1 Department of Molecular, Cellular and Developmental Biology, University of
Michigan, Ann Arbor, MI 48109-0720, USA
2 Mental Health Research Institute, University of Michigan, Ann Arbor, MI
48109-0720, USA
3 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104-6058, USA
* Author for correspondence (e-mail: kuwada{at}umich.edu)
Accepted 16 August 2004
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SUMMARY |
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Key words: Zebrafish, Accordion, Behavior, Muscle, Calcium, SERCA1, Brody diseasea
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Introduction |
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Zebrafish embryos display three stereotyped behaviors by 36 hours
post-fertilization (hpf) (Saint-Amant and
Drapeau, 1998). The earliest behavior consists of repetitive
spontaneous alternating coiling of the tail, much like a metronome. This
simple slow coiling behavior is independent of stimulation and starts at 17
hpf, reaches a peak frequency of 1 Hz at 19 hpf, and declines to less than 0.1
Hz by 26 hpf. After 21 hpf, embryos start to respond to mechanosensory
stimulation with coils that are stronger than spontaneous coils. Typically
embryos respond with two alternating coils. By 26 hpf mechanosensory
stimulation initiates swimming episodes. The frequency of muscle contractions
during swimming increases from 7 Hz at 26 hpf to 30 Hz at 36 hpf, the latter
being similar to the frequency of swimming of adult zebrafish
(Buss and Drapeau, 2001
).
The production of any touch response can be divided into several steps from
sensory perception to muscle activation (reviewed by
Roberts, 2000;
Berchtold et al., 2000
). In
zebrafish, two types of mechanosensory neurons sense touch stimuli. Head and
yolk stimulation acts through trigeminal neurons, whereas tail stimulation
activates Rohon-Beard neurons (Drapeau et
al., 2002
). Once triggered by sensory input, interneuronal
networks located in the hindbrain and spinal cord produce the appropriate
motor rhythm (Fetcho, 1992
).
Motoneurons are activated by these central networks, release acetylcholine at
neuromuscular junctions (NMJs) and depolarize the muscle membrane
(Buss and Drapeau, 2002
;
Li et al., 2003
). In muscle,
depolarization is sensed by voltage-dependent L-type Ca2+ channels,
such as dihydropyridine receptors, and activates ryanodine receptors in the
sarcoplasmic reticulum (SR) membrane to release Ca2+ from the SR to
the cytosol (Fill and Copello,
2002
). Increased cytosolic Ca2+ activates troponin C
that initiates actin/myosin sliding, thus contracting the muscles
(Gordon et al., 2000
).
Cytosolic Ca2+ levels are rapidly decreased by sarco(endo)plasmic
reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump expressed
in skeletal muscle, which enables relaxation (McLennan et al., 1997).
Any defect in the above pathway induces motor disorder in human. For
example, impairment of Ca2+ regulation by SERCA1 mutation
in human causes Brody disease, a rare inherited disorder of skeletal muscle
function (Odermatt et al.,
1996). The clinical signs and symptoms in individuals involve
exercise-induced impairment of muscle relaxation, stiffening and cramps
(Brody, 1969
;
MacLennan, 2000
). In fact, in
vitro study with cultured muscle cells from individuals with this disease
revealed slow Ca2+ reuptake and reduced Ca2+-ATPase
activity that can explain the relaxation defect (Benders, 1994).
The large-scale Tübingen mutagenesis screen isolated 63 behavioral
mutants that displayed abnormal touch responses between 48 and 60 hpf
(Granato et al., 1996). Some
of the locomotion mutants, raised in this screen as well as the others, were
further characterized. Reduced touch sensitivity mutants such as macho,
alligator and steifftier had defects of Na+ channel
function in mechanosensory Rohon-Beard neurons
(Ribera and Nüsslein-Volhard,
1998
). shocked mutants, which showed abnormal swimming,
were defective in motor processing in the central nervous system (CNS)
(Cui et al., 2004
). Reduced or
abnormal motility mutants diwanka, unplugged and space cadet
had defects in axon projections (Zeller
and Granato, 1999
; Zeller et
al., 2002
; Zhang and Granato,
2000
; Zhang et al.,
2001
; Lorent et al.,
2001
). Immotile or reduced motility mutants, such as
nic/twister, sofa potato, ache/zieharmonika and twitch once,
were defective at the NMJ (Westerfield et
al., 1990
; Lefebvre et al.,
2004
; Ono et al.,
2001
; Behra et al.,
2002
; Downes and Granato,
2004
; Ono et al.,
2002
). Another immotile mutant, relaxed, had a coupling
defect between muscle excitation and contraction
(Ono et al., 2001
). Mutants
carrying reduced striations in somatic muscles, such as fibrils
unbundeled and sapje, were shown to have abnormal myofiber
organization (Felsenfeld et al.,
1990
; Bassett et al.,
2003
). The genes mutated in the mutants nic/twister, sofa
potato, ache/zieharmonika, twitch once and sapje were,
respectively, identified as the
-subunit of the nicotinic
acetylcholine receptor, the
-subunit of the nicotinic
acetylcholine receptor, acetylcholinesterase, rapsyn and the Duchenne
muscular dystrophy gene (Sepich et
al., 1998
; Lefebvre et al.,
2004
; Ono et al.,
2004
; Behra et al.,
2002
; Downes and Granato,
2004
; Ono et al.,
2002
; Bassett et al.,
2003
).
Another zebrafish mutation, accordion (acc), exhibited
apparent simultaneous contractions of trunk muscles on both sides of the
embryo resulting in the shortening of the trunk in response to touch and was
named in analogy to the action of the musical instrument
(Granato et al., 1996). As
application of a glycine receptor blocker, strychnine, to wild-type embryos
phenocopied the abnormal behavior, it was proposed that the acc
phenotype was due to defects in glycine-mediated transmission within the CNS
(Granato et al., 1996
).
Agreeing with this anticipation, inhibitory glycine-mediated transmission is
essential to prevent simultaneous, bilateral muscle contractions in a variety
of vertebrates (Fetcho, 1990
;
Grillner, 2003
;
Kudo et al., 2004
;
Roberts, 2000
). However, the
acc phenotype can, in principle, be generated in a variety of other
ways. As muscle defects can result in abnormal behavior
(Bassett et al., 2003
;
Granato et al., 1996
;
Lefebvre et al, 2004
;
Ono et al., 2002
), one of
these ways could be via a muscle defect that increases the duration of muscle
contractions without any defects in the alternation or timing of output from
motoneurons in the two sides of the spinal cord. Such a defect would result in
overlap of contractions on the two sides and shortening of the trunk.
In this paper, we have used electrophysiology to show that the output of
the CNS and the function of the NMJ are normal in acc. However, after
active contraction, acc truck muscles stiffened for a while and
relaxation was five times slower than that in wild-type siblings. There was a
correlated slowing of the decay of cytosolic Ca2+ transients in
mutant muscles. These results suggested impaired Ca2+ re-uptake
into the SR from the cytosol in mutant muscles. Indeed, application of
thapsigargin, an inhibitor of SERCA, to wild-type embryos phenocopied
acc defects. We identified mutations in the atp2a1 gene,
which encodes SERCA1, of acc mutants. The molecular identification of
the atp2a1/acc gene was confirmed by rescue of the mutant phenotype
by injection of wild-type atp2a1 mRNA into mutant embryos and by
phenocopy of the mutation by injection of antisense atp2a1 morpholino
oligonucleotides (MO) into wild-type embryos. Mutations in ATP2A1 in
humans are known to cause Brody disease, an exercise-induced muscle relaxation
disorder (Brody, 1969;
Odermatt et al., 1996
), making
the acc mutant an attractive animal model for Brody disease.
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Materials and methods |
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Video recording of zebrafish behavior and muscle contraction
Embryonic behaviors were observed and video recorded using a dissection
microscope. Mechanosensory stimuli were delivered to the yolk with forceps.
For closer examinations of muscle contractions, 24 hpf embryos were
anaesthetized (0.02% tricaine) and pinned with tungsten wires (25 µm
diameter) through the notochord onto a silicon elastomer (Sylgard)-lined dish
in Evans solution (134 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl2, 1.2 mM
MgCl2, 10 mM glucose, and 10 mM HEPES at 290 mOsm and pH 7.8).
Tricaine was removed by replacing the tricaine/Evans solution with Evans with
no tricaine. Mechanosensory stimuli were delivered to the yolk by ejecting
bath solution (20 psi, 20 mseconds pulse) from a pipette with a 15-30 µm
tip using a Picospritzer II (General Valve Corporation). Several somites were
observed in high resolution with Hoffman modulation optics (40x water
immersion objective). Videos were captured with a CCD camera (WVBP330,
Panasonic) and a frame grabber (LG-3, Scion Corporation) and analyzed with
Scion Image on a G4 Macintosh. The contraction and relaxation durations were
measured by counting frames from the videos (1 frame=33 mseconds).
Kinetic analysis of muscle contraction
Embryos at 24 hpf were anaesthetized and pinned to a Sylgard dish in Evans
solution. Tungsten wires were put through the forebrain, because the intact
forebrain and midbrain are not necessary for the touch response and
spontaneous contractions at 24 hpf
(Saint-Amant and Drapeau,
1998). Tricaine/Evans was replaced with Evans containing no
tricaine and mechanosensory stimuli delivered to the tail for the touch
response. Videos were captured and analyzed with Scion Image. The durations
for active contraction, constant contraction and relaxation were measured by
counting frames (1 frame=33 mseconds).
Muscle recording
The dissection protocols for in vivo patch recordings have been described
previously (Buss and Drapeau,
2000). Briefly, 48 hpf zebrafish embryos were anaesthetized in
tricaine and pinned on a Sylgard dish. The skin was peeled off to allow access
to the underlying muscle cells. For electrophysiological recordings, embryos
were partially curarized in an Evans solution containing 6 µM
d-tubocurarine but not tricaine. Patch electrodes were pulled from
borosilicate silicate glass to yield electrodes with resistances of 6-10
M
. The patch electrode was visually guided to patch muscle cells using
Hoffman modulation optics (40x water immersion objective). The electrode
solution was consisted of 105 mM potassium gluconate, 16 mM KCl, 2 mM
MgCl2, 10 mM HEPES, 10 mM EGTA and 4 mM Na3ATP at 273
mOsm and pH 7.2. Recordings were performed with an Axopatch 200 amplifier
(Axon Instruments), low-pass filtered at 5 kHz, and sampled at 10 kHz. Data
was collected with Clampex 8.2 software (Axon Instruments) and analyzed with
Clampfit 9.0 software (Axon Instruments). Mechanosensory stimulation to induce
fictive swimming was delivered by ejecting bath solution (20-50 psi, 10-30
mseconds pulse) from a pipette with a 15-30 µm tip using a Picospritzer II
to the tail of the pinned embryo.
Muscle contractions by current injection
As described previously, it is possible to inject current through a patch
to evoke muscle contractions in live zebrafish and still maintain the whole
cell configuration (Buss and Drapeau,
2000). Embryos were completely curarized with an Evans solution
containing 15 µM d-tubocurarine. The current intensity and the current
pulse duration were set to 2-7 nA and 15-20 mseconds, respectively, which were
sufficient to evoke a contraction. This condition mimics the cycle duration of
motoneurons output that must be occurring for the embryo to swim at the
observed tail beat frequencies of 30 Hz
(Buss and Drapeau, 2001
). The
contractions of the muscle were observed on a video monitor and captured with
a CCD camera to analyze with a computer.
Pharmacological treatment
Thapsigargin (10 mM stock in DMSO, Sigma) was diluted to 1 µM in Evans
solution and applied to embryos 1 hour before assay.
Ca2+ imaging
We injected Calcium Green-1 dextran (10,000 Mr,
Molecular Probes) into 1- to 4-cell stage embryos from acc carrier
in-crosses. At later stages, random subsets of cells including muscles were
seen to contain Calcium Green-1 dextran. The genotype of the injected embryos
(24 and 48 hpf) was determined by responses to touch and the embryos were
anaesthetized with 0.02% tricaine and pinned to a Sylgard dish. The tricaine
was washed out and embryos were bathed in Evans solution containing a muscle
myosin inhibitor, N-benzyl-p-toluene sulphonamide at 5 mM (Sigma), which does
not affect to the Ca2+ regulation
(Cheung et al., 2001). This
drug suppressed the muscle contractions and enabled us to observe the
Ca2+ transients with a confocal microscope (LSM510, Carl Zeiss). As
plane-scanning requires too much time to adequately measure Ca2+
transients, we performed line-scanning at 132 Hz (7.6 mseconds/scan) of a
fluorescent muscle cell. The time course of the average strength of
fluorescence in the line at each scan was calculated with the maximum
fluorescence of each record equalized. The observed changes in fluorescence
indicate Ca2+ transients during spontaneous coiling at 24 hpf.
N-methyl-D-aspartate (NMDA, 50 µM) was bath applied to 48 hpf
embryos to activate swimming (Cui et al.,
2004
), as spontaneous movement was hardly observed at 48 hpf
(Saint-Amant et al., 1998).
Meiotic and physical mapping
acc carrier fish (Tübingen strain) were crossed with
wild-type WIK fish to generate mapping carriers that were crossed to identify
mutants for meiotic mapping to microsatellites and genes using PCR as
described previously
(Nüsslein-Volhard and Dahm,
2002; Gates et al.,
1999
; Shimoda et al.,
1999
). For mapping acc against netrin 1, a
polymorphism of the DraI restriction site in a 136 bp PCR product
(forward primer, 5'-GCATATGAAATATGATAGAGGAAGATT-3'; reverse
primer, 5'-TGAAACAAGTCTGCTTGAACCAG-3') found in the map crosses
was used. The LN54 radiation hybrid panel was used for the physical mapping
(Hukriede et al., 1999
).
Cloning, knockdown, mRNA rescue, and mutagenesis of ATP2A1
The following primers were used for cloning of ATP2A1 cDNA:
forward primer, 5'-GGGGCCATCTGTTTTGTCCCTATCCTTCAC-3'; reverse
primer, 5'-GGGTGATTTTTCACTGGTTCCCGGTTGG-3'. To knock down the
protein synthesis (Summerton and Weller,
1997), SERCA1 antisense MO
(5'-CGTTCTCCATCCTGTCTGCTCAAAG-3') was designed against 16
mer of 5'-UTR sequence and adjacent 9 mer coding region on mRNA. The
CAT sequence in bold corresponds to the start ATG. Standard control MO
(randomized sequence available from Gene Tools) was used for control MO.
Capped atp2a1 mRNA synthesis was performed with mMESSAGE mMACHINE T7
Ultra (Ambion). The 5'-UTR sequence of the synthesized mRNA was amended
(5'-CCTCGAGCCGCCACCATGGAGAACGCA-3') in order to avoid
knockdown by the antisense MO. The ATG sequence in bold is the start
ATG. Injections (0.25 mM for MO and 1 µg/µl for mRNA) were performed as
described previously (Xiao et al.,
2003
; Nüsslein-Volhard
and Dahm, 2002
). The Pro789Leu mutation (CCA to CTG), previously
identified as a mutation underlying Brody disease
(Odermatt et al., 2000
), was
introduced into the zebrafish atp2a1 construct by the Quick Change
method (Stratagene) with the following primers: forward primer,
5'-CCTGAGGCTCTGATCCTGGTTCAGCTGCTGTGG-3'; reverse primer,
5'-CCACAGCAGCTGAACCAGGATCAGAGCCTCAGG-3'. The CTG and
CAG sequence in bold indicates the sense and antisense codon,
respectively, of the introduced Leu residue.
Immunostaining and in situ hybridization
Immunostaining was carried out as described previously
(Devoto et al., 1996).
Briefly, embryos at 24 hpf were anesthetized (0.02% tricaine), fixed in 4%
paraformaldehyde and then washed several times in 0.1 M phosphate-buffered
saline (PBS), pH 7.4. Embryos were blocked in PBS containing 2% BSA and 2%
sheep serum, incubated in F59 antibody (anti-slow twitch myosin, 1:20,
Developmental Studies Hybridoma Bank) diluted in PBS, and washed in PBS. Alexa
488-conjugated anti-mouse IgG was used as a secondary antibody (1/1000,
Molecular Probes). In situ hybridization was performed as described previously
(Li et al., 2004
).
Mixture of two atp2a1 probes covering nucleotides 1997 (N
terminus
M4 domain in SERCA1 protein) and 2598
3220 (M7
C
terminus) were used for in situ hybridization. For sectioning after color
development, embryos were equilibrated in 15% sucrose/7.5% gelatin in PBS at
37°C and then embedded in it at 80°C. Sections (20 µm) were
cut with a cryostat (CM3050S, Leica).
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Results |
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A significant increase in the duration of relaxation in acc embryos should decrease the stimulation frequency at which tetanus occurs. To test this, we stimulated muscles by injecting current with a patch electrode and video recorded the resultant contractions in mutant and wild-type siblings at 48 hpf. Wild-type muscles followed trains of depolarizations at frequencies from 1 to 35 Hz (n=5), whereas acc muscles followed at lower frequencies but failed to follow trains of depolarizations at 15±1.4 Hz (n=5), showing that acc muscles do reach tetanus at lower stimulation frequencies compared with wild-type muscles. This result, along with the relaxation data, strongly suggests that one site of defect in the mutants is muscle and that the overlap in muscle contractions on the two sides due to the slow relaxation causes the shortening of the trunk seen in acc embryos during an escape response following a mechanosensory stimulus.
In addition to behavioral defects, acc embryos exhibited
morphological defects (Granato et al.,
1996). After 48 hpf, the trunks of acc embryos were
frequently bent (Fig. 2A,B) and
this phenotype worsened with age (not shown). Disruption of the notochord was
also observed after 36 hpf (Fig.
2D,E). Labeling of slow twitch muscle fibers in acc with
mAb F59 (Devoto et al, 1996
)
showed that they were disturbed (Fig.
2G,H), but no increased cell death assayed with TUNEL and Acridine
Orange labeling was observed (data not shown). These morphological defects
were probably secondary ones resulting from the mechanical stress caused by
the aberrant contractions. Supporting this idea, prolonged treatment with
tricaine, a weak Na+ channel inhibitor that prevented motor
behavior, abolished the morphological defects in acc mutants
(Fig. 2C,F,I). Treatment with
N-benzyl-p-toluene sulphonamide, a specific inhibitor of muscle myosin, also
prevented the morphological defects (data not shown).
|
The overall morphology of the CNS, including the hindbrain and spinal cord,
which are necessary and sufficient for the early behaviors in zebrafish, was
not obviously perturbed in acc embryos (data not shown). The pattern
of the hindbrain reticulospinal interneurons, spinal Rohon-Beard sensory
neurons, and spinal motoneurons and their axons labeled with anti-acetylated
-tubulin (which labels all axons)
(Chitnis and Kuwada, 1990
;
Bernhardt et al., 1990
) and
anti-Islet (which labels Rohon-Beard and motoneurons nuclei)
(Korzh et al., 1993
) were
normal (data not shown). Furthermore, the pattern of
-bungarotoxin-labeled acetylcholine receptor on muscles
(Ono et al., 2001
) was normal
(data not shown).
The physiological output from the CNS at the NMJ was assayed by in vivo
patch recordings from trunk axial muscle of 48 hpf embryos
(Fig. 3A). Muscle voltage
recordings from wild-type sibling embryos showed sustained episodes of
rhythmic depolarizations following mechanosensory stimulation
(Fig. 3B). This rhythmic
activity had a frequency (25 to 30 Hz) that was comparable with the swimming
frequency observed during free swimming and presumably underlies swimming
contractions (Buss and Drapeau,
2001). The average duration of the muscle response in wild-type
siblings was 1.28±0.22 seconds (n=10) and maximum amplitude
was 54.1±1.0 mV. When strychnine (2 µM) was added to the bath
solution, the pattern of activity in the muscle following mechanosensory
stimulation dramatically changed so that the responses were shorter
(0.29±0.03 seconds, n=10, Student's t-test,
P<0.01) and lost their fast rhythmic activity, although the
maximum amplitude (54.9±1.0 mV) was comparable with wild-type
siblings (Fig. 3C). Muscle
recordings from acc embryos following mechanosensory stimulation
showed a rhythmic voltage response in which the duration (1.38±0.22
seconds, n=10), maximum amplitude (54.8±0.8 mV) and
rhythmicity (25-30 Hz) were indistinguishable from that seen in wild-type
siblings (Fig. 3D). Thus, the
muscle recordings in acc embryos are similar to that in wild-type
siblings and do not resemble the output of the CNS when glycine-mediated
inhibitory transmission is blocked by strychnine. These results are consistent
with the hypothesis that the output from the CNS and the response of the NMJ
following mechanosensory stimulation is unperturbed in mutants and that the
defect resides in muscle.
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acc encodes for SERCA1
To identify the gene responsible for the acc mutation, we
meiotically mapped the mutation to two flanking markers, z4161 (6.8 cM, 246
recombinants in 3616 meioses) and netrin 1 (0.2 cM, 5 recombinants in
2448 meioses), in linkage group 3. As the acc phenotype suggested a
defect in SERCA1, we physically mapped atp2a1 gene that encodes
SERCA1 using the LN54 radiation hybrid panel and found it to be close (4.3 cR)
to netrin 1 in linkage group 3
(Fig. 5A).
|
As SERCA1 is found in the SR of muscle, atp2a1 should be expressed in muscle. Indeed, in situ hybridization showed that atp2a1 is expressed by axial muscles as early as 15 hpf, which precedes the appearance of motor behaviors (Fig. 6A,B). Cross-sections of the trunk showed that expression appears to be exclusively in muscles and not in the CNS (Fig. 6C). No expression of atp2a1 in the CNS was observed at any level of the CNS (data not shown). Although expression of atp2a1 in the CNS below the resolution of our in situ hybridizations cannot be excluded, muscle specific expression of ATP2A1is consistent with the primary acc defect found in muscles.
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Discussion |
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acc was originally proposed to have a neuronal defect because
strychnine-treated embryos displayed acc-like bilateral muscle
contractions (Granato et al.,
1996). Although the primary defect in acc mutants is a
muscle relaxation defect caused by mutations in muscle-specific SERCA1, it is
possible that there might be a secondary neuronal defect that is due to
deficient motility of mutants. So far, we have not detected a neuronal
phenotype. We found that the voltage response of wild-type muscles to touch,
when glycine-mediated inhibitory synaptic transmission is blocked by
strychnine, is highly aberrant. By contrast, acc muscles exhibited
normal rhythmic activity following mechanosensory stimulation. This along with
the fact that the morphology of the nervous system is not obviously aberrant
suggests that many features of the nervous system are normal. However, we
cannot rule out a neuronal phenotype secondary to the primary muscle defect in
acc embryos.
acc embryos also displayed morphological defects, such as a bent
trunk, disrupted notochord and disturbed slow-twitch muscle fibers. These are
secondary to the muscle relaxation defect, as paralysis by tricaine or
N-benzyl-p-toluene sulphonamide prevented these morphological phenotypes. In
fact, similar notochord and slow twitch defects are seen in another behavior
mutant, nictwister dbn12, that has a gain-of-function
mutation in the -subunit of the muscle-specific nicotinic acetylcholine
receptor (Lefebvre et al.,
2004
). In nictwister dbn12 homozygous mutant,
prolonged neuromuscular transmission causes persistent activation of axial
muscles bilaterally that results in acc-like hypercontractions and as
a consequence cause notochord and slow twitch muscle abnormalities.
acc mutations are found in functionally important regions of SERCA1
We found three missense mutations that disrupt or diminish the SERCA1
function of acc mutants. Each mutation was located in a highly
conserved -helix, which forms a membrane domain. Most mutations
introduced in M5 disrupt or severely reduce SERCA1 function in vitro,
suggesting an essential role of the M5 domain
(Sørensen, et al.,
1997
; Strock et al.,
1998
; Asahi et al.,
1999
; Guerini et al.,
2000
; Zhang et al.,
2000
; Ma et al.,
2003
; Dode et al.,
2003
). In fact, acctq206 contains a mutation
in the M5 domain that is consistent with the functional importance of the M5
for SERCA1 function. The structure and function of SERCA1 has been
characterized by crystal structures in different states and by numerous point
mutations. SERCA1 has two Ca2+-binding sites: one consists of M5,
M6 and M8; the other consists of M4 and M6
(Toyoshima et al., 2000
). In
M5, both Asn768 and Glu771 form hydrogen bond with Ca2+ by their
side-chain oxygen atoms. During the conformational change from the
Ca2+-binding state to the dissociation state, the middle part of
the M5 domain called the flexible hinge (Ile765-Asn768) tilts about 30°,
whereas the adjacent lower region of M5 (Gly 770-Leu 777) hardly moves at all.
This tilt results in turning the upper part of M5 and disrupts the hydrogen
bond for Ca2+ binding
(Toyoshima and Nomura, 2002
;
Nielsen et al., 2003
). The
mutation Ser766Phe found in acctq206 is located in the
hinge region and could account for the strongest phenotype.
During the conformational change of SERCA1, M4 inclines together with the
M5 as a rigid body to induce movement of the M2 helix
(Toyoshima and Nomura, 2002).
This movement by M2 probably regulates the binding of sarcolipin, a regulator
of SERCA1 in skeletal muscle (Odermatt et
al., 1998
; Asahi et al.,
2002
; MacLennan et al.,
2003
). Although this binding has not been studied structurally, a
binding model between SERCA1 and phospholamban, a sarcolipin-like protein
expressed in cardiac muscles (MacLennan
and Kranias, 2003
), suggests that the hydrophobic residues in the
M2, including Ile97, are important for this interaction
(Toyoshima et al., 2003
;
Asahi et al., 2003
). The
accmi25i allele has an Ile97Asn mutation in M2, suggesting
that regulation of SERCA1 by sarcolipin may be aberrant in this allele. In
contrast to the other membrane domains, M7 does not move much, because the M7
helix associates with the lower region of M5
(Toyoshima and Nomura, 2002
),
and M7 has not been well characterized by mutations. The fact that the
accmi289a allele has a Thr848Ile mutation in M7 is the
first functional evidence for a role of M7 for SERCA1 function.
acc mutants are an animal model for Brody disease
Brody disease is a rare inherited disorder of skeletal muscle function in
humans. It is characterized by painless cramping induced by exercise and
linked to an impairment of skeletal muscle relaxation
(Brody, 1969). Most families
harboring Brody disease show an autosomal recessive inheritance pattern and
carry a mutation in the ATP2A1 gene on chromosome 16
(Odermatt et al., 1996
;
Odermatt et al., 1997
;
Odermatt et al., 2000
). In a
minority of cases, Brody disease is inherited in an autosomal recessive
pattern but there are no mutations in ATP2A1
(MacLennan, 2000
;
Zhang et al., 1995
) or in an
autosomal dominant pattern with a (2;7) chromosomal translocation
(Novelli et al., 2004
). Thus,
there are at least two genes other than ATP2A1 that can give rise to
Brody disease (MacLennan,
2000
).
Among seven different SERCA1 mutations so far identified in Brody disease,
five induce a stop codon 5' to the Ca2+-binding sites and are
considered to be null mutations (Odermatt
et al., 1996; Odermatt et al.,
1997
; Odermatt et al.,
2000
). The other missense mutations are found in M6 and in a loop
between the M6 and M7 (Odermatt et al.,
2000
). Surprisingly, the individuals that carry apparent null
alleles of ATP2A1 are able to relax their skeletal muscle albeit at a
much slower rate without any obvious respiratory distress
(MacLennan, 2000
).
Interestingly, SERCA1-null mice were defective in diaphragm function and died
soon after birth, making them less attractive as an animal model for Brody
disease (Pan et al., 2003
).
Compensatory or redundant mechanism for Ca2+ removal from the
muscle cytosol in humans but not in mice, such as
Na+-Ca2+ exchangers, Ca2+-ATPase in the
plasma membrane, Ca2+ uptake by mitochondria and/or ectopic
expression of other members of the SERCA family might account for the
difference in viability between mouse and human
(Wuytack et al., 2002
).
In zebrafish, seven acc mutations were isolated in the
Tübingen screen (Granato et al.,
1996) and two in our ongoing screen. All of these mutations are
autosomal recessive, suggesting the same inheritance as ATP2A1 Brody
disease. Zebrafish acc mutants and human Brody disease share the most
crucial feature of aberrantly slow relaxation of muscles because of much
slower reuptake of Ca2+ from the muscle cytosol to the SR caused by
mutations in the atp2a1 gene. Although zebrafish acc mutants
die by day 10, their fast development and accessibility up to the time of
death allows for detailed physiological analysis of the consequences of
atp2a1 mutations. As zebrafish embryos are readily accessible to
molecular, genetic, pharmacological and physiological interventions,
acc mutants could serve as an attractive animal model for Brody
disease. For example, acc mutants could be used to screen for drugs
that relieve the muscle defect in vivo by enhancing Ca2+ re-uptake
or reducing Ca2+ release from the SR during a contraction.
Interestingly, mutations in at least six genes in addition to acc can
give rise to an acc-like behavioral phenotype in zebrafish
(Granato et al., 1996
). It
would be interesting to see if any of these genes might also be genes that are
associated with Brody disease.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5457/DC1
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REFERENCES |
---|
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---|
Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M. and MacLennan,
D. H. (1999). Transmembrane helix M6 in sarco(endo)plasmic
reticulum Ca2+-ATPase forms a functional interaction site with
phospholamban. J. Biol. Chem.
274,32855
-32862.
Asahi, M., Kurzydlowski, K., Tada, M. and MacLennan, D. H.
(2002). Sarcolipin inhibits polymerization of phospholamban to
induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPase
(SERCAs). J. Biol. Chem.
277,26725
-26728.
Asahi, M., Sugita, Y., Kurzydlowski, K., de Leon, S., Tada, M.,
Toyoshima, C. and MacLennan, D. H. (2003). Sarcolipin
regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by
binding to transmembrane helices alone or in association with phospholamban.
Proc. Natl. Acad. Sci. USA
100,5040
-5045.
Bassett, D. I., Bryson-Richardson, R. J., Daggett, D. F.,
Gautier, P., Keenan, D. G. and Currie, P. D. (2003).
Dystrophine is required for the formation of stable muscle attachments in the
zebrafish embryo. Development
130,5851
-5860.
Behra, M., Cousin, X., Bertrand, C., Vonesch, J. L., Biellmann, D., Chatonnet, A. and Strähle, U. (2002). Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nat. Neurosci. 5, 111-118.[CrossRef][Medline]
Benders, A. A. G. M., Veerkamp, J. H., Oosterhof, A., Jongen, P. J. H., Bindels, R. J. M., Smit, L. M. E., Busch, H. F. M. and Wevers, R. A. (1994). Ca2+ homeostasis in Brody's disease. J. Clin. Invest. 94,741 -748.[Medline]
Berchtold, M. W., Brinkmeier, H. and Müntener, M.
(2000). Calcium ion in skeletal muscle: its crucial role for
muscle function, plasticity, and disease. Physiol.
Rev. 80,1215
-1265.
Bernhardt, R. R., Chitnis, A. B., Lindamer, L. and Kuwada, J. Y. (1990). Identification of spinal neurons in the embryonic and larval zebrafish. J. Comp. Neurol. 302,603 -616.[Medline]
Brody, I. A. (1969). Muscle contracture induced by exercise. New Eng. J. Med. 281,187 -192.[Medline]
Buss, R. R. and Drapeau, P. (2000).
Physiological properties of zebrafish embryonic red and white muscle fibers
during early development. J. Neurophysiol.
84,1545
-1557.
Buss, R. R. and Drapeau, P. (2001). Synaptic
drive to motoneurons during fictive swimming in the developing zebrafish.
J. Neurophysiol. 86,197
-210.
Buss, R. R. and Drapeau, P. (2002). Activation
of embryonic red and white muscle fibers during fictive swimming in the
developing zebrafish. J. Neurophysiol.
87,1244
-1251.
Cheung, A., Dantzig, J. A., Hollingworth, S., Baylor, S. M., Goldman, Y. E., Mitchison, T. J. and Straight, A. F. (2001). A small-molecule inhibitor of skeletal muscle myosin II. Nat. Cell Biol. 4,83 -88.[CrossRef]
Chitnis, A. B. and Kuwada, J. Y. (1990). Axonogenesis in the brain of zebrafish embryos. J. Neurosci. 10,1892 -1905.[Abstract]
Cui, W. W., Saint-Amant, L. and Kuwada, J. Y. (2004). The shocked gene is required for the function of a premotor network in the zebrafish CNS. J. Neurophysiol. (in press).
Devoto, S. H., Melancon, E., Eisen, J. S. and Westerfield,
M. (1996). Identification of separate slow and fast muscle
precursor cells in vivo, prior to somite formation.
Development 122,3371
-3380.
Dode, L., Andersen, J. P., Leslie, N., Dhitavat, J., Vilsen, B.
and Hovnanian, A. (2003). Dissection of the functional
differences between sarco(endo)plasmic reticulum Ca2+-ATPase
(SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2)
mutants by steady-state and transient kinetic analyses. J. Biol.
Chem. 278,47877
-47889.
Downes, G. B. and Granato, M. (2004). Acetylcholinesterase function is dispensable for sensory neurite growth but is critical for neuromuscular synapse stability. Dev. Biol. 270,232 -245.[CrossRef][Medline]
Drapeau, P., Saint-Amant, L., Buss, R. R., Chong, M., McDearmid, J. R. and Brustein, E. (2002). Development of the locomotor network in zebrafish. Prog. Neurobiol. 68, 85-111.[CrossRef][Medline]
Eaton, R. C. and Farley, R. D. (1973). Development of the mauthner neurons in embryos and larvae of the zebrafish, Brachydanio rerio. Copeia 4, 673-682.
Felsenfeld, A. L., Walker, C., Westerfield, M., Kimmel, C. and Streisinger, G. (1990). Mutations affecting skeletal muscle myofibril structure in the zebrafish. Development 108,443 -459.[Abstract]
Fetcho, J. R. (1990). Morphological variability, segmental relationships, and functional role of a class of commissural interneurons in the spinal cord of goldfish. J. Comp. Neurol. 299,283 -298.[Medline]
Fetcho, J. R. (1992). The spinal motor system in early vertebrates and some of its evolutionary changes. Brain Behav. Evol. 40,82 -97.[Medline]
Fetcho, J. R. and O'Malley, D. M. (1997). Imaging neuronal networks in behaving animals. Curr. Opin. Neurobiol. 7,832 -838.[CrossRef][Medline]
Fill, M. and Copello, J. A. (2002). Ryanodine
receptor calcium release channels. Physiol. Rev.
82,893
-922.
Gates, M. A., Kim, L., Egan, E. S., Cardozo, T., Sirotkin, H.
I., Dougan, S. T., Lashkari, D., Abagyan, R., Schier, A. F. and Talbot, W.
S. (1999). A genetic linkage map for zebrafish: comparative
analysis and localization of genes and expressed sequences. Genome
Res. 9,334
-347.
Gordon, A. M., Homsher, E. and Regnier, M.
(2000). Regulation of contraction in striated muscle.
Physiol. Rev. 80,853
-924.
Granato, M., van Eeden, F. J. M., Schach, U., Trowe, T., Brand,
M., Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C. P.,
Jiang, Y. J. et al. (1996). Genes controlling and mediating
locomotion behavior of the zebrafish embryo and larva.
Development 123,399
-413.
Grillner, S. (2003). The motor infrastructure: from ion channels to neuronal networks. Nat. Rev. Neurosci. 4,573 -586.[CrossRef][Medline]
Guerini, D., Zecca-Mazza, A. and Carafoli, E.
(2000). Single amino acid mutations in transmembrane domain 5
confer to the plasma membrane Ca2+ pump properties typical of the
Ca2+ pump of endo(sarco)plasmic reticulum. J. Biol.
Chem. 275,31361
-31368.
Hukriede, N. A., Joly, L., Tsang, M., Miles, J., Tellis, P.,
Epstein, J. A., Barbazuk, W. B., Li, F. N., Paw, B., Postlethwait, J. H. et
al. (1999). Radiation hybrid mapping of the zebrafish genome.
Proc. Natl. Acad. Sci. USA
96,9745
-9750.
Korzh, V., Edlund, T. and Thor, S. (1993).
Zebrafish primary neurons initiate expression of the LIM homeodomain protein
Isl-1 at the end of gastrulation. Development
118,417
-425.
Kudo, N., Nishimaru, H. and Nakayama, K. (2004). Developmental changes in rhythmic spinal neuronal activity in the rat fetus. Prog. Brain Res. 143, 49-55.[Medline]
Lefebvre, J. L., Ono, F., Puglielli, C., Seidner, G.,
Franzini-Armstrong, C., Brehm, P. and Granato, M. (2004).
Increased neuromuscular activity causes axonal defects and muscular
degeneration. Development
131,2605
-2618.
Lewis, K. E. and Eisen J. S. (2003). From cells to circuits: development of the zebrafish spinal cord. Prog. Neurobiol. 69,419 -449.[CrossRef][Medline]
Li, W., Ono, F. and Brehm, P. (2003). Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors. J. Neurosci. 19,10467 -10474.
Li, Q., Shirabe, K. and Kuwada, J. Y. (2004). Chemokine signaling regulates sensory cell migration in zebrafish. Dev. Biol. 269,123 -136.[CrossRef][Medline]
Lorent, K., Liu, K. S., Fetcho, J. R. and Granato, M.
(2001). The zebrafish space cadet gene controls axonal
pathfinding of neurons that modulate fast turning movements.
Development 128,2131
-2142.
Ma, H., Inesi, G. and Toyoshima, C. (2003).
Substrate-induced conformational fit and headpiece closure in the
Ca2+ ATPase (SERCA). J. Biol. Chem.
278,28938
-28943.
MacLennan, D. H. (2000). Ca2+
signaling and muscle disease. Eur. J. Biochem.
267,5291
-5297.
MacLennan, D. H. and Kranias, E. G. (2003). Phospholamban: a crucial regulator of cardiac contractility. Nat. Rev. Mol. Cell Biol. 4,566 -577.[CrossRef][Medline]
MacLennan, D. H., Rice, W. J. and Green, N. M.
(1997). The mechanism of Ca2+ transport by
sarco(endo)plasmic reticulum Ca2+-ATPase. J. Biol.
Chem. 272,28815
-28818.
MacLennan, D. H., Asahi, M. and Tupling, A. R.
(2003). The regulation of SERCA-type pumps by phospholamban and
sarcolipin. Ann. N.Y. Acad. Sci.
986,472
-480.
Mullins, M. C., Hammerschmidt, M., Haffter, P. and Nüsslein-Volhard, C. (1994). Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate. Curr. Biol. 4,189 -202.[Medline]
Nielsen, G., Malmendal, A., Meissner, A., Møller, J. V. and Nielsen, N. C. (2003). NMR studies of the fifth transmembrane segment of sarcoplasmic reticulum Ca2+-ATPase reveals a hinge close to the Ca2+-ligating residues. FEBS Lett. 544,50 -56.[CrossRef][Medline]
Nüsslein-Volhard, C. and Dahm, R. (2002). Zebrafish. New York, NY: Oxford University Press.
Novelli, A., Valente, E. M., Bernardini, L., Ceccarini, C., Sinibaldi, L., Caputo, V., Cavalli, P. and Dallapiccola, B. (2004). Autosomal dominant Brody disease cosegregates with a chromosomal (2;7)(p11.2;p12.1) translocation in an Italian family. Eur. J. Hum. Genet. 12,579 -583.[CrossRef][Medline]
Odermatt, A., Taschner, P. E. M., Khanna, V. K., Busch, H. F. M., Karpati, G., Jablecki, C. K., Breuning, M. H. and MacLennan, D. H. (1996). Mutations in the gene-encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase, are associated with Brody disease. Nat. Genet. 14,191 -194.[Medline]
Odermatt, A., Taschner, P. E. M., Scherer, S. W., Beatty, B., Khanna, V. K., Cornblath, D. R., Chaudhry, V., Yee, W. C., Schrank, B., Karpati, G. et al. (1997). Characterization of the gene encoding human sarcolipin (SLN), a proteolipid associated with SERCA1: absence of structural mutations in five patients with Brody disease. Genomics 45,541 -553.[CrossRef][Medline]
Odermatt, A., Becker, S., Khanna, V. K., Kurzydlowski, K.,
Leisner, E., Pette, D. and MacLennan, D. H. (1998).
Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle
sarcoplasmic reticulum Ca2+-ATPase. J. Biol.
Chem. 273,12360
-12369.
Odermatt, A., Barton, K., Khanna, V. K., Mathieu, J., Escolar, D., Kuntzer, T., Karpati, G. and MacLennan, D. H. (2000). The mutation of Pro789 to Leu reduces the activity of the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) and is associated with Brody disease. Hum. Genet. 106,482 -491.[CrossRef][Medline]
Ono, F., Higashijima, S. I., Shcherbatko, A., Fetcho, J. R. and
Brehm, P. (2001). Paralytic zebrafish lacking acetylcholine
receptors fail to localize rapsyn clusters to the synapse. J.
Neurosci. 21,5439
-5448.
Ono, F., Shcherbatko, A., Higashijima, S. I., Mandel, G. and
Brehm, P. (2002). The zebrafish motility mutant twitch
once reveals new roles for rapsyn in synaptic function. J.
Neurosci. 22,6491
-6498.
Ono, F., Mandel, G. and Brehm, P. (2004).
Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in
zebrafish. J. Neurosci.
24,5475
-5481.
Pan, Y., Zvaritch, E., Tupling, A. R., Rice, W. J., de Leon, S.,
Rudnicki, M., McKerlie, C., Banwell, B. L. and MacLennan, D. H.
(2003). Targeted disruption of the ATP2A1 gene encoding
the sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1)
impairs diaphragm function and is lethal in neonatal mice. J. Biol.
Chem. 278,13367
-13375.
Ribera, A. B. and Nüsslein-Volhard, C. (1998). Zebrafish touch-insensitive mutants reveal an essential role for the developmental regulation of sodium current. J. Neurosci. 15,9181 -9191.
Roberts, A. (2000). Early functional organization of spinal neurons in developing lower vertebrates. Brain Res. Bull. 53,585 -593.[CrossRef][Medline]
Saint-Amant, L. and Drapeau, P. (1998). Time course of the development of motor behaviors in the zebrafish embryo. J. Neurobiol. 37,622 -632.[CrossRef][Medline]
Sepich, D. S., Wegner, J., O'Shea, S. and Westerfield, M.
(1998). An altered intron inhibits synthesis of the acetylcholine
receptor -subunit in the paralyzed zebrafish mutant nic1.Genetics 148,361
-372.
Shimoda, N., Knapik, E. W., Ziniti, J., Sim, C., Yamada, E., Kaplan, S., Jackson, D., de Sauvage, F., Jacob, H. and Fishman, M. C. (1999). Zebrafish genetic map with 2000 microsatellite markers. Genomics 58,219 -232.[CrossRef][Medline]
Sørensen, T., Vilsen, B. and Andersen, J. P.
(1997). Mutation Lys758 Ile of the sarcoplasmic
reticulum Ca2+-ATPase enhances dephosphorylation of
E2P and inhibits the E2 to
E1Ca2 transition. J. Biol.
Chem. 272,30244
-30253.
Strock, C., Cavagna, M., Peiffer, W. E., Sumbilla, C., Lewis, D.
and Inesi, G. (1998). Direct demonstration of Ca2+
binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants
overexpressed in COS-1 cells transfected with adenovirus vectors.
J. Biol. Chem. 273,15104
-15109.
Summerton, J. and Weller, D. (1997). Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7,187 -195.[Medline]
Toyoshima, C. and Inesi, G. (2004). Structural basis of ion pumping by Ca2+-ATPase of the sarcoplasmic reticulum. Annu. Rev. Biochem. 73,269 -292.[CrossRef][Medline]
Toyoshima, C. and Nomura, H. (2002). Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418,605 -611.[CrossRef][Medline]
Toyoshima, C., Nakasako, M., Nomura, H. and Ogawa, H. (2000). Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution. Nature 405,647 -655.[CrossRef][Medline]
Toyoshima, C., Asahi, M., Sugita, Y., Khanna, R., Tsuda, T. and
McLennan, D. H. (2003). Modeling of the inhibitory
interaction of phospholamban with the Ca2+ ATPase. Proc.
Natl. Acad. Sci. USA 100,467
-472.
Treiman, M., Caspersen, C. and Christensen, S. B. (1998). A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase. TiPS 19,131 -135.[Medline]
Westerfield, M. (1995). The zebrafish book. Eugene, OR: University of Oregon.
Westerfield, M., Liu, D. W., Kimmel, C. B. and Walker, C. (1990). Pathfinding and synapse formation in a zebrafish mutant lacking functional acetylcholine receptors. Neuron 4, 867-874.[Medline]
Wuytack, F., Raeymaekers, L. and Missiaen, L. (2002). Molecular physiology of the SERCA and SPCA pumps. Cell Calcium 32,279 -305.[CrossRef][Medline]
Xiao, T., Shoji, W., Zhou, W., Su, F. and Kuwada, J. Y.
(2003). Transmembrane sema4E guides branchiomotor axons to their
targets in zebrafish. J. Neurosci.
23,4190
-4198.
Zeller, J. and Granato, M. (1999). The
zebrafish diwanka gene controls an early step of motor growth cone
migration. Development
126,3461
-3472.
Zeller, J., Schneider, V., Malayaman, S., Higashijima, S. I., Okamoto, H., Gui, J., Lin, S. and Granato, M. (2002). Migration of zebrafish spinal motor nerves into the periphery requires multiple myotome-derived cues. Dev. Biol. 252,241 -256.[CrossRef][Medline]
Zhang, J. and Granato, M. (2000). The zebrafish
unplugged gene controls motor axon pathway selection.
Development 127,2099
-2111.
Zhang, Y., Fujii, J., Phillips, M. S., Chen, H. S., Karpati, G., Yee, W. C., Schrank, B., Cornblath, D. R., Boylan, K. B. and MacLennan, D. H. (1995). Characterization of cDNA and genomic DNA encoding SERCA1, the Ca2+-ATPase of human fast-twitch skeletal muscle sarcoplasmic reticulum, and its elimination as a candidate gene for Brody disease. Genomics 30,415 -424.[CrossRef][Medline]
Zhang, Z., Lewis, D., Strock, C., Inesi, G., Nakasako, M., Nomura, H. and Toyoshima, C. (2000). Detailed characterization of the cooperative mechanism of Ca2+ binding and catalytic activation in the Ca2+ transport (SERCA) ATPase. Biochemistry 39,8758 -8767.[CrossRef][Medline]
Zhang, J., Malayaman, S., Davis, C. and Granato, M. (2001). A dual role for the zebrafish unplugged gene in motor axon Pathfinding and pharyngeal development. Dev. Biol. 240,560 -573.[CrossRef][Medline]
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