T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 172, No. 5, February 27, 2006 733–745
The microtubule motor cytoplasmic dynein and its activator
dynactin, which mediate minus end–directed movement, have
important roles in both interphase and dividing cells. In inter-
phase cells, the dynein–dynactin complex is essential for ves-
icle and organelle transport, such as ER-to-Golgi vesicular
traffi cking (for review see Schroer, 2004). The dynein–dynactin
motor complex also transports RNA particles (Carson et al.,
2001), aggresomes (Johnston et al., 2002), and virus particles
along microtubules (Dohner et al., 2002). During cell divi-
sion, dynein and dynactin play a critical role in both nuclear
envelope breakdown and spindle formation (for review see
Consistent with these multiple cellular roles, dynein
and dynactin function are required in higher eukaryotes. Loss
of dynein or dynactin is lethal in Drosophila melanogaster
(Gepner et al., 1996), and mice homozygous for loss of cyto-
plasmic dynein heavy chain die early in embryogenesis
(Harada et al., 1998). Cells cultured from dynein heavy
chain–null embryos show fragmented Golgi and a disper-
sal of endosomes and lysosomes throughout the cytoplasm
(Harada et al., 1998).
Neurons appear to be particularly susceptible to defects
in dynein–dynactin complex function. The dominant-negative
mutation in D. melanogaster Glued, which encodes a truncated
form of the p150Glued subunit of dynactin, shows defects that are
most profound in neurons (Harte and Kankel, 1983). Two
N-ethyl-N-nitrosurea–induced point mutations in cytoplasmic
dynein heavy chain cause slowly progressive motor neuron dis-
ease in mice (Hafezparast et al., 2003). Legs at odd angles (Loa)
and Cramping (Cra1) mice each carry missense mutations in a
highly conserved domain of cytoplasmic dynein that mediates
subunit interactions. When homozygous, these mutations are
lethal; heterozygous mice exhibit progressive loss of motor
neurons, leading to muscle weakness and atrophy (Hafezparast
et al., 2003). A similar phenotype is observed in transgenic mice
with a targeted disruption of dynactin in motor neurons
(LaMonte et al., 2002).
<doi>10.1083/jcb.200511068</doi><aid>200511068</aid>A motor neuron disease–associated mutation
in p150Glued perturbs dynactin function
and induces protein aggregation
Jennifer R. Levy,1 Charlotte J. Sumner,2 Juliane P. Caviston,1 Mariko K. Tokito,1 Srikanth Ranganathan,2
Lee A. Ligon,1 Karen E. Wallace,1 Bernadette H. LaMonte,1 George G. Harmison,2 Imke Puls,2
Kenneth H. Fischbeck,2 and Erika L.F. Holzbaur1
1Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
2Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
pressed in eukaryotes, but a G59S mutation in the
p150Glued subunit of dynactin results in the specifi c degen-
eration of motor neurons. This mutation in the conserved
cytoskeleton-associated protein, glycine-rich (CAP-Gly)
domain lowers the affi nity of p150Glued for microtubules
and EB1. Cell lines from patients are morphologically nor-
mal but show delayed recovery after nocodazole treat-
he microtubule motor cytoplasmic dynein and its ac-
tivator dynactin drive vesicular transport and mitotic
spindle organization. Dynactin is ubiquitously ex-
ment, consistent with a subtle disruption of dynein/dynactin
function. The G59S mutation disrupts the folding of the
CAP-Gly domain, resulting in aggregation of the p150Glued
protein both in vitro and in vivo, which is accompanied by
an increase in cell death in a motor neuron cell line. Over-
expression of the chaperone Hsp70 inhibits aggregate
formation and prevents cell death. These data support a
model in which a point mutation in p150Glued causes both
loss of dynein/dynactin function and gain of toxic func-
tion, which together lead to motor neuron cell death.
J.R. Levy and C.J. Sumner contributed equally to this paper.
Correspondence to Erika L.F. Holzbaur: email@example.com
Abbreviations used in this paper: CAP-Gly, cytoskeleton-associated protein,
glycine-rich; DIC, dynein intermediate chain; PI, propidium iodide; SOD1,
The online version of this article contains supplemental material.
JCB • VOLUME 172 • NUMBER 5 • 2006
In humans, a G59S missense mutation has been iden-
tifi ed in the gene encoding p150Glued (DCTN1) in a kin-
dred with slowly progressive motor neuron disease (Puls et
al., 2003). Affected patients develop adult-onset vocal fold
paralysis, facial weakness, and distal-limb muscle weakness
and atrophy. Clinical, electrophysiological, and pathologi-
cal investigations have confi rmed the selective loss of motor
neurons in this disorder (Puls et al., 2005). p150Glued is the
dynactin subunit responsible for binding to dynein and micro-
tubules (Vaughan and Vallee, 1995; Waterman-Storer et al.,
1995). The G59S substitution occurs in the highly conserved
NH2-terminal cytoskeleton-associated protein, glycine-rich
(CAP-Gly) domain, which interacts directly with microtubules
(Waterman-Storer et al., 1995) and the microtubule plus-end
protein EB1 (Ligon et al., 2003).
In this study, we examined the biochemical and cellular
effects of the G59S substitution in p150Glued. Our data sug-
gest that the G59S mutation leads to both decreased micro-
tubule binding and enhanced dynein and dynactin aggregation,
suggesting that both loss of function and toxic gain of func-
tion contribute to the motor neuron degeneration observed in
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 affi nities of wild-type and G59S p150Glued
peptides (Fig. 1 A). The CAP-Gly domain of wild-type p150Glued,
which spans residues 1–107, bound weakly to microtubules (un-
published data). This 1–107 peptide lacks the serine-rich region of
p150Glued (111–191), which may be required for effi cient micro-
tubule 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 affi nity. 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
(Fig. 1 B). Similar results were observed in experiments with full-
length wild-type and G59S p150Glued (unpublished data).
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
contains the CAP-Gly domain, an adjacent ser-
ine-rich domain (residues 111–191), and a
small part of the fi rst predicted coiled-coil do-
main. (B) Wild-type (WT) and G59S p150Glued
were expressed in vitro and incubated with
increasing concentrations of microtubules.
The microtubule bound and unbound proteins
were separated by centrifugation and visual-
ized by SDS-PAGE and fl uorography. The frac-
tion bound, as determined by densitometry,
was plotted against the concentration of tubu-
lin ± SEM and fi tted to a rectangular hyper-
bola. (C) COS7 cells were transfected with
GFP-tagged wild-type (top) or G59S (bottom)
p150Glued. Cells were fi xed after 48 h and
stained for GFP (green) and microtubules (red).
Bar, 10 μm.
MUTANT DYNACTIN CAUSES MOTOR NEURON DISEASE • LEVY ET AL. 735
We performed sequential microtubule binding experi-
ments, in which the unbound fraction of G59S p150Glued
(1–333) protein was incubated for a second time with a satura-
ting 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
signifi cantly inhibit the binding of wild-type polypeptide
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 Fig. 1 C, 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 (Fig.
1 C). 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 microtu-
bules (unpublished data). This observation confi rms our in
vitro data that only a portion of the G59S p150Glued protein
population may be available for microtubule binding.
The NH2-terminal CAP-Gly domain of p150Glued binds
to EB1 (Ligon et al., 2003). Crystallographic studies demon-
strate 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 affi nity chromatography. The wild-type pep-
tide bound to the EB1 column and was retained until elution
with high ionic strength buffer, but the G59S peptide had de-
creased retention on the column, indicating reduced affi nity
for EB1 (Fig. 2 A).
Figure 2. The G59S mutation impairs the binding of p150Glued to EB1
and to microtubule plus ends. (A) Affi nity chromatography of in vitro–
translated wild-type (WT) or G59S p150Glued (residues 1–333) over
an EB1 column. Load (L), fl ow-through (F), wash (W), and eluate frac-
tions (E1 and E2) were analyzed by SDS-PAGE and Western blot us-
ing a polyclonal antibody to p150Glued. There is less G59S p150Glued
in the eluate fractions, in comparison to wild-type p150Glued, indicating
a de-crease in G59S p150Glued affi nity for EB1. (B and C) Live cell fl u-
ores-cence microscopy was used to observe the dynamics and localiza-
tion of p150Glued in COS7 cells expressing low levels of GFP-tagged
wild-type (B) and G59S (C) p150Glued. Bar, 10 μm. B and C show still
images from Videos 1 and 2, respectively (available at http://www.jcb.
Figure 3. Expression of G59S p150Glued does not alter the integrity of
the dynactin complex. (A) Quantifi cation of levels of p150Glued RNA in
lymphoblast and fi broblast cell lines derived from patients carrying the
G59S mutation and unaffected controls, as measured by RT-PCR. n = 3.
(B) Western blot analysis of dynactin expression levels in fi broblast and
lymphoblast cell lines from control individuals (C) and patients heterozy-
gous for the G59S mutation (P). Cell extracts were resolved by SDS-PAGE
and probed for the dynactin subunit dynamitin, as well as DIC and actin.
(C, right) An anti-p150Glued monoclonal antibody (mAb) directed against
the CAP-Gly domain does not recognize in vitro–translated (IVT) G59S
p150Glued. (left) An anti-p150Glued polyclonal antibody (pAb) recognizes
both the wild-type and mutant in vitro–translated protein. (D) Relative
levels of total and wild-type p150Glued expressed in fi broblasts isolated
from patients carrying the G59S mutation. (E) Protein extracts from G59S
and control fi broblast cell lines were sedimented on 5–25% sucrose gra-
dients. The fractions were resolved by SDS-PAGE, and Western blot was
performed using antibodies for the dynactin subunits p150Glued and DIC.
There is no peak of dynactin subunits in the lower density fractions,
indicating that these subunits are incorporated into the dynactin complex.
JCB • VOLUME 172 • NUMBER 5 • 2006
Previous studies have shown that p150Glued tracks dy-
namically 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 exog-
enous p150Glued. Wild-type p150Glued tracked dynamically with
growing microtubule ends (Fig. 2 B and Video 1, available
whereas the G59S construct showed no microtubule associa-
tion, even at tips (Fig. 2 C 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 fi bro-
blast 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 lympho-
blasts and fi broblasts, quantitative RT-PCR analysis of RNA
levels showed no difference in p150Glued transcript levels be-
tween cell lines heterozygous for the G59S mutation and con-
trol cell lines (Fig. 3 A). 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 (Fig. 3, B and D).
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 blot-
ting using both a monoclonal antibody to the microtubule bind-
ing region of p150Glued, which binds the wild-type protein with
a much higher affi nity than the mutant protein, and a polyclonal
antibody to p150Glued, which recognizes both forms equally well
(Fig. 3 C). 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
(Fig. 3 D). Western blots with the monoclonal antibody demon-
strated that patient cells express 82 ± 4% of the wild-type
p150Glued that control cells express (Fig. 3 D). 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 com-
plex, we fractionated cell extracts from the patient-derived
and control fi broblast 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 signifi cant
pool of unincorporated p150Glued subunits was observed in
the lower S value fractions from either the patient or con-
trol cells (Fig. 3 E), suggesting that expression of the mutant
polypeptide does not signifi cantly disrupt dynactin structure
and that the mutant polypeptide is incorporated into dynactin
in these cells.
Figure 4. Dynein, dynactin, and EB1 are localized normally in cells
heterozygous for the G59S mutation in p150Glued. Control fi broblasts and
fi broblasts from patients heterozygous for the G59S mutation were stained
with antibodies to tubulin (MT; red) and dynamitin (A), DIC (B), EB1 (C),
and the Golgi marker GM130 (D). We observed no difference in morphol-
ogy or microtubule organization in control and patient cells. There was a
punctate, cytoplasmic localization of dynein and dynactin and microtubule
tip localization of EB1 in both control and patient cells. The Golgi is intact
and perinuclear in control and patient cells. Bar, 10 μm.
MUTANT DYNACTIN CAUSES MOTOR NEURON DISEASE • LEVY ET AL. 737
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 fi broblasts derived from patients compared with
control fi broblasts (Fig. 4 A and Fig. S2 A, available at http://
was present diffusely in the cytoplasm in a fi ne, 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 overlap-
ping with dynactin staining in both patient and control cells (Fig.
4 B), or EB1, which was localized specifi cally to microtubule
tips (Fig. 4 C).
We examined the effects of the G59S mutation on the in-
tegrity of the Golgi and the assembly of the mitotic spindle in
the patient-derived fi broblasts. Disruption of dynactin by dyna-
mitin overexpression has been shown to disrupt the Golgi in in-
terphase cells (Burkhardt et al., 1997) and the mitotic spindle in
dividing cells (Echeverri et al., 1996). However, no gross mor-
phological defects in the organization of the Golgi or the mitotic
spindle were evident in patient-derived heterozygous cells un-
der normal growth conditions (Fig. 4 D and Fig. S2 B). In addi-
tion, no consistent defects in the growth rate were observed in
the patient fi broblasts (unpublished data).
The G59S mutation in p150Glued impairs
To test the patient fi broblasts for dynactin function, we looked
at several dynein/dynactin-dependent processes. Dynactin, as
well as dynein and the dynein-interacting protein LIS1, are nec-
essary for directed fi broblast migration (Dujardin et al., 2003).
However, wounded monolayers of patient cells recovered at the
same rate as control cells (unpublished data). Aggresome for-
mation 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 poly-
glutamine repeat that induces inclusion formation (Merry et al.,
1998) was expressed in patient fi broblasts. These fi broblasts
formed inclusions at a rate indistinguishable from control cells
Although a single wild-type copy of the gene for p150Glued
may be suffi cient to mediate dynein-dependent processes under
normal conditions, conditions of cellular stress may reveal la-
tent effects of the G59S mutation. Nocodazole, a microtubule-
depolymerizing drug, causes dispersal of the Golgi. During
recovery from nocodazole treatment, microtubules reas-
semble 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 fi bro-
blasts cultured from homozygous Loa mice. Therefore, we
assayed the cytoskeletal and organelle recovery rates in hetero-
zygous G59S and control fi broblasts after nocodazole washout.
Microtubules were depolymerized and the Golgi body dispersed
after 1 h of nocodazole treatment. 1 h after drug washout, mi-
crotubules had reassembled in both control and patient-derived
cells; however, Golgi complex morphology was signifi cantly
different in patient cells. In control cells, 75 ± 2% of cells had
an intact Golgi complex, 22 ± 3% of cells had a partially dis-
rupted Golgi complex, and 3 ± 1% of cells had completely dis-
rupted Golgi complex (Fig. 5, A and B). 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 fi broblasts (unpublished data), indicating that expres-
sion of mutant dynactin slows but does not block the minus
end–directed transport of Golgi elements toward the microtu-
bule organizing center.
We also observed that the localization of EB1 to micro-
tubule plus-end tips was altered in patient cells during no-
codazole recovery. After microtubule depolymerization with
nocodazole, EB1 demonstrated diffuse cytoplasmic staining.
After 30 min of recovery in conditioned growth media, EB1
was localized specifi cally to the plus ends of microtubules in
control cells, forming comet tails that were 1.20 ± 0.06 μm
long (Fig. 5 C). In patient-derived cells, EB1 was not limited to
microtubule tips but was also observed to localize along micro-
tubules (Fig. 5 C). EB1 tail length increased signifi cantly in pa-
tient-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 specifi c 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% (Fig. 5 E). This knockdown
caused dispersal of the Golgi throughout the cell body (Fig. 5 D).
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
(Fig. 5 F). The lengthening of EB1 comet tails is similar to what
was observed in patient fi broblasts 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 Fig. 1, we ob-
served the binding of only half of the mutant p150Glued polypep-
tide 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 fi rst coiled-coil domain of p150Glued hypo-
thesized to mediate dimerization (Schroer, 2004), we observed
no association of the T7- and His-tagged wild-type polypeptides
(Fig. 6 A). 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,
JCB • VOLUME 172 • NUMBER 5 • 2006
has a tendency to self- associate. There was no coimmuno-
precipitation 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.
We next investigated whether aberrant biochemical spe-
cies 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 (Fig. 6 B). 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
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 fi broblasts. Cells were
treated with nocodazole for 1 h, washed twice with PBS, and
returned to normal growth media. (A) After 1 h of recovery,
cells were fi xed and stained for the cis-Golgi marker GM130
(red) and microtubules (green). Control cells have compact
and perinuclear Golgi, but patient cells have partially dis-
rupted Golgi at the same time point after drug washout.
(B) Quantifi cation of Golgi morphology after 1 h of recovery,
± SD (*, P < 0.05; **, P < 0.01). n = 3. (C) After 30 min of
recovery, cells were fi xed and stained for EB1 (red) and micro-
tubules (green). Enlargements of merged images are shown
at the bottom. Control cells show distinct tip localization of
EB1, but patient cells show subtle mislocalization of EB1
along microtubules. (D) HeLa-M cells, either mock-transfected
or transfected with small interfering RNA against p150Glued,
stained with antibodies for EB1 or trans-Golgi marker 46.
(E) Knockdown of p150Glued, compared with cells transfected
with a fl uorescein-labeled, nontargeting oligo or mock-
transfected cells. (F) Quantifi cation of the length of EB1 tails,
± SD (*, P < 0.05). Bars, 10 μm.
MUTANT DYNACTIN CAUSES MOTOR NEURON DISEASE • LEVY ET AL. 739
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 (Fig. 6 B). Endogenous
p150Glued and dynamitin are not present in these fractions, indi-
cating 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).
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) fractions were resolved by SDS-PAGE
and probed with antibodies to the His tag. The G59S construct, though not the wild type, runs as a doublet. We observed coimmunoprecipitation of the
differentially tagged G59S constructs, indicating that the mutant protein self-associates. (B) Lysates from COS7 cells transfected with either GFP-tagged
wild-type (WT) or G59S p150Glued constructs were sedimented on 5–25% sucrose gradients. The fractions were resolved by SDS-PAGE and probed
for the dynactin subunits p150Glued and p50. The histogram indicates the mean percentage of protein in each fraction, as determined in three experiments,
± SEM. G59S p150Glued appears in the high-density fractions at a higher frequency than wild-type p150Glued, which indicates it is incorporated into a
high–molecular weight complex. n = 4. (C) COS7 cells transfected with GFP-tagged wild-type or G59S p150Glued and fi xed after 96 h. Bar, 10 μm.
(D) MN1 cells transfected with wild-type or G59S p150Glued. Bar, 25 μm.
JCB • VOLUME 172 • NUMBER 5 • 2006
As shown in Fig. 1 C, G59S p150Glued was cytoplasmi-
cally 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 local-
ized along microtubules in the perinuclear region (Fig. 6 C). 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 trans-
fection (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 (Fig. 6 D) 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, neu-
rofi laments, 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 micro-
tubule 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 trans-
fected with GFP-tagged full-length G59S p150Glued and labeled
with immunogold showed granular, nonfi brillar inclusions of
mutant protein (Fig. 7, A and B). Analysis of nonimmunogold-
labeled, glutaraldehyde-fi xed COS7 cells demonstrated that the
inclusions were not membrane bound (Fig. 7 C). These micro-
graphs show inclusions that look remarkably like the dynein-
and dynactin-containing inclusions seen in patient neurons by
immunohistochemistry (Puls et al., 2005).
In these ultrastructural studies, mitochondria frequently
surrounded or were contained within the G59S p150Glued inclu-
sions (Fig. 7 C). 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
Figure 7. The p150Glued inclusions are associated with
mitochondria. (A and B) Low-magnifi cation (A) and high-
magnifi cation (B) electron micrographs of MN1 cells that have
been transfected with GFP-labeled G59S p150Glued and immuno -
labeled with an antibody to GFP. Inclusions (i) and nuclei (n)
are labeled. Bars, 500 nm. Inset, immunohis-tochemistry
for DIC was performed on sections from the medulla of an
affected patient. Bar, 10 μm. (C) High-magnifi cation electron
micrograph of a COS7 cell that has been transfected with GFP-
labeled G59S p150Glued and fi xed with glutaraldahyde. No
membrane surrounds the inclusion; the visible membrane is a
nuclear envelope. Arrows indicate mitochondria surrounding
and within G59S p150Glued protein inclusions. Bar, 500 nm.
(D and E) COS7 cells transfected with GFP-tagged wild-type
(D) or G59S (E) p150Glued and fi xed and stained using anti-
bodies for p150Glued and Hsp60. Bar, 10 μm. (F) Quantifi ca-
tion of area of cells containing mitochondria in arbitrary units,
± SEM (*, P < 0.05). Wild type, n = 8; G595, n = 12.
MUTANT DYNACTIN CAUSES MOTOR NEURON DISEASE • LEVY ET AL. 741
were partially relocalized in the area of the aggregates (Fig. 7,
D and E). Quantifi cation of the cross-sectional area of the cells
that contained mitochondria demonstrated that mitochondria in
cells transfected with G59S p150Glued were less widely distrib-
uted than in cells transfected with wild-type protein (Fig. 7 F).
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 ex-
pression 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 trans-
fected with G59S p150Glued demonstrated a signifi cantly higher
percentage of cell death than cells transfected with wild-type
p150Glued or GFP alone (Fig. 8 A). Furthermore, the percentage
of cell death increased with time after transfection, correspond-
ing to an increase in the percentage of cells containing inclusions
visible by immunofl uorescence (Fig. S3). Embryonic rat motor
neurons expressing G59S p150Glued also demonstrated an in-
crease in cell death compared with motor neurons expressing ex-
ogenous 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 (Fig. 8 A).
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 (Fig. 8, B and E). However,
cells expressing both Hsp70 and G59S p150Glued exhibited a dis-
perse localization of both exogenous proteins and only 17 ± 4%
of transfected cells contained visible inclusions (Fig. 8, C and E).
Hsp70 containing the T13G mutation cannot undergo the con-
formational 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 signifi cantly different from that of cells transfected
with G59S p150Glued alone (Fig. 8, D and E). A Western blot
of the cell protein lysates showed that levels of G59S p150Glued
were decreased when wild-type, but not T13G, Hsp70 was co-
expressed (Fig. 8 F). The chaperone function of Hsp70 may
aid proper folding of G59S p150Glued, thereby avoiding the for-
mation 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 (Fig. 8 A). 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 (Fig. 8 G). This protection was not observed
when MN1 cells were cotransfected with G59S p150Glued and
either empty vector or T13G Hsp70 (Fig. 8 G). These data dem-
onstrate that expression of active Hsp70 reduces the amount of
Figure 8. Overexpression of Hsp70 decreases both aggregation of G59S
p150Glued and MN1 cell death. (A) Quantitation of cell death after transfec-
tion with wild-type (WT) or G59S p150Glued or EGFP alone, as determined
by PI exclusion. Values represent mean percentage of cell death in three
sets of transfections, ± SEM (*, P < 0.05). n = 3. (B–D) Representative im-
ages of cells transfected with G59S p150Glued alone (B), G59S p150Glued
and wild-type Hsp70 (C), or G59S p150Glued and T13G Hsp70 (D). Cells
were stained with antibodies for p150Glued (green) and the hemagglutinin
tag on the Hsp70 constructs (red). Bar, 10 μm. (E) Quantitation of the
percentage of COS7 cells containing aggregates, ± SD (*, P < 0.05).
n = 2. (F) Expression levels of p150Glued and Hsp70 in lysates from cells
that have been mock-transfected, transfected with G59S alone, and trans-
fected with wild-type or T13G Hsp70. Tubulin was used as a loading
control. (G) Quantitation of mean percentage of cell death in MN1 cells
48 h after transfection with wild-type and G59S p150Glued and wild-type
and T13G Hsp70 or empty vector, ± SEM (*, P < 0.05). Cotransfection
of G59S p150Glued and wild-type Hsp70 protects MN1 cells from death,
but cotransfection of G59S p150Glued with T13G Hsp70 or empty vector
does not. n = 3.
JCB • VOLUME 172 • NUMBER 5 • 2006
G59S p150Glued aggregates, decreases the amount of p150Glued
expressed, and protects MN1 cells from the toxicity associated
with expression of the mutant p150Glued.
A key question in the analysis of many neurodegenerative dis-
eases is the cell-type specifi city observed: why would a mu-
tation in a ubiquitously expressed protein preferentially affect
a single cell type? This question is particularly critical in the
study of motor neuron diseases, such as Amyotrophic Lateral
Sclerosis, in which multiple mutations in a ubiquitously ex-
pressed protein, SOD1, result in motor neuron– specifi c degen-
eration and cell death. Several mechanisms have been proposed,
including neuron-specifi c aggregation and defects in axonal
transport (for review see Bruijn et al., 2004).
We focus on the cellular effects of a point mutation in the
p150Glued subunit of dynactin. Dynactin is ubiquitously ex-
pressed in vertebrates, interacting with cytoplasmic dynein to
serve as the major motor for microtubule minus end–directed
transport in the cell. The dynein–dynactin complex is required
for a range of cellular functions, including mitotic spindle as-
sembly, ER-to-Golgi traffi cking, and endosome and lysosome
motility. Although complete loss of dynactin function is there-
fore likely to affect all cell types, patients expressing the G59S
mutation in the p150Glued subunit of dynactin develop an auto-
somal-dominant, slowly progressive degeneration specifi c to
motor neurons (Puls et al., 2003).
The G59S missense mutation results in a subtle impair-
ment of dynactin function. A subtle loss of function in a protein
required for retrograde axonal transport may be suffi cient to in-
duce a slow degeneration of motor neurons. Mice with a tar-
geted disruption in dynactin function or with point mutations in
cytoplasmic dynein heavy chain exhibit a slowly progressive
loss of motor neurons, resulting in muscle atrophy (LaMonte
et al., 2002; Hafezparast et al., 2003).
The G59S mutation also results in a toxic gain of function,
as the G59S polypeptide is prone to aggregate. Parrini et al.
(2005) have shown that evolutionarily conserved glycines in-
hibit aggregation because of their low propensity to form
β structure. The G59S substitution alters a highly conserved
glycine residue within the NH2-terminal CAP-Gly domain of
the protein and is predicted to result in steric crowding and
misfolding of this domain (Puls et al., 2003).
Comparisons of aggregate formation in both neuronal and
nonneuronal cells overexpressing the G59S mutation suggest
that motor neurons are uniquely vulnerable to aggregate forma-
tion, leading to enhanced cell death. One explanation for this
observation is that motor neurons may not express adequate
levels of chaperones to cope with the high levels of misfolded
protein. Recent studies have shown that motor neurons are not
able to up-regulate Hsp70 in response to cellular stress and that
they are particularly vulnerable to depletion of Hsp70 (Robinson
et al., 2005). Consistent with this mechanism, overexpression of
Hsp70 led to decreased aggregations of the G59S polypeptide
and decreased cell death.
The mechanism by which aggregate formation leads to
cell death remains to be determined. However, EM analysis of
the aggregates demonstrates the presence of trapped organelles,
including mitochondria. The aggregates may either actively trap
organelles or passively disrupt microtubule-based transport via
“organelle jams” (for review see Holzbaur, 2004). The seques-
tration of cytoplasmic dynein in these aggregates, as observed
in both transfected cells and patient motor neurons (Puls et al.,
2005), would further disrupt axonal transport. This disruption in
transport is likely to be most deleterious to motor neurons be-
cause of their overall size and extended axons.
Based on our observations, we propose the following
model to explain the motor neuron–specifi c phenotype observed
in patients expressing the G59S mutation in dynactin (Fig. 9).
The mutation leads to a decreased effi ciency in minus end–
directed transport. This subtle loss of function does not signifi -
cantly perturb nonneuronal cells but may be suffi cient to affect
the overall effi ciency of retrograde axonal transport in neurons.
However, the mutation also results in a gain of function, as the
G59S polypeptide has an enhanced propensity to misfold.
Aggregation of the misfolded protein is concentration depen-
dent, and the p150Glued polypeptide is highly expressed in motor
neurons (Melloni et al., 1995; unpublished data). Further, motor
neurons may be specifi cally vulnerable to misfolding and ag-
gregation of the G59S polypeptide because of insuffi cient
expression of molecular chaperones. Finally, both the seques-
tration of active motors and the trapping of organelles by the
p150Glued aggregates will further exacerbate the inhibition of
Together, our data provide the foundation for a testable
model for the cellular mechanisms leading to the motor
neuron–specifi c degeneration observed in patients expressing
the G59S mutation, involving both loss of function and toxic
gain of function. We anticipate that these studies will provide
further insight into the mechanisms by which a mutation in an
essential cellular protein can result in specifi c degeneration of
motor neurons in vivo.
Figure 9. Proposed mechanism of G59S p150Glued–mediated motor
neuron toxicity. G59S p150Glued expression leads to motor neuron
toxicity through three intersecting pathways that lead to cell death. The
mutation causes impaired microtubule and EB1 binding, which leads
to disrupted dynein/dynactin-based transport. In addition, the muta-
tion causes misfolding of the CAP-Gly domain, which leads to aber-
rant self-association. The large proportion of unbound p150Glued, along
with the high expression levels of p150Glued in neurons, leads to a high
cytosolic concentration of misfolded mutant protein resulting in aggre-
gates specifi cally in neurons. This gain of function may induce further
impairment in axonal transport, either by physical blockage of the axon
or by sequestration of dynein and dynactin, leading to motor neuron–
specifi c degeneration.
MUTANT DYNACTIN CAUSES MOTOR NEURON DISEASE • LEVY ET AL. 743
Materials and methods
Wild-type and G59S p150Glued were expressed and labeled with
[35S]methionine using the TNT T7 Quick system (Promega), clarifi ed by
centrifugation at 39,000 g for 30 min, incubated for 30 min at 20°C with
increasing concentrations of microtubules polymerized from purifi ed tubu-
lin (Cytoskeleton, Inc.), and stabilized with paclitaxel (Cytoskeleton, Inc.).
Microtubule bound and unbound proteins were separated by centrifuga-
tion at 39,000 g for 20 min and analyzed by SDS-PAGE and fl uorography.
Results were quantitated by densitometry using NIH ImageJ. Prism Soft-
ware (GraphPad) was used to fi t the binding data to the one-site ligand
binding equation y = Bmax × x/(Kd + x).
Affi nity chromatography and Western blot
Affi nity matrices were prepared by cross-linking recombinant EB1 to
activated CH Sepharose 4B (GE Healthcare) beads at 4 mg/ml ligand.
In vitro–expressed p150Glued was incubated with the EB1-bound beads for
30 min at room temperature. These mixtures were loaded onto a column
and washed extensively with 50 mM Tris and 25 mM KCl, pH 7.4,
with 0.1% Triton X-100. Retained proteins were eluted with 2 M NaCl.
Fractions were analyzed by SDS-PAGE and Western blot.
Cell culture, transfections, immunocytochemistry, and nocodazole
Fibroblast cell lines were derived from forearm punch skin biopsies from
two symptomatic patients with the G59S mutation and from an age-
matched, unaffected control sibling (Priest, 1997). An additional age-
matched control fi broblast line (AG02222) was obtained from the Coriell
Cell Repository. Lymphoblast cell lines were derived from blood samples
from an affected family member carrying the G59S mutation using stan-
dard techniques. Cell lines were established in the Cytogenetics Labora-
tory at Georgetown University.
Fibroblast cell lines were maintained in Hams F-10 culture media
supplemented with 15% fetal bovine serum; lymphoblast cells were
maintained in RPMI media with 10% fetal bovine serum. COS7 cells
(American Type Culture Collection) were maintained as described previ-
ously (Ligon et al., 2003). HeLa-M cells (a gift from A. Peden, Genen-
tech, South San Francisco, CA) were maintained in DME with 10% fetal
bovine serum. For immunofl uorescence assays, cells were grown to
?75% confl uence and then fi xed in –20°C methanol and processed for
immunocytochemistry. Monoclonal antibodies to tubulin (DM1A from
Sigma-Aldrich), hemagglutinin (Sigma-Aldrich), kinesin heavy chain
(Chemicon), neurofi lament (NE14 from Sigma-Aldrich), vimentin (Sigma-
Aldrich), microtubule-associated protein 2 (Sigma-Aldrich), Cu/Zn
SOD1 (StressGen Biotechnologies), survival of motor neurons (BD Bio-
sciences), Golgi protein GM130 (BD Biosciences), cytoplasmic DIC
(Chemicon), EB1 (BD Biosciences), TGN46 (Serotech), Hsp60 and -70
(StressGen Biotechnologies), and dynactin subunits p150Glued and dyna-
mitin (BD Biosciences) were purchased commercially. Affi nity-purifi ed
polyclonal antibodies to p150Glued, Arp1, and DIC have been described
previously (Holleran et al., 1996; Tokito et al., 1996; Ligon et al.,
2001). Immunostaining was visualized with Alexa 350–, 488–, and
594–conjugated secondary antibodies (Invitrogen). Images were ac-
quired on a microscope (DMIRBE; Leica) with a 63× or 100× Plan Apo
objective using OpenLab software (Improvision) and a charged-coupled
device camera (Orca ER; Hamamatsu).
MN1 cells were maintained as described previously (Brooks et al.,
1998) and fi xed in 4% paraformaldehyde at room temperature for 10 min,
permeabilized with 0.1% Triton X-100 in PBS for 10 min, and incubated
with monoclonal antibodies to α-tubulin (clone 2.1 from Sigma-Aldrich) fol-
lowed by Texas red–conjugated secondary antibodies (Jackson Immuno-
Research Laboratories). Deconvoluted images were acquired with a
microscope (Olympus) using DeltaVision software (Applied Precision) on a
Silicon Graphics workstation.
Transient transfection assays were performed using Fugene (Roche)
and plasmids encoding either wild-type or G59S full-length human
p150Glued or truncated constructs of the wild-type and G59S polypeptide
spanning residues 1–333, both fused to GFP and untagged. Hemagglutinin-
tagged Hsp70 constructs were a gift from Y. Argon (University of Pennsyl-
vania, Philadelphia, PA).
For nocodazole recovery assays, patient and control fi broblasts
were treated with nocodazole at 5 μg/ml for 1 h and then allowed to re-
cover for 0, 30, or 60 min or 24 h in conditioned culture media at 37°C
in 5% CO2 before fi xation.
Microtubule plus-end dynamics assay
Live cell time-lapse recordings were performed on transiently transfected
COS7 cells expressing either full-length wild-type or G59S p150Glued or
NH2-terminal residues 1–333 of wild-type or G59S p150Glued, all fused to
GFP. Cells on glass coverslips were sealed in an imaging chamber (FCS2;
Bioptechs) and maintained at 37°C in culture media. Sequential time-lapse
fl uorescent images were acquired at 12-s intervals.
HeLa-M cells were transfected using Oligofectamine (Invitrogen) with
100 nM of a mixture of four RNA duplexes targeting different regions of
human DCTNI (SMARTpool siRNA reagent [Dharmacon]; available from
GenBank/EMBL/DDBJ under accession no. NM_004082): 5′-gaagauc-
gagagacaguu-3′, 5′-cgagcucacuacugacuua-3′, 5′-caugagcgcuccuug-
gauu-3′, and 5′-ggagcgcuguaucguaaga-3′. Cells were methanol fi xed
after 72 h and processed for immunocytochemistry or resuspended in
denaturing sample buffer and processed for Western blot analysis.
RNA extraction and quantitative RT-PCR
RNA was extracted from cells using TRIzol (Invitrogen) and purifi ed
with the RNAeasy clean-up kit according to the manufacturer’s protocol
(QIAGEN), and cDNAs were generated using the High Capacity cDNA
Achieve kit (Applied Biosystems). Quantitative PCR reactions were run in
triplicate using the ABI Prism 7900 sequence detection system (Applied
Biosystems). A forward primer (5′-gaagggcatggcatctttgtg-3′), a reverse
primer (5′-gaagcagaagaatcaggtgtctct-3′), and a fl uorescent probe
(5′-FAM-ccagtcccagatccag-BHQ1-3′) were designed to amplify p150Glued
transcripts. Endogenous controls were simultaneously amplifi ed using com-
mercially available primers (Applied Biosystems). The reactions were per-
formed in triplicate and averaged, and p150Glued Ct values (cycle number
when signal reaches a threshold above background) were corrected for
endogenous control Ct values using the ∆∆Ct method per the Applied
Biosystems User Bulletin 2.
Sucrose density gradient centrifugation
Cells from 3–6 fl asks of patient fi broblasts and 3–6 fl asks of control fi bro-
blasts were washed in PBS, harvested, and homogenized in 20 mM Tris-
HCl, pH 7.4, 2 mM EGTA, and 1 mM EDTA with protease inhibitors
(leupeptin, peptstatin A, N-p-tosyl-L-arginine methyl ester, and PMSF). Triton
X-100 was added to 0.4%, and the homogenate was clarifi ed by low-
speed centrifugation. The resulting supernatant fraction was layered over a
5–25% linear sucrose density gradient and centrifuged at 126,000 g for
16 h. The gradients were eluted in 0.5-ml fractions, which were resolved
by SDS-PAGE, and analyzed by Western blot.
For aggregation assays, His- and T7-tagged constructs of wild-type and
G59S p150Glued were coexpressed in vitro and incubated for 2 h at 30°C.
The reactions were then incubated sequentially with protein A beads to
preclear the extracts, followed by protein A beads with bound monoclonal
antibody to T7 (Novagen). After a 1-h incubation with antibody bound
beads, the beads were isolated by centrifugation; washed four times with
50 mM Tris, pH 7.3, 50 mM NaCl, and 0.1% Triton X-100; and eluted by
boiling in denaturing gel sample buffer. The immunoprecipitates were ana-
lyzed by Western blots probed with antibodies to the His and T7 tags.
Cell death assays
A FACS-based survival assay was used to measure cell death (Taylor et al.,
2003). MN1 or COS7 cells were transfected with pEGFP wild-type and
G59S p150 constructs in 6-well plates. Each transfection condition was
performed in triplicate. After 24, 48, or 72 h, cells were harvested with
trypsin, gently pelleted with centrifugation, and resuspended in 1 ml of
PBS. Cells were stained with 2 μg/ml PI (Sigma-Aldrich) and gently
vortexed. For each sample, 50,000 nongated events were acquired using
a FACSCalibur instrument (BD Biosciences) and Cell Quest software (Becton
Dickinson). GFP fl uorescence was collected in the FL-1 channel, and PI fl uo-
rescence was collected in the FL-3 channel in dot and density blot formats.
Results were expressed as a percentage of PI-positive cells (cell death)
divided by the total number of GFP-positive cells (transfected cells).
MN1 and COS7 cells were plated on permanox chambered slides (Lab-
Tek; Nunc) and transfected with pEGFP wild-type and G59S p150 con-
structs or wild-type and G59S p150Glued (untagged) constructs. 48 h
after transfection, one set of cells (for immunogold labeling) was fi xed
with 4% paraformaldehyde in PBS for 1 h and another set was fi xed in
JCB • VOLUME 172 • NUMBER 5 • 2006
4% glutaraldehyde in cacodylate buffer. Paraformaldehyde-fi xed cells
were washed three times with PBS and then blocked and permeabilized
with 0.1% saponin for 1 h. The p150Glued-GFP–transfected cells were
then incubated with mouse polyclonal GFP antibody (Invitrogen), and
p150Glued-transfected cells were incubated in dynactin polyclonal anti-
body (UP235) followed by anti-mouse or anti-rabbit Nanogold (Nano-
probes). Slides were subjected to staining and silver enhancement as
described previously (Tanner et al., 1996). After dehydration, embed-
ding, and sectioning, samples were examined with an electron micro-
scope (1200EX; JEOL).
Online supplemental material
Fig. S1 shows sequential and mixed microtubule binding of G59S
p150Glued. Fig. S2 shows that the localization of p150Glued and the for-
mation of spindles are normal in cells heterozygous for the G59S muta-
tion in p150Glued. Fig. S3 shows that the percentage of cells containing
G59S p150Glued inclusions increases with time after transfection. Video 1
shows microtubule plus-end tracking of GFP-labeled wild-type p150Glued
in transfected COS7 cells. Video 2 demonstrates a loss of microtubule
tip localization and plus-end tracking of GFP-labeled G59S p150Glued
in transfected COS7 cells. Online supplemental material is available
We gratefully acknowledge the members of the affected family for their willing
participation. We thank Susan Cheng and Virginia Crocker in the National
Institute of Neurological Disorders and Stroke EM Core Facility, Barbara Crain
in the Johns Hopkins Department of Pathology, and Anish Patel and Maura
Strauman for technical contributions.
This work was supported by National Institutes of Health grant
GM48661 (to E.L.F. Holzbaur), National Institutes of Health/National Institute
on Aging training grant T32 AG00255 (to J.R. Levy), National Institute of Neu-
rological Disorders and Stroke Career Transition award K22-NS0048199-01
(to C.J. Sumner), funds from the Intramural Research Program of the National
Institutes of Health (to C.J. Sumner, S. Ranganathan, G. G. Harmison, I. Puls,
and K.H. Fischbeck), and a grant from the Amyotrophic Lateral Sclerosis
Association (to E.L.F. Holzbaur).
Submitted: 17 November 2005
Accepted: 31 January 2006
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