© 2000 Oxford University Press
Human Molecular Genetics, 2000, Vol. 9, No. 19 2789–2797
ARTICLE
Huntingtin: an iron-regulated protein essential for
normal nuclear and perinuclear organelles
Paige Hilditch-Maguire, Flavia Trettel, Lucius A. Passani, Anna Auerbach1,
Francesca Persichetti and Marcy E. MacDonald+
Molecular Neurogenetics Unit, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129,
USA and 1Howard Hughes Medical Institute and Skirball Institute for Biomolecular Medicine, Department of Cell
Biology, New York University School of Medicine, New York, NY 10016, USA
Received 7 July 2000; Revised and Accepted 25 September 2000
Huntington’s disease (HD), with its selective neuronal cell loss, is caused by an elongated glutamine tract in
the huntingtin protein. To discover the pathways that are candidates for the protein’s normal and/or abnormal
function, we surveyed 19 classes of organelle in Hdhex4/5/Hdhex4/5 knock-out compared with wild-type embryonic stem cells to identify any that might be affected by huntingtin deficiency. Although the majority did not
differ, dramatic changes in six classes revealed that huntingtin’s function is essential for the normal nuclear
(nucleoli, transcription factor-speckles) and perinuclear membrane (mitochondria, endoplasmic reticulum,
Golgi and recycling endosomes) organelles and for proper regulation of the iron pathway. Moreover, upmodulation by deferoxamine mesylate implicates huntingtin as an iron-response protein. However, excess huntingtin
produced abnormal organelles that resemble the deficiency phenotype, suggesting the importance of huntingtin level to the protein’s normal pathway. Thus, organelles that require huntingtin to function suggest roles
for the protein in RNA biogenesis, trafficking and iron homeostasis to be explored in HD pathogenesis.
INTRODUCTION
Huntingtin is a novel protein which was discovered because of
the elongation of an N-terminal glutamine tract of >37 residues
which triggers the loss of striatal neurons in Huntington’s
disease (HD), a dominantly inherited disorder (1,2). The
expansion confers on the mutant protein a novel attribute (3,4)
that may initiate disease by changing an activity of huntingtin
or an interacting protein, assuming that these are critical to the
targeted neurons. Alternatively, it may act independently of the
protein’s normal activity, perhaps by disrupting the function of
a cellular constituent that is not a normal interactor.
Conservation in evolution (5) suggests an essential function
for the ∼350 kDa protein, although this is not evident from its
novel sequence which features only multiple HEAT protein
interaction domains (6). A broad subcellular distribution,
however, implies a function that may involve multiple intracellular sites. The bulk of the protein resides in the cytoplasm (7–
12), where some is loosely associated with the membrane (7), but
a fraction is also found in the nucleus (11,12). Antibodies have
distinguished alternate versions of the protein that are detected in
distinct subsets of nuclear and cytoplasmic organelles (13,14),
each consistent with a different subset of huntingtin’s binding
+To
partners that have implied roles for huntingtin in RNA
biogenesis (15–18) and in vesicle trafficking (15,19–21).
Homozygous inactivation of the mouse HD gene, Hdh (22–
26) has demonstrated that huntingtin’s function is required for
normal embryonic development, during gastrulation (22–25),
for extra-embryonic tissue (25) and in neurogenesis (26). In
contrast, the protein appears to be dispensable for the growth,
viability (22,23,25) and the neuronal differentiation (27) of
cultured ‘double knock-out’ embryonic stem (ES) cells, but
intriguingly is needed for the production of hematopoietic
progenitor cells (28). Although revealing the protein’s essential nature, these analyses of the consequences of huntingtin
deficiency at the whole animal and cellular levels have not
yielded specific candidate pathways for huntingtin function.
Consequently, we have sought clues to huntingtin’s activity
by the identification of organelles that may require the protein.
We have conducted a comprehensive survey of the consequences of huntingtin deficiency at the subcellular level,
comparing Hdhex4/5/Hdhex4/5 knock-out and wild-type ES cells.
We have assessed 19 classes of organelle and have found 6 that
require huntingtin for normal morphology and function. These
organelles reveal a role for huntingtin in the response to
hypoxia and also implicate huntingtin function in specific
cellular processes that can be investigated in HD pathogenesis.
whom correspondence should be addressed. Tel: +1 617 726 5089; Fax: +1 617 726 5735; Email: [email protected]
2790 Human Molecular Genetics, 2000, Vol. 9, No. 19
Table 1. Summary of wild-type and Hdhex4/5/Hdhex4/5 ES cell organelle
survey results
Marker protein/lectin Organelles with staining pattern in wild-type
and Hdhex4/5/Hdhex4/5 ES cells that are:
Similar
Different
Fibrillarin
HYPA/FBP-11
Nucleoli
Nuclear splicing factorspeckles
40
15,16
HYPB
HYPI/symplekin
Ref.
Nuclear transcription 16
factor-speckles
Nuclear coil bodies
N-CoR
15,18
Nuclear transcription 17
factor-speckles
NuMA
Nuclear matrix
40
Lamin A, lamin B,
syntaxin 1A
Nuclear membrane
40
γ-tubulin
Centrosome
40
α-tubulin, dynein
Microtubule
cytoskeleton
40
Actin
Actin cytoskleton
40
Calveolin
Plasma membrane, nonclathrin vesicles
40
LDL receptor
Early, sorting, late
endosomes lysosomes
40
Rab5a
Early endosomes
β-COP, GM130,
VVL, Arf1
Transferrin receptor
ConA
40
Golgi apparatus
Early, sorting
endosomes
40
Perinuclear recycling 40
endosomes
Endoplasmic reticulum 40
Figure 1. Nuclear defects in Hdhex4/5/Hdhex4/5 ES cells: collapsed nucleoli and
mislocalization. Wild-type (WT) and Hdhex4/5/Hdhex4/5 (dKO) ES cells stained
with antibodies to fibrillarin (green) and nuclear envelope protein, lamin A
(red), reveal compact nucleoli in parental cells but collapsed necklaces in the
dKO cells (top). HYPB (BF-1) and NCoR antibodies (white) detect nuclear
speckles in WT and dKO cells, respectively, plus cytoplasmic puncta in dKO
cells only (arrow), denoting mislocalization of huntingtin partners (middle and
bottom). Data were collected and analyzed identically for WT and dKO ES
cells.
RESULTS
Select nuclear and perinuclear organelles are abnormal in
Hdhex4/5/Hdhex4/5 ES cells
To probe huntingtin function we have investigated whether
complete deficiency for this novel protein would perturb
organelles that may require its activity. Consequently, we
compared parental ES cells that express huntingtin by
immunoblot analysis (22) and targeted Hdhex4/5/Hdhex4/5 knockout ES cells which lack the protein (22), using confocal antibody or lectin staining with a total of 24 markers that detect 19
different classes of organelle. The results of this survey are
summarized in Table 1.
For the majority of the markers, Hdhex4/5/Hdhex4/5 and wildtype ES cells exhibited similar staining patterns. The plasma
and nuclear membranes, nuclear coil bodies or splicingspeckles detected by two huntingtin partners, HYPI/symplekin
and HYPA/FBP-11, the cytoskeleton, centrosome and distinct
endosomes (early, sorting, late) or lysosomes all appeared relatively unaffected by the absence of huntingtin. In contrast, 10
of the markers that probed six kinds of organelle exhibited
dramatically different staining patterns. Two of these were
nuclear: nucleoli and transcription-speckles; and four others
were perinuclear: mitochondrial clusters, the endoplasmic
reticulum (ER), Golgi complex and recycling vesicles.
Abnormal nuclear organelles involved in RNA biogenesis
The abnormal marker staining patterns reflected aberrant
organelle morphology, typically a reduced size or an altered
intracellular distribution. This is illustrated for the affected
nuclear organelles in Figure 1. The nucleoli were stained only
weakly for fibrillarin and were collapsed necklaces rather than
robust clusters. These were located within the nuclei bounded
by the lamin A-reactive nuclear envelope. However, transcription factor-speckles defined by huntingtin partners HYPB and
N-CoR, in each case, were abnormally localized to the cytoplasm, indicating that huntingtin is needed for the normal
nuclear localization of these complexes.
As these organelles are involved in rRNA and mRNA
biosynthesis, we tested cellular attributes that are determined
by normal gene expression and protein synthesis. Consistent
with the normal growth properties of the knock-out cells, flow
Human Molecular Genetics, 2000, Vol. 9, No. 19 2791
Figure 2. Leptomycin B blocks nuclear export of AP229-positive huntingtin.
Confocal images of STHdh+/Hdh+ cells stained with AP229 (white) before
(top) or following (bottom) leptomycin B (LMB) treatment. Treatment results
in abrogation of cytoplasmic staining (arrow) which can be seen at low laser
power but is more evident using high laser power.
cytometry indicated that huntingtin deficiency did not alter
either DNA content or cell size, although mini-nuclei were
found in rare Hdhex4/5/Hdhex4/5 but not wild-type ES cells (data
not shown). Thus, although the protein is essential for normal
nucleoli and transcription factor-speckles, huntingtin deficiency
appears not to globally disrupt nuclear function.
Exportin 1-dependent export of nuclear versions of
huntingtin to the cytoplasm
To characterize the nuclear versions of huntingtin that were
implicated by the abnormal nuclear organelles, we tested
whether the export to the cytoplasm might involve exportin
1 (crm1) by treating cells with the inhibitor leptomycin B
(29,30). To assess the nuclear amino-terminal-accessible
version of the protein (14), we first stained wild-type ES cells
with reagent AP229. However, the low level of signal in these
cells was not suited to the confocal format. Consequently, we
examined STHdh+/Hdh+ mouse striatal cells, which exhibit
readily detectable AP229-reactive N-terminal-accessible
protein in splicing-speckles (14). The results indicated that
leptomycin B prevented the cytoplasmic AP229-reactive
speckles that were evident in the untreated cells, indicating
exportin 1-dependent nuclear export (Fig. 2). This implies
nuclear export signals (NES) in huntingtin that are involved in
the nuclear–cytoplasmic localization of the 350 kDa protein.
Abnormal perinuclear mitochondrial clusters
In the cytoplasm our survey detected abnormally distributed
mitochondria in the absence of huntingtin. The results of
staining for Grp75, a mitochondrial matrix protein, are shown
Figure 3. Abnormal distribution of mitochondria in Hdhex4/5/Hdhex4/5 ES cells.
Confocal images of wild-type (WT) and Hdhex4/5/Hdhex4/5 (dKO) ES cells
stained for mitochondrial proteins, Grp75 (white) and α-tubulin (white). Perinuclear clustering of mitochondria in dKO cells is absent (top) despite comparable microtubule distribution in WT and dKO ES cells (bottom). Data were
collected and analyzed identically for WT and dKO ES cells.
in Figure 3. Perinuclear clusters, that are associated with
replication and coordinate transcription of mitochondrial and
nuclear genes involved in energy biogenesis (31), were evident
in wild-type cells. In contrast, Hdhex4/5/Hdhex4/5 ES cells did not
exhibit clusters but instead displayed linear mitochondrial
arrays. These arrays were abundant in all cells, however,
suggesting normal segregation of the mitochondria after cell
division. Consistent with this possibility, staining for α-tubulin
demonstrated that the cytoskeleton and microtubule organizing
center, which are involved in both segregation and perinuclear
cluster formation, were not noticeably altered by huntingtin
deficiency (Fig. 3). This finding implies that the normal
assembly of mitochondria around the nucleus has some
specific requirement for huntingtin.
Abnormal ER and Golgi in the absence of huntingtin
The size of each perinuclear component of the secretory
apparatus was reduced by huntingtin deficiency. Figure 4a
illustrates the hearty perinuclear Golgi clusters detected by
GM130 and by vicia villosa lectin (VVL) in the wild-type
cells. In contrast, the knock-out cells exhibited weak, disperse
Golgi membrane (cis and trans) that were, however, located
near the lamin B-stained nuclear membrane. Perinuclear
signals for the Golgi membrane fusion proteins, β-COP
coatmer protein and Arf1 ADP-ribosylation factor, were also
reduced, suggesting impaired trafficking (data not shown).
Consistent with this possibility, the Concanavalin A (ConA)‘stained’ rough ER (Fig. 4b) appeared to be diminished and did
not properly extend toward the edges of the cell. To directly
test ER–Golgi membrane trafficking, we co-stained cells that
2792 Human Molecular Genetics, 2000, Vol. 9, No. 19
Human Molecular Genetics, 2000, Vol. 9, No. 19 2793
had been treated with Brefeldin A, which is an inhibitor of Arf
activation (32). The results (Fig. 4b) revealed the expected
intermixing of Golgi and ER vesicles in wild-type cells. In
contrast, Hdhex4/5/Hdhex4/5 cells exhibited engorged ConA-filled
ER balloons, ringed by GM130-reactive dots, that indicated
abnormal ER–Golgi membrane fusion.
A re-orientation assay (33) demonstrated impaired perinuclear translocation of the Golgi apparatus in the absence of
huntingtin. The VVL signals in the wild-type ES cells
bordering a scrape in the monolayer are aligned, reflecting a
repositioning of the Golgi to a perinuclear location that is
nearest the extending edge of the cell (Fig. 4c). In contrast, the
knock-out ES cells were unable to rapidly shift their weak
Golgi, indicating that perinuclear membrane trafficking was
impaired.
Abnormal perinuclear recycling endosomes
Perinuclear recycling endosomes were detected by the transferrin receptor, and were also reduced in the cells that lack
huntingtin (Fig. 5a), although an over-abundance of signal was
found throughout the cytoplasm. Brefeldin A treatment to
inhibit membrane fusion revealed diminished perinuclear
membrane in the knock-out cells compared with the wild-type
cells.
To determine whether this deficit was restricted to the
perinuclear recycling endosomes, we tested the uptake and
transport of extracellular FITC-tagged transferrin by ES cells
that had been stimulated by the iron chelator, deferoxamine
mesylate (34). The results (Fig. 5b) confirmed trafficking of
FITC–transferrin to both the early and sorting endosomes in
the knock-out ES cells, although a weak perinuclear ligand
signal indicated impaired transport to perinuclear recycling
endosomes compared with the wild-type cells. Furthermore,
consistent with the normal low density lipoprotein (LDL)
receptor staining found in the marker survey, the receptormediated uptake and transport of extracellular Dil-tagged LDL
to the lysosomes via the early–late and sorting endosomes (34)
was indistinguishable in knock-out and wild-type ES cells. Of
the endosomal compartments, the absence of huntingtin affects
primarily the perinuclear recycling endosomes. Thus, huntingtin function is implicated in a perinuclear process that is
essential both for the normal trafficking of secretory
membrane and for the assembly of mitochondria near the
nucleus.
Huntingtin and the cellular iron pathway
The abnormalities in perinuclear transferrin receptor trafficking and mitochondrial cluster-tethering also suggested that
iron metabolism might be abnormal in the absence of huntingtin. Therefore, we tested the levels of transferrin receptor in
naïve and deferoxamine treated ES cells by immunoblot
analyses. Typical results are shown in Figure 6a; these
revealed an expected ∼3.9-fold increase (n = 3) in transferrin
receptor levels in the ‘treated’ compared with the ‘untreated’
wild-type lysate. In contrast, the naïve knock-out cell extract
exhibited a strong band that was ∼4.2 fold (n = 3) increased
compared with the naïve wild-type lysate. Furthermore, this
abnormally increased level was only marginally elevated
(∼1.3-fold; n = 3) by deferoxamine mesylate, implicating huntingtin in the normal regulation of the iron pathway.
Consequently, we assessed the iron modulation of huntingtin
itself by probing the immunoblots in Figure 6a with huntingtin
reagent monoclonal antibody (mAb) 2166 (Fig. 6). The ∼350
kDa protein was not detected in the Hdhex4/5/Hdhex4/5 ES cell
extract as expected (22). However, the huntingtin band
detected in the naïve wild-type proteins was increased with
deferoxamine mesylate treatment by ∼4.5-fold (n = 3), indicating that huntingtin was upregulated by stimulation of the
iron pathway.
We then searched the 5′ and 3′ non-coding regions of the
mouse, rat and human HD genes for canonical CAGUGX
motifs (35) but we failed to find any that were likely to form
the ‘hair-pin’ iron-responsive element (IRE) implicated in the
mRNA stabilization of iron response proteins (35). However,
searches of the MatInspector matrices (http://www.gsf.de/cgibin/matsearch ) with the promoter region sequences (36) identified a core binding site for the HIF-1 hypoxia-inducible transcription factor (AHRARNT). As shown in Figure 6b, this
sequence is conserved in the mouse, the rat and the human HD
homologs, suggesting a hypoxia response element (HRE). This
element is also found in HIF-1 target genes such as those
encoding the glucose transporter and transferrin receptor (37),
suggesting that this hypoxia transcription factor may also coordinately regulate huntingtin levels.
Overexpressed protein produces a phenotype that
resembles Hdh deficiency
To explore whether the level of huntingtin is important for its
cellular pathway in ES cells and in striatal cells that are
targeted in HD, we assessed the impact of excess protein on
organelles that were found to require its function. The wildtype and the double knock-out ES cells and the STHdh+/Hdh+
striatal cells were transiently transfected with HD1-3144Q23,
which drives expression of full-length normal huntingtin (37).
The results of co-staining of huntingtin with HF1 and either
fibrillarin or GM130 reagents to detect nucleoli and Golgi
membrane, respectively, are shown in Figure 7. These images
demonstrated that both the wild-type ES cells and the striatal
cells that overexpressed huntingtin exhibited collapsed
nucleoli and abnormal fragmented Golgi compared with their
untransfected neighbors. In addition, the overexpressed huntingtin also worsened the abnormal organelles that character-
Figure 4. Abnormal Hdhex4/5/Hdhex4/5 ER–Golgi complex reveals aberrant membranes. (a) Fragmented Hdhex4/5/Hdhex4/5 (dKO) membranes are revealed in confocal
images of perinuclear Golgi complexes in wild-type (WT) and dKO cells stained with GM130 (green) and lamin B for nuclear envelope (red) (top), and VVL (red)
(bottom). (b) Abnormal ConA-stained ER (green) in dKO cells fails to extend to the periphery, as in WT cells. Brefeldin A (BFA) treatment in dKO cells induces
aberrant ConA balls (red) ringed with green non-colocalizing GM130-positive membranes (merge). ConA-reactive and GM130-positive membranes in BFAtreated WT cells partially overlap (merge). (c) Wound-healing, VVL-reactive Golgi membranes (red) re-polarize toward the leading edge in WT cells but remain
disorganized in dKO cells. Data were collected and analyzed identically for WT and dKO ES cells.
2794 Human Molecular Genetics, 2000, Vol. 9, No. 19
Figure 6. Huntingtin modulates Tfn R and is itself upregulated by iron depletion. (a) Immunoblot analysis of extracts of wild-type (WT) and Hdhex4/5/
Hdhex4/5 (dKO) ES cells untreated (–) or treated (+) with deferoxamine
mesylate (DM). The blot was probed for transferrin receptor (Tfn R), revealing
upregulated levels in DM-treated wild-type extract. In naïve dKO extract basal
levels were abnormally high and only modestly increased by DM. Staining the
same blot for huntingtin (Httn) with mAb 2166 reveals the ∼350 kDa band in
proteins from untreated WT, but not dKO, cells. The Httn band is dramatically
augmented in extracts from DM-treated WT cells. Equal loading of proteins is
shown by detection of fodrin (Spectrin). (b) Location of a conserved HIF-1
transcription factor binding site (HBS) in the HD promotor region. Shown is
the core HBS (underlined) and preferred flanking DNA sequence identified by
MatInspector version 2.2 in the promoter region (36) upstream of the ATG start
site (+1) in the human (HD) (GenBank accession no. L12392), mouse (Hdh)
(GenBank accession no. L34008) and rat (rhd) (GenBank accession no.
AJ224197) HD genes. Functional HBS sites in the mouse glucose transporter1 gene (GLUT-1) and human and mouse transferrin receptor (TfR) genes from
Lok and Ponka (37) are given below.
Figure 5. Transferrin receptor recycling is compromised in Hdhex4/5/Hdhex4/5
cells. (a) Perinuclear recycling compartment in untreated cells (–BFA),
revealed by antibody stain of endogenous transferrin receptor (Tfn R) (green),
is robust in wild-type (WT) and diminished in Hdhex4/5/Hdhex4/5 (dKO) cells.
Brefeldin A (+BFA) swollen recycling compartment is reduced in dKO cells
compared with WT cells. (b) Functional tracking of early, late and recycling
endosomes in ES cells of Tfn R via FITC-tagged ligand (green) reveals perinuclear foci and cytoplasmic dots in WT cells but only sparse puncta in the
periphery of dKO cells. Trafficking of lysosomal-fated Dil-LDL (red) in WT
and dKO cells is similar, with numerous cytoplasmic puncta. Data were collected and analyzed identically for WT and dKO ES cells.
ized the knock-out ES cells. In these experiments staining for
perinuclear transferrin receptor also demonstrated a reduction
in the recycling endosome compartment in all the cell types
overexpressing huntingtin (data not shown). Thus, overexpressed huntingtin produced a set of nuclear and perinuclear
abnormalities that mirrored huntingtin deficiency, strongly
suggesting a dominant-negative impact on huntingtin’s
pathway that may reflect the overwhelming of a critical
limiting component.
DISCUSSION
Huntingtin’s novel sequence does not predict the protein’s
physiological role or reveal the mechanism by which the
expanded polyglutamine segment in the mutant protein
triggers the selective degeneration of striatal neurons. To
uncover these processes we have conducted a survey to determine which cellular organelles are chiefly affected by the huntingtin deficiency. Our findings indicate that huntingtin is an
iron-regulated protein that is essential for normal nuclear and
perinuclear organelles that implicate the protein in iron
homeostasis, RNA biogenesis and trafficking, providing a
variety of candidates for the protein’s normal and/or abnormal
pathway.
Although abnormal columnar epithelial cells in huntingtindeficient embryos (25) and impaired erythroid progenitors
from knock-out ES cells (28) have previously implied a
connection, our findings demonstrate an essential role for hunt-
Human Molecular Genetics, 2000, Vol. 9, No. 19 2795
Figure 7. Overexpression of huntingtin results in dominant negative phenotypes. Merged confocal images of (a) wild-type (WT) and Hdh ex4/5/Hdhex4/5 (dKO) ES
cells and (b) STHdh+/Hdh+cells, transfected with HD1-3144Q23. Typical cells overexpressing HF1-reactive full-length huntingtin with 23 glutamines (red),
costained for fibrillarin (green) and GM130 (green) to detect nucleoli and Golgi membranes, respectively. Both WT and STHdh+/Hdh+ transfectants which overexpress huntingtin (red) exhibit collapsed fibrillarin-positive nucleoli and fragmented Golgi rather than robust organelles (green) in surrounding untransfected
cells. Overexpression in dKO cells further worsens the aberrant organelle phenotypes. Data were collected and analyzed identically for all WT and dKO ES cells
and striatal cells.
ingtin function in iron homeostasis. Huntingtin was required
for normal regulation of a key iron protein, transferrin
receptor, and in response to iron need was modulated with it.
This may involve an HIF-1 binding site that suggests coordinate regulation of huntingtin with diverse hypoxia response
proteins at the transcriptional level. Interestingly, normal perinuclear mitochondrial clustering also required huntingtin function, implying a role for perinuclear versions of the protein in
properly localizing mitochondria that are importing nuclear
products for the linked energy–iron pathways.
Our survey has also revealed a role for huntingtin function in
normal membrane trafficking of perinuclear portions of the
secretory apparatus (ER, Golgi, recycling endosomes), that
may be the same activity that is involved in mitochondrial
clustering. This role is consistent with results of antibody
localization (7,14) and with a subset of huntingtin-interacting
proteins that participate in membrane function (15,19–21).
Intriguingly, a version of huntingtin with ‘internal-accessible’
epitopes that colocalizes with perinuclear membranes also
resides in the nucleolus (14) and may be important to normal
nucleolar morphology that was uncovered in our survey. Moreover, these locations imply that this form of huntingtin may be
involved in a process that is essential to nucleoli and to the
arrangement of membrane near the nucleus.
The necessity for the function of huntingtin in the normal
nuclear localization of its transcription factor partners (HYPB
and N-coR), however, may involve an alternate version of huntingtin with amino-terminal-accessible epitopes (11,12,14). This
form of the protein colocalizes with nuclear-speckles and the
nuclear matrix, consistent with huntingtin’s pre-mRNA
splicing and polyadenylation complex factors (15–17) that
have supported a role for the protein in RNA biogenesis. Our
data indicate that huntingtin is exported from the nucleus to the
cytoplasm via an exportin 1-dependent pathway. This may
entail conformational properties of huntingtin (14) that may be
involved in masking/unmasking of NES motifs, determining
the proper distribution of huntingtin in the nucleus and the
cytoplasm.
Essential nuclear and perinuclear organelles that require
huntingtin function were also disrupted by excess protein. This
finding suggests that some critical constituent of huntingtin’s
pathway is limiting, implying the importance of regulated
huntingtin levels. Exploration of the protein’s normal and
abnormal functions, therefore, may require accurately expressed
protein. Indeed, the organelles that require huntingtin function
implicate specific pathways involved in essential cellular
processes, including rRNA and mRNA biogenesis, perinuclear
membrane trafficking and iron metabolism, to be investigated
in HD patient tissue and in model systems.
MATERIALS AND METHODS
Cell culture and cell assays
R1 wild-type and Hdhex4/5/Hdhex4/5 ES cells have been described
previously (22) and were maintained on gelatinized dishes in ES
culture medium supplemented with 106 U/l leukemia inhibitory
factor (LIF) (ESGRO; Life Technologies, Gaithersburg, MD).
Twenty-four hours before an uptake experiment the medium was
replaced with fresh ES culture medium containing 4 µM deferoxamine mesylate (Sigma, St Louis, MO). Uptake of fluorescent
transferrin and LDL was performed at steady state levels over
30 min as described previously (37). Brefeldin A (Calbiochem,
La Jolla, CA) treatment (5 µM) was for 90 min. STHdh+/Hdh+
striatal progenitor cells and their growth at 33°C have been
described (14). Leptomycin B treatment was for 2 h (100 ng/ml)
2796 Human Molecular Genetics, 2000, Vol. 9, No. 19
at 33°C. Wounding–healing entailed a toothpick scrape through
a 90% confluent monolayer (33), followed by incubation at 37°C
for 1 h.
Antibodies and fluorescent labels
Ligands used in this study were as follows: fluoroscein-labeled
transferrin, Dil-labeled LDL, Texas Red-conjugated ConA and
biotinylated VVL (Molecular Probes, Eugene, OR). Antibody
reagents used in this study were as follows: GM130, caveolin,
NuMA, symplekin (Transduction Laboratories, San Diego,
CA); lamin A, lamin B, N-CoR, Rabs 1A, 5A and 6 (Santa
Cruz Biotechnology, Santa Cruz, CA); AF-1 for HYPA and
BF-1 for HYPB (16); fibrillarin, actin, α- and γ-tubulin, dynein
and β-COP (Sigma); anti-syntaxin 1A (StressGen Biotechnologies, Victoria, Canada); huntingtin mAb 2166 (Chemicon,
Temecula, CA), HF1 (37) and spectrin (Chemicon); rat mAb
transferrin receptor (Tfn R; Biosource International, Camarillo,
CA). Secondary antibodies conjugated to horseradish peroxidase
or fluorescent labels were from Amersham Lifesciences
(Piscataway, NJ) and Jackson ImmunoResearch (West Grove,
PA), respectively.
Immunofluorescence, fluorescence-activated cell sorting
(FACS) and confocal microscopy
ES cells were fixed in 4% paraformaldehyde, permeabilized
for 5 min in 0.1% Triton X-100 in phosphate-buffered saline
(PBS), treated for 10 min with blocking solution (1% bovine
serum albumin in PBS) and incubated for 90 min in blocking
solution containing primary antibodies/lectins. After several
washes in PBS, cells were incubated for a further 1 h in
blocking solution containing secondary antibodies and then
rinsed in PBS. Cells were examined with a BioRad (Hercules,
CA) MRC-1024 laser confocal microscope using 20× and 40×
objective lenses. Digitized images for each field were saved as
separate files for each channel and were merged using Adobe
PhotoShop.
Transfection
Full-length huntingtin constructs HD1-3144Q23 and HD13144Q113 (40) were introduced into ES cells seeded at densities
of ∼5 × 103 cells per 24-well plate using the SuperFect Transfection kit (Qiagen, Valencia, CA) and immunofluorescence was
examined 72 h after transfection.
Immunoblot analysis
Soluble proteins were extracted from PBS-washed ES cells by
needle sheering in buffer containing 50 mM Tris–Cl pH 7.5,
10% glycerol, 5 mM magnesium acetate, 0.2 mM EDTA and
Complete
Protease
Inhibitors
(Roche
Diagnostics,
Indianapolis, IN), followed by three freeze–thaw cycles and
centrifugation for 2 min at 17 110 g. Supernatants (25 µg) were
boiled for 5 min in Laemmli loading buffer, separated on a 6%
SDS–polyacrylamide gel and transferred onto nitrocellulose
membranes. Proteins were detected by chemiluminescence
(KPL Laboratories, Gaithersburg, MD) following incubation
with primary antibodies and horseradish peroxidaseconjugated secondary antibodies. Quantitation was by
densitometry of transferrin receptor or huntingtin band (on
non-saturating exposures) and normalization to the spectrin
(fodrin) band in the same lanes.
ACKNOWLEDGEMENTS
We thank Drs J.F. Gusella and T. Greenamyre for critical
discussion, Drs A. Bernards and B. Terns for the gifts of Ecadherin and fibrillarin antibodies and Dr M. Yoshida for
leptomycin B. L.A.P. is supported by a fellowship from the
Hereditary Disease Foundation. The research was supported
by NIH grants NS32765 and NS16367 (Huntington’s Disease
Center Without Walls), a grant from the Foundation for the
Care and Cure of Huntington’s Disease and Telethon, Italy.
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