Download Cayman ataxia protein caytaxin is transported by kinesin along

Research Article
4177
Cayman ataxia protein caytaxin is transported by
kinesin along neurites through binding to kinesin light
chains
Takane Aoyama, Suguru Hata, Takeshi Nakao, Yuka Tanigawa, Chio Oka and Masashi Kawaichi*
Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 9 September 2009
Journal of Cell Science 122, 4177-4185 Published by The Company of Biologists 2009
doi:10.1242/jcs.048579
Summary
Deficiency of caytaxin results in hereditary ataxia or dystonia
in humans, mice and rats. Our yeast two-hybrid screen
identified kinesin light chains (KLCs) as caytaxin-binding
proteins. The tetratricopeptide-repeat region of KLC1
recognizes the ELEWED sequence (amino acids 115-120) of
caytaxin. This motif is conserved among BNIP-2 family
members and other KLC-interacting kinesin cargo proteins
such as calsyntenins. Caytaxin associates with kinesin heavy
chains (KHCs) indirectly by binding to KLCs, suggesting that
caytaxin binds to the tetrameric kinesin molecule. In cultured
hippocampal neurons, we found that caytaxin is distributed in
both axons and dendrites in punctate patterns, and it colocalizes
with microtubules and KHC. GFP-caytaxin expressed in
hippocampal neurons is transported at a speed (~1 m/second)
compatible with kinesin movement. Inhibition of kinesin-1 by
Introduction
Cayman-type cerebellar ataxia is a rare autosomal recessive disease.
Patients show marked psychomotor retardation and prominent noneprogressive cerebellar dysfunction manifested by nystagmus,
intention tremor, dysarthric speech and a wide-based ataxic gait
(Brown et al., 1984; Jonson et al., 1978; Nystuen et al., 1996).
Recently, the gene responsible for this disease has been identified
and named ATCAY, which encodes a protein termed caytaxin
(Bomar et al., 2003). Three different mutant alleles, jittery, hesitant
and sidewinder, of the homologous mouse Atcay gene cause similar
neurological abnormalities. Hesitant mice show mild ataxia and
dystonia whereas jittery and sidewinder mice have severe symptoms
and die within 4 weeks of birth (Gilbert et al., 2004; Kapfhamer et
al., 1996). In rats, deficiency of caytaxin results in generalized
dystonia (dt) (Xiao and Ledoux, 2005). Cerebellectomy rescues the
severe motor symptoms and prolongs the lifespan of dt rats
(LeDoux et al., 1993). Electrophysiological and biochemical studies
of dt rats define the olivocerebellar pathway, particularly the
climbing fiber projection to Purkinje cells, as main sites of
dysfunction. The cerebellar cortex of the dt rat shows abnormal
expression of genes involved in phosphatidylinositol signaling
pathways, calcium homeostasis and extracellular matrix interaction
(Xiao et al., 2007). Caytaxin is therefore crucial for cerebellar
function. In addition, the range of symptoms seen in human
patients, including psychomotor retardation, suggests that caytaxin
is also essential for cerebral functions.
Caytaxin protein shows the highest homology to BNIP-2, which
was originally isolated as an interacting protein for the antiapoptotic Bcl-2 protein and the adenovirus E1B 19 kDa protein
dominant-negative KHC decreases the accumulation of caytaxin
in the growth cone. Caytaxin puncta do not coincide with
vesicles containing known kinesin cargos such as APP or JIP-1.
A part of caytaxin, however, colocalizes with mitochondria and
suppression of caytaxin expression by RNAi redistributes
mitochondria away from the distal ends of neurites. These data
indicate that caytaxin binds to kinesin-1 and functions as an
adaptor that mediates intracellular transport of specific cargos,
one of which is the mitochondrion.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/22/4177/DC1
Key words: Atcay, BNIP-2, Cayman ataxia, Kinesin, Kinesin light
chain, Axonal trafficking.
(Boyd et al., 1994). C-terminal regions of caytaxin and BNIP-2
contain a conserved domain known as a SEC14 domain in yeast
and a CRAL-TRIO domain in mammals. The CRAL-TRIO
domains in general bind lipophilic small molecules such as
retinal, vitamin E, squalene and phosphatidylinositol (Panagabko
et al., 2003). However, the CRAL-TRIO domain of BNIP-2, also
known as the BCH domain, has been shown to interact with Cdc42
and p50RhoGAP (Cdc42GAP) (Zhou et al., 2005). Overexpressed
BNIP-2 induces cell elongation, membrane protrusion and
subsequent apoptosis of a variety of non-neuronal cultured cells.
This activity depends on the interaction with Cdc42. Although it
is not known whether the CRAL-TRIO domain of caytaxin binds
small lipophilic molecules, the caytaxin CRAL-TRIO domain has
been reported to associate with a neurotransmitter-producing
enzyme, kidney-type glutaminase (KGA) and the peptidyl-prolyl
isomerase Pin1 in neurons (Buschdorf et al., 2006; Buschdorf et
al., 2008). The physiological and etiological significance of this
binding remains to be elucidated. To understand the function of
caytaxin, we conducted yeast two-hybrid screening and identified
kinesin light chains (KLCs) as binding partners for the N-terminal
region of caytaxin. KLC is a component of kinesin-1, which is a
tetrameric motor protein composed of two KLCs and two kinesin
heavy chains (KHCs). Kinesin-1 is involved in axonal and
dendritic transport of mitochondria; vesicles containing amyloid precursor protein (APP) or apolipoprotein E receptor 2
(ApoER2); the c-Jun N-terminal kinase interacting proteins (JIPs);
other protein complexes; and mRNA granules (Hirokawa and
Takemura, 2005; Salinas et al., 2008). Here we show that caytaxin
is transported by kinesin in neurites. Defects in axonal and
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Journal of Cell Science 122 (22)
dendritic transport could cause ataxia and dystonia in caytaxindeficient animals.
Results
Journal of Cell Science
Binding of caytaxin to kinesin light chains
Yeast two-hybrid screening using full-length caytaxin as bait
isolated KLC1 most frequently from a mouse adult brain cDNA
library (20 clones out of three million clones screened). Two clones
of KLC2 and a single clone of KLC3 were also isolated. Other
proteins isolated several times were RNA polymerase II polypeptide
G (four clones) and Bcl-2-associated transcription factor (two
clones). Considering the nuclear localization of these two proteins,
it seemed implausible that they were interacting partners for
caytaxin, which is a cytoplasmic protein (Buschdorf et al., 2006;
Hayakawa et al., 2007). Another 17 genes were isolated only once.
Quantitative two-hybrid -galactosidase assay using the caytaxin
N-terminal fragment (amino acids 1-170) [caytaxin(1-170)] as bait
showed that the most isolated clones including KLCs bound to
caytaxin(1-170). One exception was inositol-1,4,5-trisphosphate 3kinase A (IP3KA), which did not interact with the N-terminal
caytaxin fragment (data not shown) and seemed to bind to the Cterminal CRAL-TRIO domain of caytaxin.
The KLC1 cDNA clones isolated in this screen ranged in size
from those encoding full-length protein to those with various
truncations in the N-terminal heptad-repeat region, which recognizes
the KHC molecule (Hirokawa and Takemura, 2005). All the clones
retained the intact C-terminal tetratricopeptide (TPR) motifs and a
KLC2 clone contained only the TPR motifs. To confirm the binding
of caytaxin to the TPR region, the full-length KLC1, the KLC1
N-terminal 1-212 amino acid fragment and the C-terminal 177-542
amino acid fragment were produced as GST-fusion proteins in
E. coli and analyzed by GST pull-down assay for binding to fulllength caytaxin produced in HEK293T cells (Fig. 1A). Full-length
KLC1 and the C-terminal TPR motif fragment bound to caytaxin
but the N-terminal heptad repeat fragment did not. Therefore,
caytaxin binds to the TPR region of KLC1. Binding of KLC1 to
caytaxin was also confirmed in HEK293T cells expressing Myctagged KLC1 and FLAG-tagged caytaxin by immunoprecipitation
(Fig. 1B). Furthermore, when endogenous caytaxin was
immunoprecipitated from the lysate of newborn rat brains,
endogenous KHC was detected in the precipitate (Fig. 1C),
suggesting the in vivo interaction of caytaxin with tetrameric
kinesin-1. To confirm the binding of caytaxin and tetrameric
kinesin-1, full-length FLAG-KLC1and a Myc-tagged KHC
derivative (Myc-KIF5A-DN) which lacked the N-terminal motor
domain but retained the ability to associate with KLC (Kimura et
al., 2005) were produced in HEK293T cells and the call lysate was
analyzed by GST pull-down assay for binding to GST-caytaxin(1170) (Fig. 1D). Binding of caytaxin(1-170) and KLC1 was
confirmed by this assay. KIF5A-DN was pulled down only when
it was co-expressed with KLC1, indicating that caytaxin binds to
tetrameric kinesin.
To narrow down the KLC1 binding motif within caytaxin(1-170),
a series of deletion mutants were produced as GST-fusion proteins
and their binding to KLC1 was assayed by GST pull-down.
Deletion of an 18 amino acid segment from residues 109 to 126
abolished the caytaxin binding to KLC1 (Fig. 2A). Replacement of
the three successive amino acids in this segment by alanine residues
further narrowed down the KLC1 interacting region to an ELEWED
sequence. Replacement of ELE to AAA greatly diminished, and
that of WED to AAA completely abolished the binding of
Fig. 1. Caytaxin binds to the TPR region of kinesin light chain 1. (A)GST
pull-down assay of caytaxin binding to the full-length, N-terminal and Cterminal fragments of KLC1. FLAG-tagged caytaxin was expressed in
HEK293T cells. The HEK293T cell lysate was incubated with 5g GST, fulllength GST-KLC1(1-542), GST-KCL1(1-212) or GST-KCL1(177-542).
Caytaxin bound to the GST-fusion proteins was detected by western blot using
anti-FLAG antibody (middle panel). The upper panel shows the structure of
KLC1. The lower panel shows Coomassie blue staining of the gel.
(B)Caytaxin binds to KLC1 in HEK293T cells. HEK293T cells were
transfected with expression vectors of either FLAG-caytaxin, KLC1-Myc, or
both. The cell lysate was immunoprecipitated with anti-FLAG antibody (upper
panel) or anti-Myc antibody (middle panel) and the precipitates were analyzed
by western blot with anti-Myc antibody (left panels) or anti-FLAG antibody
(right panels). The lower panel shows the 2% input control. (C)Association of
KHC with caytaxin in the newborn rat brain. The V1 fraction of newborn
brains was incubated with anti-caytaxin polyclonal antibody (second lane) or
non-immune rabbit IgG (first lane) bound to Protein-A-Sepharose beads.
Proteins bound to the beads were analyzed by western blot using anti-KHC
antibody (upper panel) or anti-caytaxin antibody (lower panel). The third lane
shows the 2% input control. (D)KHC binds to caytaxin only in the presence of
KLC1. HEK293T cells were transfected with expression vectors of either
FLAG-KLC1, a Myc-tagged KHC fragment (Myc-KIF5A-DN), or both. The
cell lysate was incubated with GST protein or full-length GST-caytaxin. KLC
and KHC bound to the GST-fusion proteins were detected by western blot
using anti-FLAG antibody (upper panel) or anti-Myc antibody (middle panel),
respectively. The lower panel shows Coomassie blue staining of the gel.
caytaxin(1-170) to KLC1 (Fig. 2B). However, other substitutions
did not affect the interaction between KLC1 and caytaxin(1-170).
When a full-length caytaxin protein with the WED to AAA
mutation and KLC1 were expressed in HEK293T cells, the mutant
caytaxin did not co-immunoprecipitate with KLC1 from the lysate
Journal of Cell Science
Transportation of caytaxin by kinesin
Fig. 2. KLC1 binds to caytaxin at the N-terminal ELEWED sequence.
(A)GST pull-down assay of KLC1 binding to various deletion mutants of
caytaxin(1-170). FLAG-tagged full-length KLC1 was expressed in HEK293T
cells. The lysate of HEK293T cells was incubated with GST-caytaxin
fragments harboring various deletions as indicated in the upper panel. Proteins
bound to the GST-proteins were analyzed by western blot using anti-FLAG
antibody (middle panel). The lower panel shows Coomassie blue staining of
the gel. (B)GST pull-down assay of KLC1 binding to caytaxin(1-170) mutants
having amino acid replacements. The GST-caytaxin(1-170) fragments with
replacement of successive three amino acids between residues 109 and 126
with alanines were incubated with cell lysate of HEK293T cells expressing
FLAG-KLC1. KLC1 bound to the GST-proteins was detected by western blot
(middle panel). wt, wild-type caytaxin(1-170); GST, control GST protein. The
three letters above each lane show the amino acid sequence replaced by
alanines. (C)Binding of caytaxin and KLC1 was abolished by mutation of
WED to AAA. FLAG-tagged full-length caytaxin with the WED to AAA
mutation was expressed along with Myc-tagged KLC1 in HEK293T cells. The
HEK293T cell lysate was immunoprecipitated with anti-FLAG antibody
(upper panels) or anti-Myc antibody (middle panels) and the precipitates were
analyzed by western-blot using anti-Myc (left panels) or anti-FLAG antibody
(right panels). The lower panel shows the 2% input control.
(Fig. 2C), further substantiating the essential function of this
sequence in the interaction with KLC1. The ELEWED motif is
highly conserved among caytaxin homologues from various animals
as well as among BNIP-2 family proteins, such as BNIP-2, BNIPS (BNIPL) and BNIP-XL (Fig. 7). This motif is also conserved
in KLC binding regions of the vaccinia envelope protein A36R and
the neural vesicular transmembrane protein calsyntenin (alcadein),
which are known to be transported by kinesin-1 (Araki et al., 2007;
Konecna et al., 2006; Ward and Moss, 2004).
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Fig. 3. Effects of caytaxin expression on neurite extension. (A)Enhancement
of neurite elongation by overexpression of caytaxin. Hippocampal neurons
were transfected in suspension culture on the day of preparation with plasmid
DNA expressing GFP (upper panel) or caytaxin-GFP (lower panel) and plated
on coverslips. Fluorescent images were taken on 2 DIV. Arrows show the tips
of neurites. Scale bars: 10m. (B)Distribution of axon length of normal and
caytaxin overexpressing neurons. Transfection was carried out as described in
A, except that a plasmid expressing GFP-caytaxin was included. GFP-positive
cells were measured for the length of the longest neurite. Frequency (%) of
cells having the longest neurite of 50m or longer (black), 30-49.9m (gray)
or shorter than 30m (white) were calculated. The average length (m) of the
longest neurites was 41.0±16.5 (mean ± s.d.) for GFP-expressing neurons
(n33), 60.0±17.1 for GFP-caytaxin [n31, P<0.001 (GFP vs GFP-caytaxin)]
and 57.2±22.5 for caytaxin-GFP [n32, P<0.01 (GFP vs caytaxin-GFP)].
(C)Suppression of neurite elongation by expression of caytaxin siRNA.
Primary hippocampal neurons were transfected with a vector expressing
caytaxin siRNA Rmi647864, a control vector expressing LacZ siRNA or an
empty RNAi vector expressing GFP alone on the day of neuron preparation.
GFP-positive cells (n30 for GFP, n24 for LacZ RNAi, n33 for caytaxin
RNAi) were measured for the longest neurite length at 3 DIV. The bars
represent means of neurite length with s.d. shown as vertical lines.
Fluorescence microscope images on the right show GFP-positive siRNAexpressing cells (right panels) and caytaxin expression in these cells (left
panels). Caytaxin expression was suppressed in a GFP-positive caytaxin
siRNA-expressing neuron (arrows in lower panels) compared with
surrounding non-transformed cells, but expression of LacZ siRNA did not
interfere with caytaxin expression (arrows in upper panels). (D)Expression of
caytaxin(1-170) suppressed neurite elongation in a WED-motif-dependent
manner. Cultured hippocampal neurons at 3 DIV were transfected with
plasmid expressing FLAG-caytaxin(1-170) of either wild-type (caytaxin-N) or
mutant with the WED sequence replaced with alanines (caytaxin-N WED). As
a control, FLAG-tagged LacZ (LacZ) was also expressed. Cells were
immunostained with anti-FLAG antibody at 7 DIV and measured for the
length of the longest neurites (n30 for LacZ, n30 for caytaxin-N, and n22
for caytaxin-N WED). The bars represent means of neurite length with s.d.
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Journal of Cell Science
Effects of caytaxin on extension of neurites
Overexpression of BNIP-2 induces cell elongation and membrane
protrusion in non-neural cells (Zhou et al., 2005). Overexpression of
BNIP-S results in cell rounding and apoptosis (Zhou et al., 2006).
These effects have been attributed to the C-terminal CRAL-TRIO
domains of these proteins. We examined the effects of caytaxin
overexpression on the shape of hippocampal neurons (Fig. 3).
Primary hippocampal neurons were transfected with GFP-caytaxin
expression vectors on the day of preparation. On day 2 of in vitro
culture (2 DIV), caytaxin-expressing cells were analyzed for the length
of the longest neurites. The population of cells having longer neurites
(length ≥50 m) was increased by caytaxin overexpression from
35.5% to 71.0% or 61.3% depending on the position of the fused
GFP polypeptide (GFP-caytaxin, a caytaxin fusion with GFP at the
N-terminus; caytaxin-GFP, a caytaxin fusion with GFP at the Cterminus, respectively) (Fig. 3B). The average length was significantly
increased by the expression of caytaxin from 41.0±16.5 m (mean
± s.d.) to 60.0±17.1 m for GFP-caytaxin or to 57.2±22.5 m for
caytaxin-GFP (Fig. 3B). Overexpression of caytaxin did not affect
the number of neurites or their characteristics as axons or dendrites
(data not shown). By contrast, knockdown of caytaxin expression by
RNAi-inhibited elongation of neurites (Fig. 3C). Two RNAi
molecules designed from different regions of caytaxin had essentially
the same effect (data not shown), and an unrelated RNAi for the LacZ
gene had no effect on the neurite extension (Fig. 3C).
So far, no biological activities have been assigned to the Nterminal regions of caytaxin, BNIP-2 and BNIP-S (Buschdorf et
al., 2006; Qin et al., 2003; Zhou et al., 2005; Zhou et al., 2006).
However, if the binding of caytaxin to KLCs has physiological
significance, overexpression of the caytaxin N-terminal fragment
might exert dominant-negative effects on the endogenous caytaxin.
Actually, when caytaxin(1-170) was overexpressed in primary
hippocampal neurons, neurite extension was compromised (Fig.
3D). However, the WED-to-AAA mutant of caytaxin(1-170) did
not show this inhibitory activity. All these data indicate that
caytaxin has a supportive effect on the elongation of neurites and
binding to KLCs is essential for this activity, suggesting that the
transportation by kinesin-1 is an integral feature of caytaxin.
Transport of caytaxin by kinesin in rat primary hippocampal
neurons
To determine whether caytaxin was transported by kinesin-1, we
expressed caytaxin fused with GFP in rat primary hippocampal
neurons and analyzed cells by time-lapse fluorescence microscopy
(Fig. 4). Expressed GFP-caytaxin protein showed a granular
distribution along the neurites with accumulation at the distal region
of neurites (Fig. 4A). A typical movement of a caytaxin-containing
granule along an axon is shown as a movie in supplementary
material Movie 1. During most of the observation period, the granule
in an axon moved in the anterograde direction, but sometimes the
granule stopped moving and then changed direction (Fig. 4B). For
~72% of the time, migrating granules moved in the anterograde
direction and for 13% of the time they moved in the retrograde
direction (Fig. 4C). The average velocity of the anterograde
movements was 1.23±0.87 m/second (Fig. 4D), with the most
frequent velocity range around 1.0-1.5 m/second (Fig. 4E). The
retrograde velocity (0.842±0.51 m/second) did not differ very
much from the anterograde velocity. These velocities were consistent
with the speed of the fast axonal flow as well as with the speed of
microtubule-dependent movement of kinesin motors (Hirokawa and
Takemura, 2005).
Fig. 4. Caytaxin is transported in neurites by kinesin-1. (A)Distribution of
GFP-caytaxin expressed in hippocampal neurons. Neurons were transfected
with the GFP-caytaxin expression plasmid at 2 DIV. Neurons were examined
24 hours after transfection. GFP-caytaxin showed granular pattern and
concentrated at the distal part of the neurites (arrows). (B)Time-lapse images
of a GFP-caytaxin-containing granule in an axon. Primary hippocampal
neurons were transfected with the GFP-caytaxin plasmid on 3 DIV and
observed with a time-lapse microscope 24 hours later. Pictures were taken at 5
second intervals. Scale bar: 50m. (C)Frequency (%) of caytaxin granules
moving in axons anterogradely and retrogradely, or in stationary phase. Time
duration of caytaxin granules moving anterogradely and retrogradely, or
staying in stationary phase between the movements were measured.
(D)Average velocity of caytaxin particles moving anterogradely and
retrogradely. (E)Distribution (%) of anterograde and retrograde transport
velocities. (C-E)Fifteen time-lapse images taken at 2 second intervals of each
moving caytaxin granule (n30 granules) were analyzed for the time periods
and velocity of the granule moving anterogradely (open bars) and retrogradely
(closed bars), or staying in stationary phase (shaded bars). (F)A dominantnegative KHC inhibits accumulation of caytaxin in the growth cone. The
primary neurons were transfected with pCAGGS-Myc-KIF5A-DN at 2 DIV.
At 4 DIV, cells were triply stained for expression of KIF5A (rhodamine),
endogenous caytaxin (FITC) and the cytoplasm (CMAC). Fluorescence
intensities of FITC and CMAC in the growth cone were measure for a KIF5Aexpressing cell (n18) and the ratio was calculated. Similarly, the ratio of
FITC and CMAC intensities were measured for a non-transformed cell in the
same field. The ratio for the KIF5-DN expressing cell was divided by the ratio
for the non-transformant (NT). The value (KIF5A-DN/NT) was compared
with the value (LacZ/NT) obtained similarly with the neurons transfected with
-galactosidase (n16) instead of KIF5A-DN. The data are shown as mean ±
s.d. with the value of LacZ/NT set as one. On the right are typical images of
LacZ-expressing (upper three panels) and KIF5A-DN-expressing (lower three
panels) neurons. Insets depict magnified view of the neurite tips indicated by
arrowheads. Scale bars: 20m.
To prove that the transport of caytaxin is mediated by kinesin-1,
we overexpressed a dominant-negative form of KLC1 (KIF5A-DN)
(Kimura et al., 2005) and measured endogenous caytaxin
Transportation of caytaxin by kinesin
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Journal of Cell Science
The well-known kinesin-1 cargo protein APP (amyloid
precursor protein) and the adaptor protein JIP-1b did not colocalize
with caytaxin-positive granules (Fig. 6A-H), indicating that
caytaxin is transported in structures other than vesicles containing
APP or JIP-1b. Other cargoes of kinesin-1 are membranous
organelles such as the endoplasmic reticulum and mitochondria.
To visualize the endoplasmic reticulum, hemagglutinin-epitope
(HA)-tagged JPDI was expressed in hippocampal neurons (Hosoda
et al., 2003). Double staining with anti-HA and anti-caytaxin
antibodies showed that caytaxin did not colocalize with the
endoplasmic reticulum (Fig. 6I-L). By contrast, some of the
caytaxin-positive puncta colocalized with mitochondria, which
were stained with MitoTracker (Fig. 6M-P). We found that the
inner plexiform layer of mouse retina expressed the highest level
of caytaxin (T.A., unpublished results). Immunoelectron
microscopy of the inner plexiform layer showed localization of
caytaxin on the surface of mitochondria (Fig. 6Q), suggesting that
caytaxin tethers mitochondria to kinesin. In fact, when caytaxin
expression was suppressed by RNAi expression, the number of
MitoTracker-stained particles in the distal part of neurites within
25 m of the neurite tip was significantly diminished (Fig. 6R,S).
These data suggest that caytaxin is involved in the distribution of
mitochondria to the distal ends of neurites.
Discussion
Binding of the caytaxin WED motif to the TPR domain of KLC
Fig. 5. Distribution of endogenous caytaxin in hippocampal neurons. Primary
hippocampal neurons at 7 DIV were doubly stained with anti-caytaxin and
anti-MAP2 (A-C), anti-Tau-1 (D-F), anti-tubulin (G-J), anti-KHC (K-N) and
anti-synapsin (O-R). The neurons were transfected with a vector expressing a
postsynapse marker, GFP-tagged shank3, at 5 DIV and stained with anticaytaxin antibody at 7 DIV (S-V). Boxed regions in G,K,O and S indicate the
field enlarged in the three panels on the right. Scale bars: 50m (A-F) and
10m (G-V).
accumulated in growth cones (Fig. 4F). Caytaxin in growth cones
was significantly decreased by the expression of KIF5A-DN.
Expression and localization of caytaxin in neurons
To elucidate the destination and cargos of caytaxin transportation,
colocalization of endogenous caytaxin with intracellular structures
in neurites was analyzed by immunofluorescence staining of
primary hippocampal neurons. Endogenous caytaxin was distributed
both in axons that were stained with anti-tau-1 antibody, and in
dendrites stained with anti-MAP2 antibody (Fig. 5A-F). Within the
axonal growth cone, part of endogenous caytaxin colocalized with
microtubules, which serve as tracks for kinesin motors (Fig. 5G-J).
Caytaxin also colocalized with KHCs in the neurite and the growth
cone (Fig. 5K-N). All these data support the idea that caytaxin is
transported by kinesin.
The presynaptic marker synapsin colocalized with caytaxin (Fig.
5O-R). We therefore were transfected hippocampal neurons with
GFP-shank3, which specifically localizes in the postsynapse
(Boeckers et al., 2002). In contrast to the presynaptic marker
synapsin, the shank-3-positive postsynapses were localized in close
proximity to the caytaxin puncta but they did not exactly coincide
(Fig. 5S-V). We can thus confirm that caytaxin is preferentially
concentrated in the presynaptic region, as reported previously
(Hayakawa et al., 2007).
We have shown that caytaxin is bound to KLCs, transported along
the neurites, and partially concentrated in the presynapse. The KLC
TPR domain, which interacts with cargos of kinesin (Hirokawa and
Takemura, 2005), recognizes the ELEWED sequence in the Nterminal region of caytaxin. The ELEWED sequence is highly
conserved among caytaxin, BNIP-2, BNIP-S and BNIP-XL in a
wide range of animals (Fig. 7). Considering the high homology of
the CRAL-TRIO domains, it is quite reasonable to conclude that
these four proteins belong to the same family of proteins whose
functions depend on the interaction with the kinesin motor protein.
Sequences similar to ELEWED are also conserved in other proteins
that are known to bind KLC1 and are transported by kinesin-1
(Fig. 7). Mouse calsyntenin, a neural vesicular membrane protein,
has two cytoplasmic KLC1-TPR-binding segments (KBS1 and
KBS2) that contain amino acid sequences EMDWDD (KBS1) and
QLEWDD (KBS2) (Konecna et al., 2006). Konecna and coworkers showed that amino acid residues M, W and two Ds
following W in KBS1 were crucial for the interaction with KCL1,
but the first E and the third D were less important. Vaccinia virus
A36R protein has a SLIWDN sequence within the KLC1-TPR
interaction domain (Ward and Moss, 2004). Taken together with
the conserved motif in the four BNIP-2 family members, a novel
consensus motif for interaction with the TPR regions of KLCs may
be defined as xxW(E/D)x where  is a hydrophobic amino acid
and x is a preferably acidic amino acid. This ‘WED’ motif could
be reminiscent of WD40 motifs, which also recognize TPR motifs
of various proteins (van der Voorn and Ploegh, 1992).
A TPR-containing ubiquitin E3 ligase, CHIP, interacts with
caytaxin and efficiently ubiquitylates caytaxin, at least in vitro
(Grelle et al., 2006). It has not yet been determined which domains
of caytaxin and CHIP interact each other, but it is likely that the
WED motif of caytaxin recognizes the TPR region of CHIP. If that
is the case, it raises the possibility that the competition between
KLC and CHIP and the consequent ubiquitylation and degradation
of caytaxin exert an intriguing regulation of caytaxin functions.
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Journal of Cell Science
Fig. 6. Colocalization of caytaxin with cargos of
kinesin. Cultured hippocampal neurons at 7 DIV were
stained with anti-caytaxin antibody along with antiAPP (A-D) or MitoTracker (M-P). Hippocampal
neurons at 5 DIV were transfected with plasmid
expressing T7-tagged JIP-1b (E-H) or HA-tagged JPDI
(I-L) and doubly stained with anti-caytaxin and
corresponding anti-tag antibodies at 7 DIV. Scale bars:
10m. (Q)Immunoelectron microscopy of the retinal
inner plexiform layer (right panel). Caytaxin
immunoreactivity was localized on or near the
mitochondrial surface, which consists of two layers of
membranes (arrowheads). The left panel shows the
structure of the area corresponding to that of the right
panel as revealed with an ultra-thin section without
antibody treatment. (R,S)Suppression of caytaxin
expression redistributes mitochondria away from the
distal regions of neurites. Caytaxin siRNA Rmi647864
and control LacZ siRNA were expressed. Neurons were
stained with MitoTracker at 3 DIV and the number of
MitoTracker-positive particles in the distal neurite
region within 25m from the neurite tip were counted
in GFP-positive siRNA-expressing cells (white bars,
n22 for LacZ RNAi, n21 for caytaxin RNAi) and
surrounding non-transfectants (black bars, n22 for
LacZ RNAi, n21 for caytaxin RNAi). The data are
shown as mean with s.d. indicated as vertical lines.
Typical images are shown in S. A GFP-positive
caytaxin siRNA-expressing cell (arrowheads) in the
right panels showed diminished MitoTracker-positive
particles in the region within 25m of the neurite tip
(middle and lower panels in the right) compared with a
GFP-negative neuron (arrows, middle and lower panels
on the left). Scale bars: 25m.
No biological activities had been assigned to the N-terminal
regions of caytaxin, BNIP-2 or BNIP-S (Buschdorf et al.,
2006; Qin et al., 2003; Zhou et al., 2005; Zhou et al., 2006).
When overexpressed in non-neural cells such as MCF-7 cells,
BNIP-2 and BNIP-S induce dramatic morphological changes
and apoptosis (Zhou et al., 2002; Zhou et al., 2005; Zhou et al.,
2006). The C-terminal CRAL-TRIO domains of these proteins
are thought to be solely responsible for these activities.
Overexpression of caytaxin in MCF-7 cells also induced cell
elongation that was indistinguishable from that caused by BNIP2 (Hayakawa et al., 2007). A caytaxin C-terminal fragment that
contained only the CRAL-TRIO domain showed virtually the
same effects (T.A., unpublished data). These results had
suggested seemingly dispensable function of the N-terminal
region of the BNIP-2 family proteins. However, it should be
noted that all these data were derived from overexpression of the
BNIP-2 family proteins, and exact physiological functions of
endogenous BNIP-2 and BNIP-S have been largely unknown.
Overexpression of proteins could complement the requirement
for intracellular trafficking mediated by kinesin motors. We
showed that the N-terminal fragment of caytaxin inhibited
neurite extension of primary hippocampal neurons in a WEDmotif-dependent manner (Fig. 3D), suggesting that binding of
caytaxin to KLCs should have physiological significance.
Another recent finding supports the importance of the Nterminal region of caytaxin. A newly found chemically induced
Atcay mutation allele, wobbly, which shows mild neurological
symptoms, has a T to A transversion in the splice donor site of
intron 4. This wobbly mutation is assumed to cause alternative
splicing that deletes the exon 4 of the Atcay gene and causes inframe splicing between exon 3 and exon 5, resulting in deletion
of amino acids 46-119 (see http://mutagenetix.scripps.edu/).
Because this deletion removes the ELEWED sequence but keeps
the CRAL-TRIO domain intact, the wobbly phenotype could be
caused by aberrant interaction with kinesin that results in
impairment of axonal and dendritic transport.
Transportation of caytaxin by kinesin
4183
suggesting that caytaxin-containing structures are specific vesicles
or unique large protein complexes.
An adaptor, a scaffold or a cargo
Journal of Cell Science
Fig. 7. Conservation of the WED motif among the BNIP-2 family, and
proteins that are bound to KLCs and transported by kinesin. The WED motif is
commonly shared by a group of proteins that are bound to KLCs and
transported by the kinesin-1 motor. The CRAL-TRIO domain of caytaxin (see
Fig. 2A for schematic structure of caytaxin) might bind to protein cargos or to
membranous organelles including transport vesicles and mitochondria directly
through the hydrophobic pocket of the domain (Bomar et al., 2003) or
indirectly through intrinsic membrane proteins of these organelles.
Considering the interaction of the caytaxin CRAL-TRIO domain
with seemingly unrelated proteins such as KGA and Pin1, and the
association of caytaxin directly or indirectly with mitochondria, it
is probable that caytaxin functions as an adaptor that tethers various
cargos to KLCs for transport by kinesin-1. In our two-hybrid
screening, we isolated IP3KA that seemed to bind to the caytaxin
CRAL-TRIO domain (T.A., unpublished data). Differential
expression analysis between dt and wild-type rats identified various
genes that encode proteins in the phosphatidylinositol-calcium
signaling pathways, such as a phosphatidylinositol-binding protein,
inositol-polyphosphate phosphatases and calcium-dependent
ATPase-4, being dysregulated in the cerebellar cortex of the dt rats
(Xiao et al., 2007). Taken together, caytaxin might function as a
scaffold for signaling proteins in the phosphatidylinositol-calcium
signaling pathways. In this sense, caytaxin might have functions
similar to those of JIPs, which also bind to KLCs. JIP-1 associates
with ApoER2 and APP and tethers vesicles containing these
proteins to kinesin-1 via KLC. JIP-1 is also known to function as
a scaffold for kinases of the JNK MAP kinase cascade (Whitmarsh
and Davis, 1998). To clearly understand the physiological function
of caytaxin, it will be essential to determine the complete repertoire
of factors that interact with the caytaxin CRAL-TRIO domain.
Transport of caytaxin by kinesin-1
We showed co-immunoprecipitation of KHCs with caytaxin from
rat brain homogenate (Fig. 1C) and indirect binding of caytaxin to
KHCs in vitro (Fig. 1D), suggesting the binding of caytaxin to
tetrameric kinesin-1, which is the entity that moves along
microtubules. Caytaxin colocalized with both microtubules and
KHCs in the growth cone (Fig. 5G-N), and moves in neurites, mostly
in the anterograde direction at a speed that is compatible with kinesin
transport (Fig. 4B-D). Furthermore, the dominant-negative KHCs
inhibit accumulation of caytaxin in the neurite tips (Fig. 4F). These
data indicate that caytaxin is transported by kinesin-1. In support
of this, Bushdorf and colleagues reported that overexpression of
caytaxin in neurons relocalized KGA from mitochondria to neurite
terminals (Buschdorf et al., 2006). Since KGA is a mitochondrial
enzyme (Curthoys and Watford, 1995), relocalization of this enzyme
could be accounted for by the transport of mitochondria harboring
this enzyme with the caytaxin-kinesin complex. We showed that a
proportion of caytaxin in neurites is colocalized with mitochondria,
which are major cargos of kinesin-1 (Hirokawa and Takemura,
2005) (Fig. 6M-Q). Knockdown of caytaxin expression by RNAi
redistributed mitochondria away from the distal part of neurite (Fig.
6R and S). It is therefore possible that caytaxin binds to mitochondria
directly or indirectly via mitochondrial membrane proteins and
tethers them to kinesin for transport.
Caytaxin-containing granules moved along the neurites mostly
in the anterograde direction, but occasionally they stopped moving
and switched to the retrograde direction. This behavior is compatible
with that observed for calsyntenin-containing organelles (Araki et
al., 2007; Konecna et al., 2006). The distribution (%) of caytaxin
granules moving anterogradely, retrogradely and those in the
stationary phase was also congruous with that reported for
calsyntenin-containing organelles (Araki et al., 2007).
APP and JIP-1 are other major cargos for kinesin-1 (Hirokawa
and Takemura, 2005). Caytaxin did not colocalize with APP or JIP1, nor with calsyntenin-containing vesicles (T.A., unpublished data),
Materials and Methods
Plasmid construction
Mouse and human caytaxin cDNAs were obtained from the mammalian gene
collection, NIH (cDNA clone MGC:56757) and Kazusa DNA Research Institute
(KIAA1872), respectively. The full-length caytaxin cDNA was modified by PCR
amplification using primers that added a BamHI site before the initiation codon. The
resulting fragment was cloned into a pcDNA3 derivative to make pcDNA3-FLAGcaytaxin, which expresses caytaxin protein with a FLAG epitope tag at the N-terminus.
The same cDNA was cloned into pEGFP-C1 (Clontech) to make a vector that
expresses a GFP-caytaxin fusion protein with GFP at the N-terminus of caytaxin.
The full-length caytaxin cDNA was also amplified by PCR using primers that changed
the termination codon into an XhoI site. The resulting fragment was cloned into a
pcDNA3 plasmid derivative that contained GFP cDNA. The product plasmid
expresses a caytaxin-GFP fusion protein with GFP at the C-terminus of caytaxin.
A fragment of cDNA encoding a mouse caytaxin N-terminal 1-170 amino acid
fragment was PCR amplified using primers that replaced codon 171 into a stop codon
and added a SalI site downstream of the new stop codon. The amplified fragment
was cloned into pcDNA3 to make pcDNA3-FLAG-caytaxin(1-170). The same
fragment was cloned into pGEX-5X-3 to express caytaxin(1-170) as a GST-fusion
protein. Caytaxin (1-150), (1-126), (1-108) or (1-90) were similarly amplified by
PCR. A fragment encoding caytaxin(55-170), (91-170), (109-170) or (127-170) was
amplified using primers that introduced a new ATG codon with a BamHI site upstream.
These deletion mutant fragments were cloned in pGEX-5X-3. Mutation of successive
three amino acids in the region of residues 109-126 of caytaxin was achieved with
QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the
manufacturer’s protocol.
pcDNA3-FLAG-KCL1 and pcDNA3-FLAG-TPR that express full-length KLC1
(1-542) and a KCL1 C-terminal fragment (177-542) containing the TPR region,
respectively, as FLAG-tagged proteins, were kind gifts from Katsuji Yoshioka
(Kanazawa University, Kanazawa, Japan). An N-terminal KLC1 fragment encoding
residues 1-212 was PCR-amplified using pcDNA3-FLAG-KLC1 as template and
cloned into pGEX-5X-3. To add Myc epitopes at the C-terminus of KLC1, the KLC1
cDNA was PCR amplified using primers that changed the stop codon to a BamHI
site. The resulting fragment was inserted into a pcDNA3 derivative that contained
three tandem sequences encoding the Myc epitope. The final plasmid product
expressed the full-length KLC1-Myc, which has three Myc epitopes at the C-terminus.
pCAGGS-Myc-KIF5A-DN, which expresses a dominant-negative form of KIF5A,
was obtained from Kozo Kaibuchi (Nagoya University, Nagoya, Japan).
Antibodies
Rabbit polyclonal anti-caytaxin antibody was raised against a human caytaxin Nterminal fragment (residues 1-131). cDNA encoding the caytaxin(1-131) fragment
was inserted in frame into pET32-b (Novagen) and the recombinant protein was
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Journal of Cell Science 122 (22)
expressed in E. coli BL21 cells. The protein was purified with a Ni-NTA agarose
column (Qiagen). The purified protein was digested with 1 unit of thrombin per 100
g of protein to remove the histidine tag. The digested sample was applied to a NiNTA agarose column. The pass-through fraction was collected and dialyzed against
PBS. New Zealand White rabbits were injected subcutaneously with the purified
protein. After three boosts, the whole blood was withdrawn to prepare serum. The
serum was affinity purified using the GST-caytaxin(1-131) protein immobilized on
a HiTrap NHS-activated HP column (Amersham) according to the manufacturer’s
protocol.
Monoclonal antibodies against KHC (H2), Tau-1 and APP were purchased from
Chemicon; anti-MAP2 and anti-FLAG (M2) were from Sigma; anti-T7-tag was from
Novagen; anti-synapsin was from StressGen. Rhodamine- and FITC-conjugated
antibodies against mouse and rabbit immunoglobulin (Ig) were from Wako Chemicals
and Jackson ImmunoResearch. Horseradish-peroxidase-conjugated antibodies against
mouse and rabbit Ig were from GE Healthcare.
Journal of Cell Science
Yeast two-hybrid screen
Yeast CG1945 cells were transformed with the pGBT9 vector (Clontech) containing
the full-length mouse caytaxin cDNA and DNA prepared from an adult mouse brain
MatchMaker cDNA library in pACT2 (Clontech). Yeast two-hybrid screening of three
million yeast clones was carried out according to the Clontech manual. Clones growing
on (–) histidine SD plates in the presence of 10 mM 3-amino-1,2,4-triazole were
picked and analyzed for -galactosidase activity. Plasmid DNA were recovered from
45 -galactosidase-positive clones, transformed into E. coli HB101 cells to select for
pACT2 plasmid and purified for DNA sequencing by ABI310 sequencer. To select
for proteins binding to the caytaxin N-terminal region, the HF7c yeast cells were
transformed with the pGBT9-caytaxin(1-170) plasmid and a pACT2 clone DNA
isolated by the screening. The transformed cells were selected on SD selection plates
containing 10 mM 3-amino-1,2,4-triazole and transferred to a liquid SD medium.
After culturing overnight, the cells were harvested, disrupted by glass beads, and the
cell extract was measured for -galactosidase activity according to the Clontech liquid
culture protocol.
Cell culture and transfection of HEK293T cells
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% FBS and antibiotics in 10 cm dishes. After 24 hours of plating
1.2⫻106 cells per dish, cells were transfected with 20 g plasmid DNA by the calcium
phosphate precipitation method (Chen and Okayama, 1987). Cells were disrupted 2
days later with 1 ml per dish of lysis buffer [50 mM HEPES, pH 7.4, 150 mM NaCl,
5 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100 and a protease inhibitor
cocktail (2 g/ml leupeptin, 2 g/ml pepstatin, 2 mM benzamide, 40 g/ml bestatin,
1 mM phenylmethylsulfonyl fluoride)]. The lysate was centrifuged at 100,000 g for
30 minutes and the supernatant recovered for further analysis by immunoprecipitation
and GST pull-down assay.
GST pull-down assay
KLC1, caytaxin(1-170), and their derivatives were produced as GST-fusion proteins
in E. coli BL21 cells and purified by glutathione-Sepharose 4B (GE Healthcare). The
purified GST-protein (5 g) was mixed with 200 l cell lysate of HEK293T cells
expressing either FLAG-KLC1, Myc-KKIF5A-DN, caytaxin or their derivatives and
incubated for 4 hours at 4°C. Then, 10 l glutathione-Sepharose 4B beads were added
to the mixture and the incubation continued for 4 hours at 4°C. The beads were
collected and washed with lysis buffer five times. The proteins bound to the beads
were separated by SDS-acrylamide gel electrophoresis, transferred to PVDF
membrane and subjected to western blot analysis. The membrane was blocked with
TBST [20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1 % (v/v) Tween20] containing
5% skimmed milk for 1 hour at room temperature and then incubated with the antiFLAG antibody 2000-fold diluted in TBST containing 5% skimmed milk for 3 hours
at room temperature. After washing extensively with TBST, membrane was incubated
with horseradish-peroxidase-conjugated anti-mouse Ig antibody (GM Healthcare,
1:2000 dilution). The bound peroxidase was detected with ECL (GM Healthcare).
Subcellular fractionation of rat brain
The V1 membrane fraction was prepared from newborn rat brains by differential
centrifugation as described previously (Konecna et al., 2006; Morfini et al., 2001).
In brief, brains were homogenized in two volumes of a solution containing 0.32 M
sucrose, 10 mM HEPES buffer, pH 7.4, 5 mM EDTA, 4 mM N-ethylmaleimide and
the protease inhibitor cocktail. The homogenate was centrifuged at 12,500 g for 8
minutes. The supernatant was centrifuged at 40,000 g for 40 minutes. The resulting
supernatant was centrifuged at 100,000 g for 1 hour. The pellet (V1 membrane fraction)
was suspended in the IP buffer (50 mM Tris-HCl, 75 mM NaCl, 5 mM EDTA, 10
mM CHAPS, 5 mM N-ethlymaleimide and protease inhibitor cocktail).
Immunoprecipitation
Protein-A-Sepharose (20 l) was incubated with 1 g anti-FLAG, anti-Myc (9E10.2)
or purified anti-caytaxin antibody for 30 minutes at 4°C. The beads were washed
with PBS and then mixed with the HEK293T cell lysate (400 l) or the V1 membrane
fraction (400 l). After 3 hours of incubation at 4°C, the beads were washed with
lysis buffer five times. Proteins bound to the beads were resolved on SDS
polyacrylamide gel and electrotransferred to PVDF membrane. The membrane was
subjected to western blot analysis using the following mouse monoclonal antibodies:
anti-FLAG (1:2000 dilution), anti-Myc (1:2000 dilution) and anti-KHC (1:1000
dilution) antibodies. The membrane was incubated with horseradish-peroxidaseconjugated anti-mouse Ig antibody (GM Healthcare, 1:2000 dilution). The bound
peroxidase was detected with ECL (GM Healthcare).
Primary culture and transfection of hippocampal neurons
Preparation of hippocampal neurons from embryonic day 18 (E18) rat embryos was
carried out as described (Inagaki et al., 2001). In short, hippocampi were resected
from E18 rat embryo brains and cells were dispersed by treatment with 0.5 mg/ml
papain. Dispersed cells were suspended in Neurobasal medium (Gibco) supplemented
with 10% FBS, and plated on poly-D-lysine-treated laminin-coated coverslips placed
in wells of a 24-well plate at 0.5⫻105 cells/well. Three hours after plating, the medium
was replaced with 0.4 ml/well complete neuron medium (Neurobasal medium
supplemented with B-27 supplement (Gibco), 1 mM glutamate and 2.5 M cytosine
-D-arabinofuranoside). Half of the medium was changed every 3 days. One hour
before transfection, the coverslips were transferred to new wells containing 0.5 ml
fresh complete neuron medium. Lipofectamine 2000 (2 l) was diluted with 50 l
of Neurobasal medium. Plasmid DNA (0.5 g total) was mixed with 50 l Neurobasal
medium. These two solutions were mixed and incubated for 20 minutes at room
temperature. The mixture was added to the well and the cells were incubated for 90
minutes. The cells were then washed twice with DMEM and culture continued in
the complete neuron medium (0.5 ml/well).
For observation of hippocampal neurons earlier than 3 days in vitro (3 DIV),
neurons were transfected before plating as follows. Dispersed neurons (6⫻105 cells)
were suspended in 0.3 ml Neurobasal medium supplemented with 10% FBS. Plasmid
DNA (1 g) and Lipofectamine 2000 (2 l) were mixed as described above and
added to the cell suspension. The cell suspension was placed in a CO2 incubator
for 90 minutes. The cells were washed with Neurobasal medium containing10%
FBS and the cells (1.5⫻105 cells) were plated on a coated coverslip. After 90
minutes, the medium was changed to complete neuron medium. To obtain efficient
expression of RNAi, the transfected neurons washed with Neurobasal medium
containing 10% FBS were suspended in 1.7 ml complete neuron medium and
cultured as floating cells in an uncoated well for 24 hours. Cells were then plated
on a coated coverslip.
For examination of caytaxin localization in neurons expressing the dominantnegative KHC (KIF5A-DN), neurons were transfected with pCAGGS-Myc-KIF5ADN as described above on 2 DIV. An expression vector for FLAG-tagged galactosidase (LacZ) was used for control. After 48 hours of culture, the cells were
incubated with 5 M CellTracker blue CMAC (Invitrogen) for 2 hours at 37°C just
before fixation. Then, the cells were fixed and stained with anti-Myc or anti-FLAG
antibody using rhodamine-conjugated anti-mouse Ig as the secondary antibody or
with anti-caytaxin antibody using FITC-conjugated anti-rabbit Ig as the secondary
antibody.
Immunostaining
Cultured hippocampal neurons were fixed with 4% paraformaldehyde in PBS for 30
minutes at room temperature. The cells were washed with PBS and permeabilized
with methanol for 15 minutes at –20°C. After washing with PBS, the cells were
incubated with a primary antibody diluted with PBS containing 1% bovine serum
albumin for 1 hour at room temperature. After washing with PBS, the cells were
incubated with a diluted FITC- or rhodamine-conjugated secondary antibody for 1
hour at room temperature. After washing with PBS, the coverslip was mounted on
a glass slide with PermaFluor (Beckman Coulter). Mitochondria were stained with
MitoTracker Orange CMTMRos (Invitrogen).
RNAi
Knockdown by plasmid-based RNAi was carried out using the pcDNA6.2GW/EmGFP-miR vectors (Invitrogen) that expressed two different rat caytaxin RNAi
sequences purchased from Invitrogen (Rmi647864 and Rmi647866). The pcDNA6.2GW/EmGFP empty vector and the vector expressing -galactosidase (LacZ) RNAi
were used as controls. The efficiency of RNAi was assayed by measuring the intensity
of endogenous caytaxin immunofluorescence in GFP-positive hippocampal neurons
in culture. Expression of Rmi647864 and Rmi647866 siRNAs suppressed endogenous
caytaxin levels to 29±7.5% (s.d., n12) and 35±9.5% (n26), respectively, compared
with cells expressing GFP alone (100±2.8%, n16) or LacZ siRNA (100.4±10.7%,
n22). These two caytaxin siRNAs gave essentially the same results and the results
with Rmi647864 were described in the text.
Immunoelectron microscopy
Eyes were removed from 4-week-old ICR mice which were perfusion-fixed with 4%
paraformaldehyde and 0.3% glutaraldehyde in PBS. The retina was resected and
further fixed overnight with the same solution. The retina was immersed in PBS
overnight on ice and then dehydrated with an ethanol series and embedded in LR
white (Nisshin EM Corporation). Ultrathin sections 70 nm thick were mounted on
uncoated Ni #300 grids. The sections were treated with 3% H2O2 for 5 minutes,
Transportation of caytaxin by kinesin
rinsed with H2O and blocked with 2% normal goat serum and 2% normal donkey
serum in PBS for 30 minutes. Sections were incubated with anti-caytaxin antibody
and then reacted with 15-nm-gold-conjugated goat anti-rabbit IgG (Biocell Research
Laboratories). After immunolabeling, sections were stained with 2% hafnium chloride
in methanol.
To observe fine structures, the fixed retina was treated with 1% OsO4 in BPS and
dehydrated with an ethanol series and embedded in Spurr resin (Nisshin EM
Corporation). Ultrathin sections of 70 nm were mounted on Cu/Rh #300 grids and
stained with 2% hafnium chloride followed by staining with a mixture containing
1% lead (II) citrate, 1% lead (II) nitrate, 1% lead (II) acetate, 2% Na citrate and
0.18 M NaOH. Samples were visualized with a transmission electron microscope
(H-7100, Hitachi).
Image analysis and statistical methods
Journal of Cell Science
Fluorescence images were captured with an Olympus BX50 fluorescence microscope
equipped with a Nikon DS-2MBWc CCD camera using NIS-elements software
(Nikon) or with a Carl Zeiss LSM510 confocal microscope. The images were
processed by Photoshop (Adobe). The length of the longest neurite and the
fluorescence intensity were measured using ImageJ (NIH). Time-lapse images were
taken with Deltavision 2 (Applied Precision) equipped with Axiovert S100 (Carl
Zeiss) or with a fluorescence microscope (IX71, Olympus) equipped with a UIC-GE
CCD camera (MDS Analytical Technologies) using MetaMorph software. The images
were taken at 2- to 5-second intervals and compiled into a movie using Windows
movie maker (Microsoft). To measure the anterograde and retrograde velocities, the
images of GFP-caytaxin granules (n30) were recorded at 2 second intervals for 30
seconds and the distance that the granule moved between two frames were divided
by two. Statistical significance was analyzed by Student’s t-test. The results shown
in figures represent means ± s.d.
We thank Naoyuki Inagaki, Nara Institute of Science and Technology
(NAIST) for kind help in experiments with primary culture of
hippocampal neurons and observation with time-lapse fluorescent
microscopes; Megumi Iwano, NAIST, for the help in immunoelectron
microscopy; Kenji Kono, NAIST, for providing us with JPDI cDNA;
Tobias M. Boeckers, University of Ulm, for Shank3 cDNA; Hisashi
Koga and Takahiro Nagase, Kazusa DNA Research Institute, for mouse
calsyntenin and caytaxin cDNAs, respectively; Katsuji Yoshioka,
Kanazawa University and Kozo Kaibuchi, Nagoya University, for
kinesin plasmids.
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