doi: 10.1161/hh0701.090462
© 2001 American Heart Association, Inc.
Response to Research Commentary |
Microtubule Disruption by Colchicine Reversibly Enhances Calcium Signaling in Intact Rat Cardiac Myocytes
From the Physiopathologie Cardiovasculaire, INSERM U-390, Montpellier, France.
Correspondence to Ana M. Gómez, INSERM U-390, CHU Arnaud de Villeneuve, 34295 Montpellier, France. E-mail agomez{at}montp.inserm.fr
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Abstract |
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Abstract—Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (ICa) and [Ca2+]i transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 µmol/L colchicine on Ca2+ signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR) activity by analyzing the characteristics of spontaneous Ca2+ sparks. Colchicine treatment decreased Ca2+ spark amplitude (F/F0: 1.78±0.01, n=983, versus 1.64±0.01, n=1660, recorded in control versus colchicine-treated cells; P<0.0001) without modifying the sarcoplasmic reticulum Ca2+ load and enhanced their time to peak (in ms: 6.85±0.09, n=1185, versus 7.33±0.13, n=1647; P<0.0001). Microtubule disruption also induced the appearance of Ca2+ sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca2+ signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca2+]i transient evoked by field stimulation. Cells were loaded with the fluorescence Ca2+ indicator, Fluo-3 cell permeant, and stimulated at 1 Hz. [Ca2+]i transient amplitude was greater and its decay was accelerated in colchicine-treated, field-stimulated myocytes. This effect is reversible. When colchicine-treated myocytes were placed in a colchicine-free solution for 30 minutes, tubulin was repolymerized into microtubules, as shown by immunofluorescence, and the increase in [Ca2+]i transient was reversed. In summary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca2+ signaling in cardiac cells with intact sarcolemma. The full text of this article is available at http://www.circresaha.org.
Key Words: microtubules • calcium transient • calcium sparks • adenylyl cyclase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microtubules, components of the cytoskeleton, are involved in cell locomotion and mitosis. In adult cardiac cells, which do not undergo cell division, the role of microtubules has long been ignored, although interest began to focus on them after Tsutsui et al1 showed that microtubule content is increased in pressure-overload cardiac hypertrophy. Since the publication of that study, successive reports from the same team2 3 have reinforced the view that microtubule disruption increases contraction in failing myocytes. Following these observations, it was proposed that the increased microtubule network enhances the viscosity and/or rigidity of the failing myocyte.4 In contrast to the findings of Cooper and colleagues over the years, other investigators have not found an effect of colchicine treatment on contraction in either normal or failing hearts.5 6 7 The different experimental conditions and durations of colchicine exposure may account for these discrepancies. Even if one assumes that microtubule disruption will have more pronounced effects in cells with increased microtubule content, an effect, albeit smaller, should also be seen in normal myocytes. In fact, microtubule disruption has been shown to modulate beating rate and contraction,8 9 10 11 12 Ca2+ current,13 14 ß-adrenergic response,15 16 and [Ca2+]i transient amplitude17 in normal cells. Although the former studies on adult cells1 2 3 4 5 6 7 did not analyze [Ca2+]i transient, contraction measurements should follow [Ca2+]i transient unless microtubule disruption modifies myofilament Ca2+ sensitivity. To our knowledge, the only publications presenting an analysis of [Ca2+]i transient during microtubule alteration in adult cardiac myocytes are our recent report, in which an increase in [Ca2+]i transient after longer colchicine treatment was demonstrated,18 and one by Howarth et al,19 who showed a decrease in [Ca2+]i transient after microtubule stabilization with taxol, suggesting that microtubules may be important regulators of Ca2+ handling in the heart.
The Research Commentary from Calaghan et al20 analyzes the effects of colchicine treatment on contraction, [Ca2+]i transient, and ICa in adult rat cardiac myocytes in various experimental settings. Their results confirm our data obtained on myocytes under whole-cell or ruptured patch-clamp. However, under perforated patch-clamp, they found no effect of colchicine. Therefore, they suggested that membrane rupture under the patch pipette tip had influenced the colchicine effects.
In the present study, we demonstrate that colchicine treatment modulated Ca2+ signaling in freshly isolated intact cardiac myocytes: the characteristics of spontaneous Ca2+ sparks were altered in a way that is consistent with ryanodine receptor (RyR) phosphorylation. Moreover, the increase in the [Ca2+]i transient induced by microtubule disruption was reversible on colchicine washout.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Preparation
Adult male Wistar rats (250 to 350 g) were anesthetized with sodium pentobarbital (100 mg/kg IP). Hearts were removed, and single ventricular myocytes were enzymatically (collagenase Worthington II, 1 mg/mL) isolated as previously described.21 Myocytes were stored in a Tyrode solution containing 1 mmol/L CaCl2 and maintained at room temperature. Some cells were supplemented with 1 µmol/L colchicine (dissolved in DMSO at 0.01% final DMSO concentration) at least 2 hours until used for experiments (2 to 6 hours). Experiments were performed on rod-shaped quiescent Ca2+-tolerant myocytes at room temperature (24°C to 25°C).
For experiments, myocytes were perfused with a Tyrode solution containing, in mmol/L: NaCl 140, MgCl2 0.5, KCl 5, glucose 5.5, HEPES 5, and CaCl2 1.8; pH was adjusted to 7.4 with NaOH.
Imaging of Ca2+
Sparks
Imaging was performed on Fluo-3–loaded myocytes
using a laser scanning confocal microscope (Zeiss LSM 510), coupled to
an inverted microscope (Axiovert 100M, Zeiss) and equipped with a x63
water immersion objective, 1.2 numerical aperture (C-Apochromat,
Zeiss). Fluo-3 fluorescence was excited with the 488-nm line of
an argon ion laser. Emitted fluorescence was measured at
wavelengths over 515 nm. Image acquisition was made in the line-scan
mode. A single myocyte was scanned repetitively along a line parallel
to the longitudinal cell axis every 1.5 ms. Image processing and
analysis of Ca2+ sparks were
performed using IDL software (Research Systems), as previously
described.22 Briefly, each
image was background-subtracted. The fluorescence transient was
obtained by averaging the fluorescence values in a 1.4-µm
frame over time. Amplitude was measured as the maximum value of
F/F0, where F is the fluorescence signal
and F0 is the basal fluorescence
(measured as the average of the 50 lowest values on the
fluorescence transient). Decay time was calculated by fitting
the descending phase of the fluorescence trace to a single
exponential. Time to peak was measured in two ways: (1) absolute
time to peak was measured as the time between the maximum of the second
derivative and the peak F/F0 and (2) time to
first peak was measured as the time between the maximum and minimum
values of the second derivative. Half-width was measured from a
transversal fluorescence transient obtained by averaging the
fluorescence values in 15 ms over space and fitted to a Gauss
function. These IDL routines allowed us to analyze a large
number of Ca2+ sparks. When failure to
converge occurred, the parameter was
disregarded.
Field-Stimulation Experiments
Myocytes were loaded with the fluorescence
membrane-permeant Ca2+ dye Fluo-3 AM, as
previously described.21
Cells were placed on a Nikon microscope fitted with an
epifluorescence attachment. Excitation was provided with a
xenon lamp as previously
described.18 The
fluorescence signal was digitized (TL-1, Axon Instruments) and
acquired at a sampling rate of 200 µs using pClamp 6. Cells were
stimulated at a 1.5 excitation threshold through two platinum
electrodes at 1 Hz. Data were acquired after 2 to 3 minutes of
stabilization.
Immunolabeling
Single ventricular myocytes were
permeabilized in a microtubule protective buffer,
followed by 3% formaldehyde
fixation.23 Antibodies were
used as previously
described.18 Confocal images
were acquired with the Lasersharp program. Analysis was
performed with IDL (Research Systems). 3D reconstruction of the Z-stack
was achieved with Imaris (Bitplane).
Statistics
Data are presented as mean±SEM. An unpaired
Student’s t test was performed
to compare control and colchicine-treated cells, and a paired
Student’s t test was used to
compare drugs effects within each group.
P<0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of Microtubule Disruption on Ca2+ Sparks
Cardiac excitation-contraction (EC) coupling is activated by the calcium-induced calcium release mechanism24 : membrane depolarization during each action potential activates sarcolemmal L-type calcium currents (ICa), leading to a local increase in [Ca2+]i around the neighboring sarcoplasmic reticulum (SR) Ca2+ release channels (RyRs). The activation of RyRs by Ca2+ triggers a large release of Ca2+ that activates cell contraction. The global [Ca2+]i transient is constituted by the spatial and temporal summation of its elementary units, Ca2+ sparks.25 Ca2+ sparks are localized Ca2+ transients that can be visualized in cardiac myocytes by using confocal imaging and appropriate Ca2+ dyes such as Fluo-3. They correspond to the synchronous activation of a cluster of RyRs. Consequently, in intact cells, RyR activity can be studied by analyzing Ca2+ spark properties.
Spontaneous Ca2+ sparks were
analyzed in colchicine-treated myocytes and compared with those
recorded in control myocytes.
Figure 1
shows averaged line-scan images corresponding to
Ca2+ sparks recorded in a cell stored in
the absence
(Figure 1A) or presence
(Figure 1B) of 1 µmol/L colchicine. To provide a better
impression of average Ca2+ spark
fluorescence intensity before and after colchicine treatment,
each image is also represented by its 3D projection. It
is clear that the fluorescence intensity decreased after
microtubule disruption.
Figure 1C shows the bar graph of the averaged
Ca2+ spark amplitude. After colchicine
treatment, Ca2+ spark amplitude was
significantly smaller. The frequency histogram
(Figure 1D) shows a shift toward
Ca2+ sparks of lesser amplitudes. The
decrease in Ca2+ spark amplitude could be
due to a smaller SR Ca2+ load. We
analyzed the SR Ca2+ load by rapid
caffeine application.21
Figure 2A shows line-scan images of caffeine-induced
Ca2+ release in a control (top) and a
colchicine-treated cell (bottom). Averaged data
(Figure 2B) show that microtubule disruption did not
significantly modify SR Ca2+ content in
quiescent cells, although there is a tendency to increase. Fluo-3 is a
highly sensitive Ca2+ dye, and the
fluorescence changes begin to be nonlinear at very high
[Ca2+]. Therefore, it is possible that
even if the fluorescence ratio increase did not reach
statistical significance after colchicine treatment, there may be an
increase in SR Ca2+ load, but in no case
would there be a decrease. Thus, the decrease in
Ca2+ spark amplitude was not due to a
reduction in the SR Ca2+ content.
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The characteristics of Ca2+
sparks are given in the
Table.
In addition to Ca2+ spark amplitude, the
only other parameter significantly altered by microtubule
disruption was the time to peak, which was prolonged
(Figure 3C). Further analysis of the lengthening in
time to peak after colchicine treatment led us to observe
[Ca2+]i peaks in
doublets
(Figures 3A and 3B). Then, when we analyzed the time
to the first peak instead of the time to the absolute peak, no
significant difference was found
(Table).
More than one peak (generally two) was observed in 9.6% of the
Ca2+ sparks recorded after microtubule
disruption. The frequency histograms corresponding to the distance and
time between the first and second peaks are shown in
Figure 3D. Most of the Ca2+
sparks in doublets occurred at the same spot or were very close to each
other. The second peak rose 
15 to 20 ms after the first one. Thus,
colchicine applied to intact quiescent cells significantly modulated
Ca2+ spark characteristics, without altering
the SR Ca2+ load.
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Field-Stimulated
[Ca2+]i
Transients
We previously reported that microtubule disruption by
colchicine increases global
[Ca2+]i transient
under whole-cell
patch-clamp.18 This result
has been challenged by Calaghan et
al.20 They suggested that
the increase in
[Ca2+]i transient
is related to the whole-cell patch-clamp technique, which implies the
rupture of the membrane under the pipette tip. To further examine
whether microtubule disruption could modulate
Ca2+ signaling in intact myocytes, we
analyzed
[Ca2+]i transients
in field-stimulated myocytes.
Figure 4 compares sample traces of fluorescence
[Ca2+]i transients
obtained in a control and a colchicine-treated myocyte. Isoproterenol
application increased the
[Ca2+]i transient
in the control but was without effect on the colchicine-treated myocyte
that originally demonstrated a larger
[Ca2+]i transient.
Pooled data are shown in
Figure 4C. On average,
[Ca2+]i transient
amplitude increased 1.4-fold after microtubule disruption, whereas
isoproterenol (1 µmol/L) significantly increased
[Ca2+]i transient
in control but not in colchicine-treated cells. These experiments were
performed in a standard solution (see Materials and Methods) containing
1.8 mmol/L Ca2+. Although this is a
Ca2+ concentration generally used to mimic
physiological conditions, in some investigations
1 mmol/L Ca2+ is used. In this latter
condition, the
[Ca2+]i transient
amplitude of 9 colchicine-treated cells was 1.34-fold that recorded
in 10 control cells.
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The decay time (
) of the
[Ca2+]i transient
was estimated by fitting to a first-order exponential function.
Similarly to the decay of the
ICa-evoked
[Ca2+]i
transient,18 the
field-stimulated
[Ca2+]i transient
decay was accelerated in colchicine-treated cells (reduced to 75%)
(Figure 4D
). This is consistent with the reduction to
80% reported by Calaghan et
al20 at the same
[Ca2+]o (see their
Table). Sarcoplasmic reticulum (SR)
Ca2+-ATPase (SERCA) is the main factor
contributing to
[Ca2+]i transient
decay in rat cardiac
myocytes.26 Its activity is
enhanced after phospholamban phosphorylation by protein
kinase A (PKA).26 The faster
[Ca2+]i transient
decay after colchicine treatment is in line with such a PKA activation.
Furthermore, as is shown in
Figure 4D, isoproterenol reduced the
[Ca2+]i transient
decay time in control cells, but it had no significant effect after
microtubule disruption.
These results are consistent with our previous report on patch-clamped myocytes showing both an increase in [Ca2+]i transient and a loss of ß-adrenergic effect after colchicine treatment, as well as with the proposal that PKA is activated after Gs protein stimulation by free tubulin.18
Rescue of the Microtubule Network
It is often assumed that microtubule disruption by
colchicine is poorly reversible; thus, most experiments are performed
in control conditions on colchicine-treated cells.
Figure 5A shows anti–ß-tubulin–labeled images of a
control (top) and a colchicine-treated (middle) myocyte. Colchicine
treatment markedly disrupted the microtubules, which was reflected by a
decrease in fluorescence
(Figure 5B). However, the immunolabeling of cells that were
similarly treated by colchicine and then allowed a 30-minute washout
demonstrated polymerized microtubules
(Figure 5A, bottom), which was reflected by an increase in
fluorescence
(Figure 5B).
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Microtubule repolymerization after colchicine removal may
have functional consequences. This is illustrated by the comparison of
[Ca2+]i transients
evoked by field stimulation on a colchicine-treated cell at the
beginning of the experiment
(Figure 5C, a1) and after a 30-minute
colchicine washout
(Figure 5C, a2). After this delay,
[Ca2+]i transients
had diminished toward control values. However, when colchicine was
present throughout the experiment, the fluorescence
[Ca2+]i transient
remained about constant over the 30-minute period
(Figure 5C, b). Average variations are presented in
Figure 5C, bottom.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microtubules, which are components of the cytoskeleton, have a structural role and are generally acknowledged to be important to cell locomotion and mitosis. In adult cardiac myocytes, their role has been neglected because of their relatively low abundance. Tubulin concentration in these cells represents
0.01% of total
myocyte
protein.27 28
However, a role as a second messenger could be important, because free
tubulin has been shown to bind and activate G
proteins.29 30 31 32 33
In general, the biological function of microtubules is based on the
ability of tubulin to polymerize and depolymerize. Proper control of
microtubule dynamics is essential for many microtubule-dependent
processes. The present work extends our previous
data.18 In the present
study, we demonstrate that microtubule disruption modulates
Ca2+ signaling in intact rat
ventricular myocytes. Microtubule
depolymerization by colchicine modulates
spontaneous Ca2+ spark characteristics in
quiescent cells and increases
[Ca2+]i transient
in field-stimulated cells. Furthermore, we show that the colchicine
effects are partially reversible.
We previously reported that adenylyl cyclase (AC) is
involved in the colchicine-induced alteration of
Ca2+ signaling. The resulting PKA activation
induces phosphorylation of the L-type
Ca2+ channel. This would account for the
increase in the whole-cell
ICa and
global [Ca2+]i
transient.18 PKA has also
been shown to phosphorylate
RyRs,34 which dissociates
them from FKBP12.635 and
sorcin.36 Furthermore,
dissociation of FKBP12.6 from RyR channels incorporated into planar
bilayers results in subconductance states because of uncoupling of the
RyRs that will not open
synchronously.35 The
decrease in Ca2+ spark amplitude found in
cardiomyocytes after microtubule disruption may well
reflect these alterations. Moreover, assuming the
Ca2+ spark amplitude is decreased because
fewer RyR channels open simultaneously to produce one
spark, the increase in
[Ca2+]i around the
quiescent subset of RyRs on the same cluster may activate them,
producing a second increase in
[Ca2+]i. This
possibility may underlie the increase in time to peak after microtubule
disruption presented in
Figure 3C. Another possibility is the reported acceleration
of RyR adaptation after
phosphorylation.34
The finding that after microtubule disruption many
Ca2+ sparks are in doublets, together with
the decrease in their peak amplitude, may indicate
phosphorylation of the RyRs. This is consistent
with our initial proposal that PKA is activated after
microtubule disruption and is schematized in
Figure 6.
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At first glance, our results in field-stimulated myocytes
(Figure 4) are inconsistent with the data
presented by Calaghan et
al.20 Our two sets of
results differ in two aspects: the maintenance or
nonmaintenance of the full ß-adrenergic response and the
similarity or difference of the
[Ca2+]i transient
amplitude in control and colchicine-treated cells. As shown in their
Table, Calaghan et al20
observed that colchicine, at 10 µmol/L, induced a significant
acceleration of the
[Ca2+]i transient
decay in intact myocytes, as we did. Also, from the 30 cells
presented in their Table, Calaghan et al studied the
isoproterenol response in a subset of 9 cells. This subpopulation
exhibited [Ca2+]i
transient characteristics, such as the decay time constant, that were
significantly different from those of the whole population (compare
Table and Figure 3 in Calaghan et
al20 ). Their conclusion
should thus be taken with caution. Furthermore, the bathing solution
used by Calaghan et al contained insulin, which is known to increase
ICa in
rat cardiac myocytes.37
Insulin effects on rat ventricular myocytes are only seen
in cells with an intact microtubule
network.38 It is thus
possible that
[Ca2+]i transients
were similar in control and colchicine-treated cells because
ICa was
increased by insulin only in the control myocytes, whereas it was
increased by microtubule disruption in the colchicine-treated cells. An
alternative explanation would be the low-stimulation frequency used by
Calaghan et al,20 twice as
slow as the stimulation frequency they used in another study on the
decrease in [Ca2+]i
transient after microtubule
stabilization.19
Despite the difficulty, or impossibility, of accurately measuring ICa in rat ventricular myocytes without blocking K+ currents, Calaghan et al20 studied ICa under two variations of the same technique: conventional whole-cell patch-clamp and perforated patch-clamp. These investigators have obtained different results with these two techniques and suggested two major mechanisms to account for the different results: (1) the loss of cellular tubulin activates AC and (2) microtubule disruption renders cells more susceptible to mechanical stimulation.
The first suggestion is weakened by the following
observations: (1) If free tubulin acts by binding and activating
Gi protein in cardiac myocytes, after
microtubule stabilization with taxol less tubulin will be free to
activate Gi. Thus, no change or increase
in [Ca2+]i
transient would be expected after taxol application to intact cells.
However, the opposite was demonstrated by Howarth et
al.19 (2) Perforated
patch-clamp was initially developed to reduce dialysis in small and
spherical cells.39 Given the
cardiac myocyte shape and the molecular weight of the complex
ß-tubulin–Gi protein, it is unlikely that
free tubulin is rapidly lost. Indeed, we recorded an increased
ICa in
colchicine-treated ventricular myocytes even just after
rupture of the patch (see Figure 7 in Reference 1818 ) that argues against
the dialysis dependence of microtubule-disruption effects. (3) It is
widely accepted that carbachol activates
Gi protein and thus inactivates AC
and decreases
ICa,
only if this pathway has been previously stimulated by PKA
activation.40 After
microtubule disruption, muscarinic stimulation effectively decreases
ICa on
whole-cell patch-clamped myocytes (Gallo MP, Malan D, Bendeti I, Biasin
C, Alloatti G, Levi R, unpublished data, 2000) and isoproterenol
recovers its activity (data not shown). This demonstrates both that the
lack of isoproterenol effect is not due to intracellular dialysis and
that Gi protein is not lost by dialysis. The
Gs protein pathway is predominant in adult
cardiac myocytes, whereas the Gi protein is
predominant in neonatal
myocytes.41 The entire set
of data presented in this report and observed in intact cells
argues against this hypothesis.
The finding by Calaghan et
al20 that after 13 minutes
of ruptured-patch achievement colchicine-treated myocytes are
shrunk—compared with what occurs in control myocytes after 10
minutes—is surprising assuming no osmolarity differences between
solutions and no negative pressure inside the pipette. Nevertheless,
the suggestion that AC is activated as a result of mechanical
stimulation, if any, in patch-clamp does not hold because the family of
AC that is activated by cell deformation is not present in
heart. Cardiac myocytes contain mainly type V and VI of AC, which are
colocalized with the L-type Ca2+
channel42 and inhibited by
low
[Ca2+].43 44 45 46
The results presented in the present report were performed
in myocytes with intact sarcolemma (neither ruptured nor perforated)
and were consistent with the activation of AC after microtubule
disruption we reported earlier. We observed that a 30-minute washout
period in a colchicine-free solution allows for rescue of the
microtubule network in colchicine-treated myocytes
(Figure 5). Furthermore, together with tubulin
repolymerization,
[Ca2+]i transient
elicited in field-stimulated myocytes is decreased. Other authors have
also reported tubulin repolymerization on cardiac
myocytes10 and isolated rat
hearts.47
The discordance between the results of Calaghan et
al20 obtained under two
variations of the patch-clamp technique is unexpected. The strength of
their argument is, however, weakened first by the technical
difficulties related to the large series resistance in the perforated
mode and, more importantly, by the maze of steps required to measure
ICa in
rat ventricular myocytes without effectively blocking
K+ currents. Rat cardiocytes exhibit
several outward K+ currents, including a
large transient one
(Ito),
in a range of potentials overlapping with the one inducing activation
of the inward
ICa.48 49
Furthermore, the data of Calaghan et al in intact cells is not
internally consistent (compare Table and Figures 3D and 2C in
Calaghan et al20 under the
same conditions). They do find an effect of colchicine in intact cells:
an increase in
[Ca2+]i
(P<0.01) and an acceleration
of [Ca2+]i
transient decay (P<0.001).
This last effect is characteristic of AC activation. Colchicine, in
their hands, blocks some response to isoproterenol (their Figure 3C).
The presence of insulin in their bathing solution and the selection of
cells to analyze ß-adrenergic response (see previous
discussion) may account for the apparent discrepancy with our results.
In any case, they do observe some effects of colchicine on intact
cardiomyocytes that would instead suggest that the
perforated patch introduces some pitfalls, leading to a masking of
colchicine effects.
Taken together, the present results demonstrate that microtubule disruption by colchicine does modulate Ca2+ handling in intact ventricular myocytes. Both Ca2+ spark characteristics in quiescent myocytes and Ca2+ transients in field-stimulated cardiomyocytes are altered by microtubule disruption. Microtubules in heart may thus have important implications in physiological and pathological conditions in which the level of tubulin polymerization is altered.
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Acknowledgments |
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The Zeiss confocal microscope was bought thanks to financial support from INSERM and IFR3 (Institut National de la Santé et la Recherche Médicale, Institut Fédératif de Recherche No. 3), an HMR (Hoechst-Marion-Roussel) research grant, the AFM (Association Francaise contre les Myopathies), and the Région Languedoc-Roussillon. B.G.K. is a fellow from GRRC (Groupe de Réflexion sur la Recherche Cardiovasculaire), and A.M.G holds a CNRS (Centre National de la Recherche Scientifique) researcher position. We are indebted to Dr J.-L. Samuel for advice on ß-tubulin immunolabeling and Dr E. Scarfone for help with confocal imaging. We also thank Dr J.-P. Bénitah, Dr C. Delgado, and M.P. Heredia for helpful comments.
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Footnotes |
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Original received December 18, 2000; revision received March 22, 2001; accepted March 22, 2001.
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References |
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