1 Programme in Cell Biology, Insulin stimulates glucose uptake into muscle
and fat cells via recruitment of the glucose transporter 4 (GLUT-4)
from intracellular store(s) to the cell surface. Robust stimulation of
glucose uptake by insulin coincides with the expression of GLUT-4
during differentiation of muscle and fat cells, but it is not known if
GLUT-4 expression suffices to confer insulin sensitivity to glucose
uptake. We have therefore examined the effect of expression of a myc
epitope-tagged GLUT-4 (GLUT-4myc) into L6 myoblasts, which do not
express endogenous GLUT-4 until differentiated into myotubes. Ectopic
expression of GLUT-4myc markedly improved insulin sensitivity of
glucose uptake in L6 myoblasts. The GLUT-4myc protein distributed
equally to the cell surface and intracellular compartments in
myoblasts, and the intracellular fraction of GLUT-4myc further
increased in myotubes. In myoblasts, the intracellular GLUT-4myc
compartment contained the majority of the insulin-regulatable amino
peptidase (IRAP) but less than half of the GLUT-1, suggesting
segregation of GLUT-4myc and IRAP to a specific cellular locus. Insulin
stimulation of phosphatidylinositol 3-kinase and protein kinase B-
GLUT-4 translocation; GLUT-4 vesicles; insulin action; muscle cells
INSULIN PROMOTES the rapid uptake of glucose from the
circulation into muscle and fat. These two tissues are unique in their expression of the glucose transporter 4 (GLUT-4) isoform of facilitated hexose transporters (2, 3, 6, 15, 16). It is now widely accepted that
insulin stimulates glucose uptake into muscle and fat cells via the
recruitment of GLUT-4 glucose transporters from an intracellular store
to the cell surface (2, 5, 13, 15, 22). The development of muscle and
fat cell lines has facilitated our understanding of this process,
specifically the rat L6 myogenic line and the mouse 3T3-L1 adipogenic
line, which are unique in their expression of the GLUT-4 protein (9,
28). Both of these cell lines undergo differentiation in culture, from myoblasts into myotubes in the case of L6 and from fibroblasts into
adipocytes in the case of 3T3-L1. In both models, GLUT-4 expression
occurs on and after differentiation into myotubes (27) or adipocytes
(7). Similarly, the expression of GLUT-4 has been shown to be
developmentally regulated in rodent skeletal muscle (33). Both L6
myoblasts and 3T3-L1 fibroblasts display only a minor stimulation of
glucose uptake on an insulin challenge (7, 21, 27). In contrast, both
L6 myotubes and 3T3-L1 adipocytes mount a robust stimulation of glucose
uptake in response to the hormone. The magnitude of the maximum
stimulation of glucose uptake by insulin relative to the basal-state
uptake is smaller for L6 myotubes (2-fold) than for 3T3-L1 adipocytes
(~10-fold). This correlates with the much smaller response to the
hormone displayed by isolated skeletal muscles (2- to 5-fold) (12, 31,
42, 45) relative to isolated adipocytes (>20-fold; Refs. 17, 34, 35,
40). Differences in species also contribute to the range of insulin
responsiveness, rodent tissues being more responsive than their human counterparts.
The stimulation of glucose uptake coincides with the expression of
GLUT-4 in muscle and fat cells, leading to the suggestion that GLUT-4
may be a determinant of the maximum insulin response. However, this
scenario has been challenged by the observation that ectopic expression
of GLUT-4 into 3T3-L1 fibroblasts or C2C12 myoblasts and myotubes did
not elevate the responsiveness of glucose uptake to insulin (11, 14,
23). Therefore, it has been hypothesized that other factors that appear
during the differentiation of these cells contribute to conferring
insulin responsiveness to the newly expressed GLUT-4 protein.
In the present study, we examined the effect of ectopic expression of a
myc epitope-tagged GLUT-4 (GLUT-4myc) into L6 myoblasts on insulin
action. We report that the expression of this protein alone confers
insulin sensitivity to glucose uptake, of a similar magnitude to that
attained during normal differentiation of the cells into myotubes. We
further demonstrate that the magnitudes of insulin stimulation of the
lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) and of the
serine/threonine kinase protein kinase B- Cell culture and glucose uptake. L6
myoblasts expressing a GLUT-4 protein with an exofacial myc epitope
tag (L6-GLUT-4myc) were constructed with a clone of L6
myoblasts selected for high fusion (27), as described by Kishi et al.
(20). L6-GLUT-4myc or parental L6 myoblasts were grown in Cell surface detection of GLUT-4myc.
Cell surface detection of GLUT-4myc was carried out as previously
described (43) with slight modifications. Briefly, after incubations
with or without insulin at 37°C as indicated, cells were washed
twice with PBS at 4°C, incubated with 3% BSA for 10 min, and then
reacted with the anti-myc antibody 9E10 (Santa Cruz, CA) in 10% goat
serum + 3% BSA in PBS for 1 h at 4°C. After being washed four
times with ice-cold PBS, cells were fixed with 3% paraformaldehyde for 3 min at 4°C. After the fixation, cells were incubated with 0.1 N
glycine in PBS for 10 min at 4°C, washed with PBS, and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG
(Jackson Laboratories) for 30 min at 4°C. The cells were
extensively washed with PBS, and then 1 ml/well of 0.4 mg/ml
o-phenylenediamine dihydrochloride
reagent (Sigma), prepared according to the instructions of the
manufacturer, was added for 30 min at room temperature. The reaction
was stopped by addition of 0.25 ml of 3 N HCl, the supernatant was
collected, and the optical absorbance was measured at 492 nm. To
measure total cellular GLUT-4myc, serum-starved cells were washed twice
with PBS and fixed with 3% paraformaldehyde for 3 min at 4°C and
then incubated with 0.1 N glycine in PBS for 10 min, followed by a
15-min incubation with 0.1% (vol/vol) Triton X-100 at 4°C. After
being washed with PBS, the cells were blocked with 10% goat serum and
3% BSA for 30 min and then incubated with the anti-myc antibody (9E10)
for 1 h at 4°C. After being washed with PBS, the cells were
incubated with HRP-conjugated donkey anti-mouse IgG as above, followed
by reaction with o-phenylenediamine dihydrochloride reagent as described previously.
PI 3-kinase and PKB- Characterization of GLUT-4- or IRAP-containing
vesicles. Immunoisolation of GLUT-4- or IRAP-containing
compartments ("vesicles") from the light-density microsome
fraction of L6-GLUT-4myc myoblasts were prepared as previously
described for parental L6 cells (37). Briefly,
L6-GLUT-4-myc myoblasts were fractionated to remove nuclei, mitochondria, and plasma membranes (30). The supernatant containing low-density microsomes was adjusted to 100 mM potassium phosphate, pH
7.4, and the protein concentration was measured with the bicinchoninic acid reagent (Pierce Chemical, Rockford, IL) according to the instructions of the manufacturer. An aliquot of 50 µg protein of the
microsome sample was incubated with 28 µl of anti-IRAP monoclonal
antibody precoupled to M-500 magnetic Dynabeads (Lake Success, NY)
coated with sheep anti-mouse IgG (ICN). Alternatively, an aliquot of
200 µg protein of the microsome sample was incubated with 2 µg of
anti-GLUT-4 monoclonal antibody 1F8 (Biogenesis, Sandown, NH)
precoupled to M-500 magnetic Dynabeads coated with sheep anti-mouse
IgG. Vesicles attached to the beads (termed Pt for immunocomplex
pellet) were concentrated via a magnet and washed three times with PBS.
The supernatants were subjected to centrifugation at 200,000 g for 60 min, and the sedimented
membranes (termed SN for immunocomplex supernatant) were resuspended in
Laemmli's sample buffer (24) and analyzed by SDS-PAGE on 7.5%
polyacrylamide gels to detect contents of IRAP, GLUT-1, and GLUT-4.
Immunoblotting was carried out with polyclonal anti-serum against
GLUT-4 (1:1,000), GLUT-1 (1:2,000; Biogenesis, Sandown, NH), or
monoclonal antibody mixture against IRAP (1:1,000; raised to 110 amino
acids from NH2-terminus fused GST-IRAP, a kind
gift from Dr. M. Birnbaum, University of Pennsylvania, Philadelphia,
PA). Immunoreactive bands were visualized by enhanced chemiluminescence
with appropriate secondary antibodies coupled to HRP. The bands on
X-ray films were quantified within the linear densitometry range with
National Institutes of Health Image software.
We have previously reported that, in contrast to L6 myotubes, L6
myoblasts do not respond significantly with an increase in glucose
uptake to an acute exposure to insulin (21). The low responsiveness and
sensitivity to insulin of parental L6 myoblasts suggest that the
endogenous GLUT-1 and GLUT-3 transporters present in these cells (1,
27) are largely unresponsive to the hormone at this cellular stage. The
insulin-stimulated glucose uptake dose-response curves of the parental
L6 myoblasts and myotubes are illustrated in Fig.
1A. The
results are expressed as hexose uptake per unit time per well of
confluent myoblasts or myotubes to compare uptake into the same number
of cells. Parental L6 myoblasts only began to display an insulin
response when exposed to 100 nM concentrations of the hormone, whereas
the parental myotubes showed a statistically significant response
beginning at 1 nM insulin. These parental L6 myotubes showed a maximal
response of twofold at 100 nM insulin, a concentration typically used
in studies of insulin action in L6 myotubes and 3T3-L1 adipocytes. The
rate of glucose uptake in response to 100 nM insulin was statistically different in parental myoblasts and in myotubes
(P < 0.05).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
activities was similar for L6-GLUT-4myc myoblasts and myotubes. At both
stages, GLUT-4myc responded to insulin by translocating to the cell
surface. These results suggest that GLUT-4myc segregates into a
specific compartment in L6 myoblasts and confers insulin sensitivity to these cells. L6-GLUT-4myc myoblasts, which are easily transfectable with various constructs, are a useful resource to study insulin action.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
(PKB-
/Akt1)
are similar in myoblasts as in myotubes. Furthermore, the ectopically
expressed GLUT-4myc protein is targeted to both an intracellular
compartment and the plasma membrane in myoblasts, and the intracellular
localization of GLUT-4myc is further accentuated in myotubes. The
intracellular GLUT-4myc compartment of myoblasts contains all of the
intracellular insulin-regulatable aminopeptidase (IRAP) but excludes,
in part, the endogenous glucose transporter isoform GLUT-1. Finally,
insulin causes a twofold increase in the amount of GLUT-4myc at the
myoblast cell surface, matching the extent of stimulation of glucose
uptake. We conclude that L6-GLUT-4myc myoblasts are a useful cell
system to study insulin regulation of GLUT-4 traffic.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
-MEM
containing 2% FBS. Myoblasts were used as they reached confluence
before any evidence of differentiation (cell fusion). For all
experiments, L6-GLUT-4myc or parental L6 cells were serum depleted for
5 h in
-MEM culture medium before insulin stimulation. Hexose uptake
into L6-GLUT-4myc or parental L6 myoblasts and myotubes was
measured as previously described (19).
/Akt1 activity
assays. The in vitro activity of PI 3-kinase associated
with IRS-1 immunoprecipitates, and of PKB-
/Akt1 in PKB-
/Akt1
immunoprecipitates, was assayed exactly as described previously (19,
36).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Dose-response curve of insulin on 2-deoxyglucose uptake in parental L6
and L6-GLUT-4myc myoblasts and myotubes. L6 parental
(A) or L6-GLUT-4myc
(B) myoblasts ( ) and myotubes
(
) were serum deprived for 5 h before treatment with various
concentrations of insulin for 30 min. Cells were washed twice with
HEPES-buffered saline, pH 7.4, and the rate of
2-[3H]deoxyglucose
uptake was measured as described in
METHODS. Uptake rates are expressed in
pmol · min
1 · well
1.
Data points are means ± SE of triplicate determinations for 2 or 3 independent experiments in the case of L6 parental cells or
L6-GLUT-4myc cells, respectively. Statistical analysis was performed
with paired Student's t-test.
* P < 0.05 vs. basal rate for
myotubes; # P < 0.05 vs. basal
rate for myoblasts.
In contrast to the lack of response of L6 parental myoblasts to hexose uptake, L6-GLUT-4myc myoblasts displayed higher insulin-responsive glucose transport (Fig. 1B). L6-GLUT-4myc myoblasts showed statistically significant increases in glucose uptake on exposure to 1 nM insulin, and at 100 nM insulin showed a gain in glucose uptake of 103% above the basal value (i.e., 2-fold stimulation). The sensitivity to insulin displayed by L6-GLUT-4myc myoblasts was similar to that of L6-GLUT-4myc myotubes (Fig. 1B). Considering the highest stimulation values shown in Fig. 1 as maxima, the concentrations of insulin yielding half-maximal stimulation of glucose uptake were different for parental L6 myoblasts (80 nM) than for any of the other cells (18 nM for parental L6 myotubes, 13 nM for L6-GLUT-4myc myoblasts, and 20 nM for L6-GLUT-4myc myotubes).
The results in Fig. 1 are expressed per well of confluent myoblasts or myotubes in an attempt to compare the same number of cells in each state. When expressed per unit protein, the myotubes appear to have a lower rate of transport given that they have a higher amount of protein than the myoblasts (see next paragraphs). Reported per well, the responsiveness of the L6-GLUT-4myc myotubes was somewhat higher than that of the parental L6 myotubes, possibly due to the contribution of both the endogenous and transfected versions of the glucose transporter. Indeed, we have recently reported that roughly similar levels of protein expression of the endogenous and the transfected GLUT-4 are attained in the L6-GLUT-4myc myotubes (43). These results also indicate that the level of expression of GLUT-4myc in the myoblasts resembles the endogenous level of GLUT-4 in the myotubes, minimizing the risk of saturating the elements involved in GLUT-4 sorting.
The exofacial location of the myc epitope on GLUT-4myc allowed us to estimate the proportion of GLUT-4myc exposed at the cell surface or the total cellular content of GLUT-4myc by analyzing either intact or permeabilized cells, respectively. The amount of myc epitope exposed at the surface of nonpermeabilized cells was determined by a quantitative assay based on the colorimetric detection of anti-myc antibody bound to a monolayer of L6 cells. The amount of myc epitope present in the entire cells was determined by permeabilization of the cells with 0.1% Triton X-100 before immunolabeling with anti-myc antibody. The intracellular content was then estimated by subtracting the amount present on the cell surface from the amount in the entire cell. In the myoblast stage, 55 ± 6% (n = 3) of the total GLUT-4myc content was found to be exposed at the cell surface, and the remainder 44 ± 6% (n = 3) was intracellularly located. This suggests that retention mechanisms exist in L6 myoblasts, which effectively hold GLUT-4myc at intracellular locations in the steady state. In the myotube stage, the distribution of GLUT-4myc changed such that only 37 ± 1% (n = 4) remained exposed at the cell surface and 63 ± 1% (n = 4) was retained intracellularly. At the myotube stage, the amount of GLUT-4myc present in total membranes was 70% higher than in myoblasts, expressed per unit of protein (data not shown). Because the total protein content of myotubes was about twice that of the same number of myoblasts, the amount of GLUT-4myc protein may rise by as much as three- to fourfold in myotubes. We speculate that the gain in GLUT-4myc expression might be caused by translational and/or posttranslational mechanisms (e.g., ribosomal transit and/or protein stabilization), given the fact that the transcription of the GLUT-4myc cDNA is determined by the cytomegalovirus promoter, which would presumably have the same activity in myoblasts and myotubes. The amount of GLUT-4myc expressed at the surface of L6-GLUT-4myc myotubes was very similar to that present at the surface of L6-GLUT-4myc myoblasts (1.14 ± 0.03 in myotubes relative to a value of 1.00 ascribed to myoblasts). This result suggests that the power of retention mechanisms, probably in the form of proteins interacting with GLUT-4, increases on cellular differentiation. The GLUT-4 polypeptide expresses retention-endocytosis motifs in both its NH2-terminal and its COOH-terminal sequences (4, 8, 10, 25, 29, 41). Based on the results of GLUT-4myc distribution discussed above, the functionality of these motifs in myotubes does not appear to be markedly compromised by the insertion of the myc epitope.
In an attempt to understand if the signals emanating from the insulin
receptor thought to lead to the mobilization of GLUT-4 also mature
during myogenesis, the activation by insulin of the lipid kinase PI
3-kinase and the serine/threonine kinase PKB-/Akt1 was measured in
both myoblasts and myotubes. We have previously shown that PI 3-kinase
activation by insulin is a prerequisite for insulin-dependent
translocation of GLUT-4 to the cell surface in parental L6 myotubes
(38, 39). Recently, we have shown that activation of
PKB-
/Akt1 by the hormone appears to be required for the
translocation of GLUT-4myc (44) detected by a single-cell immunofluorescence approach. Therefore, we compared the activation of
PI 3-kinase and of PKB-
/Akt1 in L6-GLUT-4myc myoblasts and myotubes.
Table 1 shows that IRS-1-associated PI
3-kinase activity was stimulated by approximately sevenfold upon rapid
exposure to insulin of both GLUT-4myc myoblasts and myotubes.
Similarly, the extent of the response of PKB-
/Akt1 activity to
insulin was comparable (~6-fold) in L6-GLUT-4myc myoblasts and
myotubes (Table 1). This stimulation is equivalent to that reported
earlier for parental L6 myotubes (19). Moreover, the phosphorylation of Ser 473 in PKB-
/Akt1 in response to insulin was virtually identical in the parental as in the L6-GLUT-4myc myoblasts (results not shown).
The equivalent responsiveness of L6 myoblasts relative to L6 myotubes
is in contrast to the poorer response of 3T3-L1 fibroblasts relative to
adipocytes (7).
|
The comparable insulin-dependent stimulation of PI 3-kinase,
PKB-/Akt1, and glucose uptake observed in L6-GLUT-4myc myoblasts and
myotubes led us to compare the extent of GLUT-4myc translocation to the
cell surface at the two stages of differentiation. Table 1 shows that
the hormone doubled the exposure of GLUT-4myc at the surface
of both myoblasts and myotubes, matching the stimulation of glucose
uptake caused under the same conditions. These observations raise the
possibility that L6 myoblasts have the cellular machinery and signaling
pathways required for GLUT-4 translocation, although they do not
naturally express this transporter isoform. Consistent with this
scenario, transfection of untagged GLUT-4 has been reported to increase
insulin-dependent glucose uptake in parental L6 myoblasts (32). This
appears to be a property of rat L6 cells, because GLUT-4 transfection
into mouse C2C12 muscle cells, which do not express endogenous GLUT-4, did not improve their low insulin response of glucose uptake even when differentiated into myotubes (23). It may
be that C2C12 cells do not have the requisite
machinery to mobilize GLUT-4 even in the myotube stage. In contrast,
transfection of GLUT-4myc has conferred an insulin response to
glucose uptake in Chinese hamster ovary cells and
increased the response of 3T3-L1 adipocytes, but the response of
GLUT-4myc- transfected 3T3-L1 fibroblasts was not examined
(18). Neither was the extent of insulin signaling nor the GLUT-4myc
compartment identified in that study.
The ability of GLUT-4myc to lodge in an intracellular location and to
respond to insulin prompted us to characterize this compartment.
Membranes containing GLUT-4myc were immunopurified out of the light-
density microsomes of L6-GLUT-4myc myoblasts with the monoclonal
antibody 1F8 directed to the COOH-terminal domain of GLUT-4. The
immunoisolated vesicles contained the vast majority of the
intracellular GLUT-4myc (Fig. 2).
In contrast, they only contained 40 ± 2%
(n = 3) of the intracellular GLUT-1. These results suggest that GLUT-4 is located in a compartment that in
part excludes GLUT-1. This segregation of the two transporters is
reminiscent of their distribution in 3T3-L1 adipocytes, in which the
oxidative ablation of the endosomal pool obliterated the
immunoreactivity of the majority of the GLUT-1 but of only half of the
GLUT-4 complements (26). Substantial exclusion of GLUT-1 from the
endogenous GLUT-4 pool was also observed in parental L6 myotubes (37).
A similar segregation of GLUT-1 and GLUT-4 was reported for cardiac
myocytes with graded immunopurification of the GLUT-4 compartment (46).
Figure 2 also shows that the immunoisolated vesicles containing
GLUT-4myc also contain the vast majority (96 ± 2%) of the
intracellular IRAP. This suggests that structural motifs present in
both GLUT-4myc and IRAP localize these proteins to the same
intracellular compartment. We have previously shown that IRAP
expression is lower in parental L6 myoblasts compared with L6 myotubes
(37). In L6-GLUT-4myc myoblasts, IRAP expression increased 3.5 ± 0.2-fold on differentiation into myotubes (data not shown).
|
The complete recovery of IRAP in the GLUT-4 intracellular pool suggests
that there is no IRAP in other membranes but does not prove that all
GLUT-4-containing membrane structures also contain IRAP. To examine
this possibility, intracellular membranes containing IRAP were
immunopurified with anti-IRAP specific antibodies. Figure
3 shows that the purification of
IRAP-containing vesicles was complete, as there was no IRAP left in the
supernatant. It also shows that virtually all of the intracellular
GLUT-4 copurified with the IRAP compartment(s). In contrast, the IRAP
compartment contained only 42% of the GLUT-1, the remainder being
recovered in the supernatant. These results corroborate the overlap of
the compartment(s) containing IRAP and GLUT-4 and their segregation from that containing GLUT-1.
|
In conclusion, ectopic expression of GLUT-4myc markedly improved
insulin sensitivity and responsiveness of glucose uptake in L6
myoblasts. The GLUT-4myc protein distributed equally to the cell
surface and intracellular compartments in myoblasts. On differentiation
into myotubes, GLUT-4myc content increased, as did its retention in the
intracellular compartment. In myoblasts, this compartment contained the
majority of the IRAP protein but less than half of the GLUT-1 protein,
suggesting segregation of GLUT-4 and IRAP to a specific cellular locus.
Similar insulin-dependent stimulation of PI 3-kinase or PKB-/Akt1
activities was observed in myoblasts and myotubes. In both myoblasts
and myotubes, GLUT-4myc responded to insulin by translocating to the
cell surface. These cells therefore constitute a cellular system in
which GLUT-4 translocation can be measured in intact cells via
detection of the exofacial myc epitope. Moreover, the cells retain the
ability to differentiate into myotubes, allowing comparisons of
GLUT-4myc translocation in both stages. Collectively, these results
suggest that L6 muscle cells expressing GLUT-4myc are a useful system
for studies of insulin action on glucose transport and that the
myoblast stage, being easily transfectable with various constructs, may
be a useful tool in the analysis of insulin action. In this regard, we
have recently used L6-GLUT-4myc myoblasts to transiently express
dominant-negative versions of Akt to assess the role of this kinase in
GLUT-4myc translocation (44).
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ACKNOWLEDGEMENTS |
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We thank Dr. Philip J. Bilan for helpful discussions and input throughout this study and Dr. Morrie Birnbaum (University of Pennsylvania) for the kind gift of the anti-IRAP monoclonal antibody.
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FOOTNOTES |
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A. Ueyama is a visiting research fellow from Otsuka Pharmaceutical, Tokushima City, Japan. K. Yaworsky was supported by a Hospital for Sick Children Foundation Scholarship at the University of Toronto. Q. Wang was supported by a personnel fellowship award from the Eli Lilly Canada/Banting and Best Diabetes Centre. This work was supported by a grant from the Canadian Diabetes Association to A. Klip.
Permanent address for A. Ueyama: Department of Advanced Pharmacology, Otsuka Pharmaceutical, 463-10 Kagasuno Kawachi-cho, Tokushima 771-0192, Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Klip, Programme in Cell Biology, The Hospital for Sick Children, 555 Univ. Ave., Toronto, Ontario M5G 1X8, Canada (E-mail: amira{at}sickkids.on.ca).
Received 28 February 1999; accepted in final form 24 May 1999.
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