1 Department of Pathology, College of Physicians and Surgeons, Columbia
University, New York, NY 10032, USA
2 Department of Development and Differentiation, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto 606-8507 Japan
3 Department of Cell Biology, Neurobiology and Anatomy, Medical College of
Wisconsin, Milwaukee, WI 53226, USA
4 Department of Developmental Neurobiology, Eunice Kennedy Shriver Center,
University of Massachusetts Medical School, Waltham, MA 02452, USA
* Author for correspondence (e-mail: rv2025{at}columbia.edu)
Accepted 12 September 2002
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Summary |
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Key words: Dynein, Cilium, Microtubule, Retina, Light intermediate chain, Brain
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Introduction |
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A second cytoplasmic dynein HC was identified by PCR-based screening, and
referred to as dhc1b (Gibbons et al.,
1994), dlp4 (Tanaka et al.,
1995
), dhc2 (Vaisberg et al.,
1996
) or dnchc2 (Vaughan et
al., 1996
). It was initially found to be upregulated during
ciliogenesis in sea urchins (Gibbons et
al., 1994
). However, in cultured mammalian cells it was localized
to the Golgi apparatus, which could also be dispersed by microinjection of
anti-dynein 2 HC antibody (Vaisberg et
al., 1996
). Dynein 2 was subsequently implicated in IFT, a
phenomenon discovered in the green alga Chlamydomonas
(Kozminski et al., 1993
). In
IFT, assembled `rafts' of protein complexes (IFT particles) composed of some17
polypeptides are translocated within the space between the plasma membrane and
outer doublet microtubules of cilia and flagella
(Cole et al., 1998
;
Rosenbaum et al., 1999
). A
mutation in the dynein 2 HC gene interferes with retrograde movement of the
IFT particles toward the base of the flagella
(Pazour et al., 1999
;
Porter et al., 1999
). In
C. elegans dynein 2 was identified as the CHE-3 gene product
(Wicks et al., 2000
). CHE-3
was also implicated in retrograde IFT within sensory cilia
(Signor et al., 1999a
). In
both systems the heterotrimeric kinesin, kinesin II, is responsible for
anterograde transport of IFT particles
(Kozminski et al., 1995
;
Beech et al., 1996
;
Muresan et al., 1997
;
Nonaka et al., 1998
;
Signor et al., 1999b
).
Here we report the full-length primary structure of rat dynein 2 HC. We also report an association of dynein 2 HC with LIC3, a homologue of LIC1 and LIC2. Dynein 2 was most abundant in ciliated epithelia and in the connecting cilia of photoreceptor cells. Immunocytochemistry of cultured cells revealed a clear staining of primary cilia, but no specific association with the Golgi apparatus. These data favor a predominant role for dynein 2 in transport within ciliated structures in the brain and elsewhere, and indicate that the function of dynein 2 is evolutionarily conserved between vertebrates and invertebrates.
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Materials and Methods |
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Antibodies
Rabbit antidynein 2 HC antibody was generated using a bacterially expressed
fragment consisting of the N-terminal 269 amino acids purified by SDS-PAGE.
Rabbit antibody against amino acids 1776 to 2242 of rat brain dynein 1 HC
(Mikami et al., 1993;
Zhang et al., 1993
) was
generated using bacterially expressed polypeptide. For the anti-LIC3
antibodies, the soluble recombinant protein was purified by Ni-NTA column
chromatography (Qiagen, Valencia, CA). For the anti-CGI-53 antibody, the
inclusion body was solubilized with 8 M urea and was purified using a Ni-NTA
column. Anti LIC1 to detect LIC1 and LIC2
(Tynan et al., 2000a
) was
described previously. The K26 monoclonal antibody is directed at an
unidentified epitope of the bovine photoreceptor connecting cilium
(Horst et al., 1990
).
Anti-dynein IC Ab was purchased from Chemicon, Temecula, CA. Rabbit
polyclonal anti-detyrosinated tubulin Ab
(Chapin and Bulinski, 1991) was
kindly provided by Steven J. Chapin and J. Chloë Bulinski (Columbia
University, New York, NY). Anti-Golgi 58K protein mAb and anti-acetylated
-tubulin mAb were purchased from Sigma (St Louis, MO). Anti-digoxygenin
antibody conjugated with alkaline phosphatase was purchased from Roche
Diagnostics, Mannheim, Germany. Cy3-conjugated anti-rabbit IgG for
immunohistochemistry or immunocytochemistry were purchased from Roche
Molecular Biochemicals, Indianapolis, IN or Jackson ImmunoResearch
Laboratories, West Grove, PA, respectively. Alexa 488-conjugated anti-mouse
IgG was purchased from Molecular Probes, Eugene, OR.
Biochemical analysis
Cytosolic extracts were prepared from fresh rat testis after homogenization
in 50 mM PIPES-NaOH, 50 mM HEPES, pH 7.0, containing 2 mM MgCl2, 1
mM EDTA, 1 mM AEBSF, 2 µg/ml leupeptin, 5 µg/ml soybean trypsin
inhibitor, 2 µg/ml aprotinin, and 1 mM benzamidine (extraction buffer) as
modified from Paschal et al. (Paschal et
al., 1987). Immunoprecipitation was carried out using Protein A
Sepharose Beads (Pharmacia, Piscataway, NJ) at 4°C overnight and analyzed
by SDS-PAGE followed by immunoblotting. For sucrose density gradient
centrifugation the cytosolic extract was loaded on a 11 ml 5-20% sucrose
gradient in the extraction buffer without protease inhibitors and centrifuged
for 22 hours at 175,000 g in a Beckman SW41 rotor.
Quantitative image processing was done with ImageJ software (Wayne Rasband,
NIH).
In situ hybridization
In situ hybridization of frozen sections of mouse embryo was performed as
described previously (Saito et al.,
1996). Briefly, after pretreatment (proteinase K, postfixation,
and acetic anhydride), sections were prehybridized for 3 hours and hybridized
with digoxygenin-labelled cRNA probes (1 µg/ml) for 15 hours. Probes used
for in situ hybridization were: an EcoRI(402)-EcoRI(3534)
fragment of dynein 1 cDNA (GenBank accession no. L08505); a
SacI(473)-XbaI(4065) fragment of dynein 2 cDNA; a
full-length SCG10 cDNA (Stein et al.,
1988
). Signals were visualized using anti-digoxygenin antibody
conjugated with alkaline phosphatase and NBT/BCIP (BioRad, Hercules, CA).
Adjacent sections used for sense and anti-sense cRNA probes of dynein 2 were
treated under the same conditions.
Immunohistochemistry
For immunohistochemistry of mouse brain, anesthetized mice were perfused
intracardially with saline and then with ice-cold 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.3). The brains were postfixed 6-15 hours at 4°C,
immersed in 30% sucrose phosphate buffer, and 40 µm thick frozen sections
were cut in the coronal plane with a sledge microtome. Sections were collected
in 0.05 M phosphate buffered saline (PBS) at pH 7.4, washed and incubated
15-18 hours at 4°C in PBS with 2% normal goat serum (NGS-PBS) and 0.3%
Triton X-100 containing diluted primary antisera. Sections were washed with
PBS, incubated with Cy3-conjugated anti-rabbit IgG for 60-90 minutes (1:50 in
NGS-PBS). Sections were mounted onto gelatin-coated slides, counterstained
with bisbenzimide, and placed on coverslips for observation with fluorescence
microscopy.
For immunohistochemistry of the retina, fresh mouse or bovine retinal tissue was placed in Tissue Freezing Medium (Triangle Biomedical Sciences, NC) and quickly frozen in liquid nitrogen with or without prior fixation in 4% paraformaldehyde. Primary antibodies were detected with goat anti-rabbit or goat anti-mouse IgG conjugated with Cy3 (Jackson Laboratories) or Alexa 488. Discrimination of signals for K26 versus dynein 2 HC involved use of conjugated anti-mouse and anti-rabbit antibodies, respectively. For discrimination of two monoclonal antibodies (K26 versus tubulin), we labeled with one monoclonal antibody and a fluorescent anti-mouse antibody, and then repeated the procedure for the second monoclonal antibody using a different fluorophore. Images of cells labeled with more than one fluorophore were pseudocolored and merged using Adobe Photoshop.
Immunocytochemistry
Normal rat kidney epithelial cells (NRK cells) on coverslips were fixed
with 4% paraformaldehyde in 137 mM NaCl, 5 mM KCl, 1.1 mM
Na2HPO4, 0.4 mM KH2PO4, 2 mM
MgCl2, 10 mM EGTA, 5 mM PIPES, and 5.5 mM glucose, pH 6.1
(Wheatley and Wang, 1998) at
37°C, permeabilized on ice in the same buffer containing 0.5% Triton
X-100, air-dried, then blocked in 0.5% BSA in PBS (blocking buffer) for 1 hour
at room temperature. Coverslips were incubated with blocking buffer containing
primary rabbit sera (anti-dynein 2 HC, anti-LIC3 or anti-detyrosinated
tubulin) and either anti-Golgi 58K protein or anti-acetylated
-tubulin
mAb for 1 hour at 37°C, then with Cy3-conjugated anti-rabbit IgG and Alexa
488-conjugated anti-mouse IgG. After two washes in blocking buffer, coverslips
were mounted using Antifade Kit (Molecular Probes) for fluorescent
microscopy.
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Results |
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In the course of our previous studies of LIC1 and LIC2 function
(Tynan et al., 2000a;
Tynan et al., 2000b
), we
identified a related sequence in the database, CGI60
(Lai et al., 2000
;
Mikami et al., 2001
), which we
have termed LIC3. During the late stages of the current project the same
sequence was reported under the name D2LIC
(Grissom et al., 2002
). LIC3
is similar to a C. elegans sequence F02D8.3 throughout its length
(Fig. 1B, right box). LIC3 (350
amino acids) is much shorter than LIC2 (497 amino acids) and exhibits only 24%
identity. However the Smith-Waterman score
(Pearson, 1991
) for LIC3 vs.
LIC2 is 200, highly significant (1.6x10-7) relative to the
score vs. the shuffled LIC2 sequence. Both shared a P-loop motif in the
N-terminus and similarity was observed around this site
(Fig. 1B, left box). In
contrast, a P-loop motif was not observed in the C. elegans gene and
the corresponding region revealed no sequence similarity.
To test which form of dynein LIC3 might associate with, we compared the
behavior of LIC3 with that of dynein 1 and 2. Antibodies prepared against the
dynein 2 HC and LIC3 each recognized a single major band of the expected size
(Fig. 2A). By sucrose density
gradient centrifugation, dynein 1 HC, the 74 kDa dynein 1 IC, and LICs 1 and 2
sedimented very similarly, showing a sharp peak at 20S. Dynein 2 HC and
LIC3 showed a broader distribution with a peak in fractions 6 to 8 (
17S)
and faster sedimenting material extending to the bottom of the gradient,
perhaps associated with membranous or axonemal material
(Fig. 2A; see below). The
profile of LIC3 was similar to that of dynein 2 HC, though a small amount of
free LIC3 could be seen at low S-value. A component of the IFT particles
(CGI-53) had a single major peak (
19S) between those of dynein 1 and
2.
|
We tested further for an interaction between LIC3 and dynein 2 HC by co-immunoprecipitation from testis cytosolic extracts. LIC3 was found at high levels in dynein 2 HC immunoprecipitates (Fig. 2B, right panel). A low, but detectable level of dynein 2 HC was observed in antiLIC3 immunoprecipitates (Fig. 2B, left panel), suggesting that the LIC3 antibody may partially interfere with dynein 2 HC-LIC3 interaction.
Cellular and subcellular localization of the dynein 2 complex
To gain insight into the different functions attributed to dynein 2, and in
view of its relative abundance in juvenile rodent brain
(Tanaka et al., 1995), we
examined its distribution in the nervous system. In newborn and adult mice,
the most striking immunohistochemical staining for dynein 2 HC was observed in
the ependymal layer lining the lateral ventricles
(Fig. 3A,D). Anti-LIC3 Ab
stained the same region (Fig.
3B), consistent with our biochemical evidence for an association
with dynein 2. A low level of staining for both dynein 2 HC and LIC3 was also
observed outside the ependymal layer. Prominent staining of CGI-53 was
observed in the ependymal layer (Fig.
3C).
|
We also studied the distribution of dynein 2 HC mRNA. In developing neural
tube dynein 2 HC mRNA was detected most intensely in the ependymal layer
(Fig. 4A,B), and weaker in the
mantle layer distinguished with the neuronal marker SCG10
[Fig. 4D
(Anderson and Axel, 1985)].
Dynein 1 HC mRNA was detected in the mantle layer and in a narrow band lining
the surface of the central canal (Fig.
4C). Therefore dynein 2 mRNA appears to be expressed not only in
the ependymal cells but also in the neuronal/glial progenitor cells in this
layer.
|
We also examined the expression of dynein 2 HC
(Fig. 4E), dynein 1 HC
(Fig. 4F) and SCG10
(Fig. 4G) mRNA in the olfactory
epithelium. At higher magnification, the distribution of the dynein HC species
differed (Fig. 4H). Dynein 2 HC
mRNA was clearly detected not only in the receptor cells but also in the basal
cells containing precursors of the receptor cells. Dynein 1 HC mRNA was also
detected in the receptor cells and apical region of the supporting cells, but
was absent in the basal cells. The SCG10 mRNA was detected in basal half of
the receptor cells layer, in accordance with recent finding of restricted
expression in immature and olfactory marker protein-negative cells
(Pellier-Monnin et al., 2001).
These data show that dynein 2 HC is expressed in ciliated cell species in the
brain and also in neuronal precursors.
We examined the bovine retina by immunohistochemistry to determine the
association of dynein 2 HC with the immotile connecting cilia. A substantial
level of traffic is thought to occur through these structures, with an
important potential role for dynein 2 in the retrograde direction
(Rosenbaum et al., 1999).
Dynein 2 HC staining was prominent on the proximal side of the connecting
cilium, the structural link between the inner and outer segments
(Fig. 5A,E), often extending
further into the inner segment (Fig.
5D). LIC3 also showed increased staining at the proximal side of
the connecting cilium (Fig.
5B,F). Staining within the connecting cilia was punctate, which
appears consistent with an association with IFT particles (yellow overlap).
Staining sometimes extended into the outer segment
(Fig. 5F), where
anti-acetylated
-tubulin staining revealed the axonemal microtubules to
extend (Fig. 5C).
|
We also conducted immunofluorescence microscopy of NRK cells, which contain
primary cilia (Wheatley and Wang,
1998). This structure (arrows in
Fig. 5G-L) was readily detected
with anti-detyrosinated (Fig.
5G) and anti-acetylated
-tubulin
(Fig. 5H) Abs, which preferably
stain stable microtubules (Chapin and
Bulinski, 1991
; Wheatley,
1995
). Anti dynein 2 HC (Fig.
5I) and anti LIC3 (Fig.
5K) Abs also stained the primary cilia, but in a punctate manner.
Outside the primary cilia, dynein 2 HC was detected diffusely throughout the
cytoplasm at low levels, but clear colocalization with Golgi 58K protein
(Bashour and Bloom, 1998
) was
not observed (Fig. 5M,N).
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Discussion |
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The dynein 2 complex exhibited a peak at 17S
[Fig. 2A
(Vaisberg et al., 1996
)], an
S-value lower than that of the dynein 1 complex, suggesting that the dynein 2
could be single-headed or partly dissociate during purification. That LIC3 is
a subunit of dynein 2 is indicated by the efficiency of cosedimentation
(Fig. 2A) and
co-immunoprecipitation (Fig.
2B), and colocalization with dynein 2 HC (Figs
3,
5). That LIC3 is
physiologically related to dynein 2 is also suggested by recent evidence that
the C. elegans homologue of LIC3 (F02D8.3) and the IFT particle
subunit OSM-6 [39% identical to CGI53
(Collet et al., 1998
)] are
regulated by a common transcription factor DAF-19
(Swoboda et al., 2000
).
Dynein 2 function
Our data favor a role for dynein 2 in IFT in vertebrates. High levels of
expression were observed in the ependymal cells (Figs
3,
4) which bear motile cilia.
Clear staining of immotile connecting cilia
(Fig. 5A,B,D-F) and primary
cilia (Fig. 5I,K) was also
observed. These observations indicate that the dynein 2 complex is generally
present in axonemal structures, and argue against a potential role for dynein
2 in axonemal bending motility. The punctate staining we observe within cilia
is consistent with images from nonvertebrates
(Pazour et al., 1999;
Wicks et al., 2000
), and
suggests an association with IFT rafts, which appear to represent collections
of IFT particles. The presence of dynein 2 in photoreceptor connecting cilia
suggests a role in retrograde transport from the outer to the inner segment.
Recent work has, indeed, demonstrated rapid and massive light-induced
retrograde translocation of transducin between these compartments
(Sokolov et al., 2002
). In
addition, because we also detect dynein 2 in the inner segment, it is possible
that this form of cytoplasmic dynein, like dynein 1
(Tai et al., 2001
), also
participates in apical sorting of rhodopsin to the base of the connecting
cilium.
We did not observe clear evidence for Golgi staining with anti-dynein 2 or
anti-LIC3 antibody even in cells where the antibody clearly recognized cilia
(Fig. 5M,N). We do not
understand the basis for the difference with other studies
(Vaisberg et al., 1996;
Grissom et al., 2002
), which
potentially could reflect differences in sample preparation. Alternatively,
the reported Golgi localization (Vaisberg
et al., 1996
; Grissom et al.,
2002
) and apical staining in cultured tracheal epithelial cells
(Criswell et al., 1996
) could
be related to the accumulation of dynein 2 at the base of axonemal structures
seen in the retina (Fig.
5A,D,E) or Chlamydomonas
(Pazour et al., 1999
).
A role for dynein 2 in undifferentiated cells still remains a possibility.
Dynein 2 mRNA was more abundant in newborn than in adult murine brain
(Tanaka et al., 1995),
suggesting a potential role during development. We observed dynein 2 mRNA
within the neuroblasts of the developing neural tube
(Fig. 4A) and in the basal
cells of olfactory epithelium (Fig.
4E,H). What the function of dynein 2 might be in these cells is
uncertain. It could participate in Golgi organization, some novel form of
intracellular protein transport, or early mitotic events. It is possible that
some of the staining in the developing layers could be of epithelial cells
prior to ciliation. In support of this possibility, expression of dynein 2
mRNA has been reported in tracheal epithelial cells prior to induction of
ciliogenesis (Criswell et al.,
1996
).
LIC3 now emerges as a potential candidate for cargo binding during IFT. The
dynein 1 ICs and LICs were absent in dynein 2
(Fig. 2A), as were at least two
dynein LCs, LC8 and Tctex-1 (our unpublished observations). LIC1 has been
implicated in cargo binding during centrosomal assembly, binding to
pericentrin (Tynan et al.,
2000b), which migrates along microtubules toward the centrosome in
a dynein-dependent manner (Young et al.,
2000
). By analogy, LIC3 might interact with a component of the IFT
particles to mediate retrograde transport. Whether this is the case, and how
the interaction with cargo is regulated remain important questions for future
study.
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Acknowledgments |
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