glued
and stained with an antibody to mitochondrial chaperone Hsp60. Mitochondria were partially relocalized in the area of the aggregates (). Quantification of the cross-sectional area of the cells that contained mitochondria demonstrated that mitochondria in cells transfected with G59S p150Glued
were less widely distributed than in cells transfected with wild-type protein (). It may be that mitochondria cannot be transported to the cell periphery because of aberrant interaction with the aggregated G59S p150Glued
. Alternatively, it is possible that loss of dynein/dynactin transport causes mitochondrial mislocalization, as expression of dynamitin has also been shown to cause an inward collapse of the mitochondrial array (Varadi et al., 2004
Expression of G59S p150Glued induces death in neuronal cells
The expression of the G59S polypeptide led to an increase in cell death in MN1 cells, as determined by propidium iodide (PI) exclusion. Cells were transfected with GFP-tagged wild-type p150Glued, G59S p150Glued, or GFP alone. The MN1 cells transfected with G59S p150Glued demonstrated a significantly higher percentage of cell death than cells transfected with wild-type p150Glued or GFP alone (). Furthermore, the percentage of cell death increased with time after transfection, corresponding to an increase in the percentage of cells containing inclusions visible by immunofluorescence (Fig. S3). Embryonic rat motor neurons expressing G59S p150Glued also demonstrated an increase in cell death compared with motor neurons expressing exogenous wild-type p150Glued in a time-dependent manner (Kalb, R.G., personal communication). Neuronal cells may be uniquely sensitive to the G59S polypeptide, as the expression of G59S p150Glued does not increase cell death in COS7 cells ().
Figure 8. Overexpression of Hsp70 decreases both aggregation of G59S p150Glued and MN1 cell death. (A) Quantitation of cell death after transfection with wild-type (WT) or G59S p150Glued or EGFP alone, as determined by PI exclusion. Values represent mean percentage (more ...)
Overexpression of Hsp70 inhibits formation of G59S p150Glued aggregates and prevents cell death
Overexpression of the chaperone Hsp70 has been reported to suppress protein aggregate formation and prevent cell death in several protein misfolding disease models (Barral et al., 2004
). 56 ± 6% of COS7 cells expressing the G59S p150Glued
protein for 2 d contained visible inclusions (). However, cells expressing both Hsp70 and G59S p150Glued
exhibited a disperse localization of both exogenous proteins and only 17 ± 4% of transfected cells contained visible inclusions (). Hsp70 containing the T13G mutation cannot undergo the conformational change necessary for chaperone activity (Sousa and McKay, 1998
). In cells cotransfected with G59S p150Glued
and T13G Hsp70, the proportion of cells containing inclusions was not significantly different from that of cells transfected with G59S p150Glued
alone (). A Western blot of the cell protein lysates showed that levels of G59S p150Glued
were decreased when wild-type, but not T13G, Hsp70 was coexpressed (). The chaperone function of Hsp70 may aid proper folding of G59S p150Glued
, thereby avoiding the formation of inclusions and allowing effective degradation of the mutant protein by the ubiquitin–proteasome pathway.
Transfection of G59S p150Glued into MN1 cells led to an increase in cell death compared with cells transfected with wild-type p150Glued (). However, coexpression of G59S p150Glued and wild-type Hsp70 reduced the percentage of MN1 cell death to levels similar to those of cells transfected with wild-type p150Glued (). This protection was not observed when MN1 cells were cotransfected with G59S p150Glued and either empty vector or T13G Hsp70 (). These data demonstrate that expression of active Hsp70 reduces the amount of G59S p150Glued aggregates, decreases the amount of p150Glued expressed, and protects MN1 cells from the toxicity associated with expression of the mutant p150Glued.