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Department of Structural and Cellular Biology, University of South Alabama, Mobile, Alabama 36688
Address all correspondence and requests for reprints to: Dr. Ron Balczon, Department of Structural and Cellular Biology, University of South Alabama, Mobile, Alabama 36688.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A potential target for either the Ca2+ or ATP signals that are generated during glucose-stimulated insulin secretion is the microtubule cytoskeleton. Pharmacological studies using various microtubule poisons suggest that an intact microtubule cytoskeleton is essential for insulin secretory events (7, 8, 9). However, the level of involvement of microtubules during the insulin secretion process remains to be elucidated. According to one hypothesis (7), the microtubules within the ß-cell may serve as a system for the transport of secretory granules from deep within the ß-cell to the plasma membrane where exocytosis occurs. Evidence supporting this proposal was provided by inhibition studies performed using microtubule poisons (7, 8, 9) as well as studies demonstrating that insulin granules were able to bind to microtubules in an in vitro assay (10). However, detailed microscopic analyses of ß-cells have failed to provide convincing evidence of insulin granule association with microtubules (11, 12). Therefore, it still is not possible to define the role of the microtubule cytoskeleton in the insulin secretory process.
A strategy for determining the role of microtubules in the insulin secretion events is to identify the components of the ß-cell microtubule cytoskeleton and then to investigate the roles of these proteins individually. In a previous study, a ß-cell form of the microtubule adenosine triphosphatase (ATPase) kinesin was identified and purified (13). Kinesin is one member of a family of high mol wt microtubule-associated proteins exhibiting microtubule-stimulated ATPase activity. Kinesin originally was identified in squid axoplasm as a fraction capable of moving either vesicles or fluorescent beads along microtubules in vitro (14, 15, 16, 17). Microtubules are polar structures with one end, the minus end, located at the cell center in association with the centrosome and the other end, the plus end, located toward the cell periphery. In vitro motility assays using asymmetric microtubule constructs with known polarity demonstrated that kinesin is a plus end-directed microtubule motor protein, meaning that kinesin might be responsible for moving vesicles from the cell center toward the cell cortex (16, 18, 19). Kinesin isolated from brains has been the most extensively characterized form of the enzyme, and each molecule of brain kinesin has a mass of approximately 400 kDa consisting of two heavy chains (110–130 kDa each) and usually two light chains of variable mol wt (20, 21). The two kinesin heavy chains (KHCs) contain both the ATPase activity for generating motile force as well as sites of interaction with microtubules and membranous cargo (22, 23, 24). The function of kinesin in neuronal cells and tissues has been investigated, and localization and activity inhibition studies performed using kinesin antibodies and antisense oligonucleotides demonstrated the role of kinesin in fast axonal transport (25, 26, 27, 28, 29). Evidence from these studies suggests that kinesin serves as a motor for anterograde rapid transport of synaptic vesicle precursors and plasma membrane components from the cell body along the axon to the presynaptic terminals in neurons. However, there have been few studies of the role of this motor protein during vesicle movement in nonneuronal mammalian cell types, as little research has been performed to address this issue.
As stated previously, a pancreatic ß-cell kinesin was purified from rat insulinoma tumors and was partially characterized in vitro (13). This novel islet ß-cell form of kinesin showed microtubule-stimulated ATPase activity and was able to translocate microtubules in an in vitro motility assay. Therefore, it is conceivable that this ß-cell form of kinesin can serve as a motor in vivo to generate the locomotive forces responsible for the translocation of insulin storage granules and other intracellular components along microtubules to the cell surface during the insulin secretion process. For the work described in this manuscript, molecular techniques were used that allowed the suppression of KHC expression in primary cultures of ß-cells and an assessment of the role of the KHC in the insulin secretion process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chymotrypsin,
0.03% deoxyribonuclease, and 4% FBS. The ß-cell preparations were
enriched by centrifugation at 2500 x g for 10 min
through a 25.5/20.4/0% discontinuous Ficoll gradient, plated into
multiwell plates (12 wells/plate; Falcon, Becton Dickinson, Lincoln
Park, NJ), and then maintained in medium 199 (Life Technologies,
Gaithersburg, MD) containing 10% FBS, 16.5 mM glucose, and
50 µg/ml gentamicin sulfate. The ß-cells were further purified by
treatment with cysteine-free MEM medium containing 4 µg/ml iodoacetic
acid. The insulin-secreting NIT-1 cell line was maintained as described
previously (13).
Construction and screening of complementary DNA (cDNA)
libraries
cDNA library NIT-la was a generous gift from Dr. Michael Appel
(CytoTherapeutics, Providence, RI). cDNA library NIT-1b was constructed
from NIT cell polyadenylated RNA using the ZAP-cDNA synthesis kit
(Stratagene, San Diego, CA) following the manufacturer’s instructions.
The resulting unamplified library contained 1.04 x
106 plaque-forming units (not shown).
The NIT-la cDNA library was screened by PCR using primers encoding cDNA
regions that were conserved among previously cloned KHC cDNAs, and
clones 1, 2, 3, and 4 were obtained (Fig. 1
). The NIT-1b
cDNA library was screened by plaque hybridization using standard
methods (31) to obtain clone 6. Rapid amplification of cDNA ends using
NIT-1 cell total RNA was performed to obtain clone 5 (32).
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For antisense experiments, oligonucleotides were added at the indicated concentrations (see text) in water directly to the culture medium. Primary ß-cells were prepared and cultured as described above, and the cells were incubated with ATG-antisense, Inner-antisense, or Re-antisense oligonucleotides, respectively. Cells not supplemented with any oligonucleotide also served as a control. Cell morphology was monitored daily, and cell viability was determined by trypan blue dye exclusion. Fresh oligonucleotides were added every 24–36 h. Oligonucleotide uptake and stability were measured according to procedures reported by Wickerstrom et al. (34).
Indirect immunofluorescence
Cells were fixed by immersion in methanol for 6 min at -20 C.
Cells were then permeabilized with 0.1% Triton X-100 in PBS and
blocked with 1% BSA in PBS. For immunofluorescence labeling, cells
were processed through the following steps: 1) incubation for 60–120
min at room temperature with primary antibodies (undiluted or diluted
with 1% BSA in PBS), 2) washing with 1% BSA three times for 15 min
each, 3) incubation with fluorescein isothiocyanate-conjugated
antispecies IgG for 45 min at room temperature, 4) washing with PBS
three times for 5 min each, and 5) mounting the coverslip onto a glass
slide and then sealing with nail polish. Primary antibodies were
monoclonal anti--tubulin (Sigma), SUK 4 monoclonal anti-KHC
(Developmental Studies Hybridoma Bank, Iowa City, IA), or antiinsulin
(Linco Research, St. Charles, MO). Secondary antibodies were obtained
from Boehringer Mannheim (Indianapolis, IN).
Quantification of mKHC expression
The relative level of mKHC expression was quantitated by
measuring the fluorescence intensities within cells using an ACAS 570
Interactive Laser Cytometer (35, 36). Primary cultures of ß-cells
were cultured on coverslips in 12-well plates and were either untreated
or treated with antisense or control oligonucleotides. Cells were
stained as described above using the SUK 4 monoclonal antibody (37),
and immunofluorescence was quantified by comparing fluorescence
intensities collected at identical settings on the scanning laser
confocal microscope. Data representing total fluorescence intensity and
area (square microns) of the cytoplasm across the equatorial plane of
the nucleus of each cell were determined using the DASY 9000 Master
Computer Program. The intensity per µm2 was calculated
for each cell by dividing the total intensity by the total area of
cytoplasm. The background intensity from secondary antibody staining
alone was subtracted from the sample values.
Electron microscopy
Treated and control ß-cells were fixed and processed for
electron microscopy using previously reported procedures (38).
RNA purification and analysis
Total RNA was isolated from monolayers of primary cultures of
ß-cells using the RNA Stat-60 reagent (Tel-Test "B", Friendswood,
TX). Northern blot analysis was performed using slight modifications of
previously reported procedures (38). Briefly, RNA was denatured with
glyoxal and fractionated on 1.0% agarose gels containing 10
mM sodium phosphate. RNA was then transferred to a nylon
membrane and immobilized by UV cross-linking. Hybridization with either
the ß-cell KHC cDNA or the antisense oligonucleotide probe was
carried out at 37 C in a solution of 6 x SSPE, 1% SDS, 10%
dextran sulfate, 1 x Denhardt’s solution, and denatured salmon
sperm DNA (120 µg/ml) with a final stringency wash in 3 x SSC
(standard saline citrate), and 0.25% SDS at 42 C. The KHC cDNA was
labeled using procedures that were reported previously (38), and the
antisense oligonucleotide probe was 5'-end labeled (32) with
bacteriophage T4 polynucleotide kinase (Promega, Madison, WI).
Insulin RIA
Insulin released into the culture medium was determined by RIA
using either a forward assay (39) or a back titration assay (40).
Guinea pig antirat insulin serum,
mono-[125I]Tyr14-insulin, and goat antiguinea
pig IgG used in the assays were purchased from Linco Research. For both
assays, the radioactivity in the pellet was determined using a Tracor
analytic 
-counter. The coefficients of variation were 0.04–5.9%
for intraassays and 8.1–8.6% for interassays, respectively.
Statistics
Each experiment was performed in triplicate. Unless specifically
indicated, all results are expressed as the mean ±
SEM with each experiment repeated at least three times.
One-way ANOVA was used to determine differences among the three
antisense oligonucleotide exposures and untreated cells followed by
Tukey’s honestly significant difference post-hoc test for
multiple comparisons. Two-way ANOVA was used to determine repeated
measurements (i.e. comparison for multiple time points in
multiple groups). The Systat statistics software package (Systat,
Evanston, IL) was used for statistical analysis. Differences were
considered statistically significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The isolated mKHC cDNA
sequences were 3791 bp in length, excluding the polyadenylated tail.
The protein-coding sequence encoded a primary translation product of
963 amino acids with a molecular mass of 109,549 daltons. The amino
acid sequence of the mKHC displayed considerable identity (70.8%)
throughout its length to the mouse brain KHC (41), with the homology
being lost in the 5'- and 3'-untranslated regions. Analysis of the
deduced amino acid sequence determined that the mKHC exhibited the
characteristics of a classical KHC with an N-terminal globular head
containing a conserved microtubule-binding domain, an -helical stalk
region, and a globular C-terminal domain.
Oligonucleotide uptake and stability
Antisense inhibition of gene expression involves cellular uptake
of the antisense oligonucleotide followed by hybridization of the
exogenous nucleotide sequence to the target messenger RNA (mRNA).
Exogenously introduced oligonucleotides would not be effective agents
for hybridization arrest if they were rapidly hydrolyzed. To examine
the stability of phosphorothioate-modified oligonucleotides after
uptake by primary cultures of mouse ß-cells, aliquots of labeled
anti-mKHC oligomer were added to ß-cells in culture medium, and the
cells were incubated for up to 40 h. Cells were collected at
various times after oligonucleotide addition, and DNA was isolated from
the cells. The amount of labeled oligonucleotide taken up by the cells
and the stability of the probe then were determined. As shown in Fig. 2, labeled oligonucleotides were found associated with
cell pellets after 16 h, and by 40 h up to 1–2% of the total
oligonucleotide was present in the cells. At 40 h, no additional
labeled oligo could be detected in the medium (not shown), and the
remainder of the probe presumably was degraded by nucleases present in
the medium and/or cells.
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). Compared to untreated cells, treatment with 1
µM ATG-antisense lowered kinesin expression by 10%,
whereas treatment with 2 µM of this oligonucleotide had a
more profound reduction of kinesin expression (42%). Treatment with 4
µM of the ATG-antisense oligo resulted in a general toxic
effect, as determined by cell viability studies (data not shown).
Therefore, the concentration of antisense oligonucleotide used in
experiments to study the effect of antisense treatment on kinesin
expression and insulin secretion was 2 µM. Importantly,
the effect of the ATG-antisense oligo in suppressing levels of kinesin
expression was specific. As shown in Fig. 3B, the amount of mKHC
protein expression in cells treated by either Inner-antisense or
Reversed-antisense alone was not significantly changed whereas
antikinesin immunofluorescence levels were decreased by 40–50% in
cells treated with the ATG-antisense oligonucleotide.
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), and the size of the transcript
hybridized by the antisense oligonucleotide corresponded well to that
of the mKHC transcript identified using the mKHC cDNA as a probe (Fig. 4).
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). The effects of
the Inner-antisense and Re-antisense oligonucleotides on intracellular
insulin content were also studied, and no significant effects were
observed.
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, antisense-treated cells exhibited no obvious
disruption of cell morphology and were indistinguishable from untreated
cells after fixation and processing for electron microscopy. Electron
microscopic investigation of ATG-antisense-treated cells demonstrated
that the cells were well granulated and contained well organized
intramembranous organelles.
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demonstrates that when cells were
maintained in 5.5 mM glucose-containing medium, the level
of insulin secretion decreased to 50% by 84 h of treatment with
mKHC ATG-antisense, and the decrease was progressive and corresponded
well with the time course of treatment. In contrast, there was no
significant difference in the amount of insulin secreted from cells
that were treated with control oligonucleotide compared to the relative
amounts of insulin that were released from untreated control cells.
Moreover, the effects of the ATG-antisense treatment were not due to
toxic effects of the oligonucleotide. No decrease in cell viability was
detected when treated cells were examined by trypan blue analysis, and
no morphological changes were detected in treated cells that were
observed by phase contrast microscopy.
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, treatment with the ATG-antisense decreased
glucose-stimulated insulin secretion by almost 70%, whereas
glucose-induced insulin secretion from cells receiving the control
oligonucleotide and from cells in the untreated group was unaffected.
The effect of the Inner-antisense oligonucleotide on glucose-stimulated
insulin secretion was also studied, and no significant effect was
found.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß-Cells express a distinct isoform of kinesin
The cDNA encoding the mKHC has been cloned and sequenced. The mKHC
cDNA shares approximately 71% sequence identity with the sequence of a
previously reported mouse brain kinesin heavy chain cDNA (41),
demonstrating that at least two isoforms of the KHC are expressed in
mouse. Moreover, Northern blotting has determined that only a single
isoform of kinesin is expressed in the ß-cells. Analysis of the
deduced amino acid sequence of the mKHC suggests that it is a classical
plus end-directed microtubule motor protein with a globular
amino-terminus that contains both microtubule- and ATP-binding sites.
The sequence of the mKHC is almost identical (96%) to that of a
previously reported human placental kinesin cDNA (42), and it is
possible that the reported mKHC is the mouse homolog of the human
placental KHC.
mKHC ATG-antisense oligonucleotide selectively inhibits kinesin
expression in primary cultured mouse pancreatic ß-cells
The cDNAs encoding several kinesins and kinesin-like molecules
have been characterized (43). To understand the functions of the
microtubule cytoskeleton, it is essential to ascribe functions to the
specific kinesin isoforms and to elucidate the mechanisms of regulating
the expression of KHC genes in biological systems. To approach the
problem of investigating the role of the mKHC protein, antisense
oligodeoxyribonucleotides offered the opportunity to create either a
null cell type or a cell exhibiting reduced concentrations of KHC
protein. In this study, it is shown that the administration of mKHC
antisense oligonucleotides induced a profound decrease in the level of
mKHC protein in primary cultured mouse ß-cells. However, as shown in
these studies, antisense treatment was unable to abolish kinesin
expression completely, and future studies will focus on developing
methods to eliminate kinesin entirely to assess the role of this
protein in the synthesis and secretion of insulin.
To study ß-cells deficient in kinesin, synthetic phosphorothioate-modified oligodeoxynucleotides were used to repress kinesin expression. Culturing ß-cells for 84 h with mKHC ATG-antisense oligonucleotide, which is complementary to the translation initiation codon of the mKHC mRNA, caused a 40–50% reduction in the level of cellular kinesin protein as quantified by confocal immunofluorescence microscopy. Several results indicate the specificity of the inhibition. First, the Re-antisense and Inner-antisense appeared to have no effect on kinesin expression. In addition, in Northern blot studies, the 15-mer ATG-antisense oligonucleotide hybridized with a single species of transcripts from pancreatic ß-cells identical in size to the mRNA that was recognized by the labeled KHC cDNA probe. Studies have shown that hybridization of an oligonucleotide to a target sequence can be abolished by a single base pair mismatch (44, 45). Through computer database searching, no sequence has been found that has identity to the mKHC ATG-antisense oligonucleotide, except for the human placental KHC (42). Furthermore, treatment for 84 h with 2 µM of mKHC ATG-antisense oligonucleotide, Inner-antisense, or Re-antisense oligonucleotides did not interrupt intracellular insulin production and did not cause damage to the cells as appraised by phase contrast microscopy, electron microscopy, and cell viability studies. Finally, 84-h treatment with either Re-antisense or Inner-antisense did not affect either basal or glucose-stimulated insulin secretion from cultured ß-cells, and the levels of released insulin were comparable to the amount of insulin released from untreated cells. In summary, the observed reduction of kinesin protein within ß-cells after exposure to mKHC ATG-antisense oligonucleotides suggests that this effect was due mainly to selective inhibition of KHC gene expression. The exact mechanism of the inhibitory effect of the ATG-antisense oligonucleotide on kinesin expression remains to be established, although Chiang et al. (46) have found that phosphorothioate oligonucleotides directed against the translation initiation codon primarily interfered with translation.
The finding that the Inner-antisense oligonucleotide, which targeted a sequence within the coding region of the mKHC mRNA, had no significant inhibitory effect on kinesin expression agrees with results reported by others. It has been shown that in the absence of ribonuclease H, antisense oligonucleotides that hybridized to their complementary target in the coding region of a mRNA were removed by a ribosome-associated unwinding activity during translation (47, 48). Furthermore, Kozak (49) demonstrated that a stable stem-loop structure inserted 5' to the AUG initiation codon inhibited translation of a chloramphenicol acetyltransferase construct. However, translation proceeded if the same stem-loop structure was inserted 3' to an AUG initiation codon. These data suggest that once the 80S ribosomal complex was formed, it was capable of unwinding RNA secondary structure. Presumably, antisense oligonucleotides that hybridize to target mRNAs at a position 3' to the AUG translation initiation codon, such as the Inner-antisense oligonucleotide, may be melted off by the 80S ribosomal complex as it translates the mRNA.
ß-Cell kinesin plays an important role in insulin secretion
The evidence supporting a role for kinesin in insulin secretion
comes from the demonstration that levels of both basal and
glucose-stimulated insulin secretion from primary cultures of mouse
ß-cells were inhibited due to the suppressed mKHC expression
resulting from the mKHC ATG-antisense oligonucleotide treatment. The
principal experimental observation supporting this conclusion is that
when cells were treated with either mKHC ATG-antisense or control
oligonucleotides, the level of insulin released correlated well with
the level of the mKHC expression, i.e. when KHC expression
in ATG-antisense-treated cells was decreased by 40–50% after 84
h of treatment, the level of basal insulin secretion from those cells
was reduced by 50%, and the glucose-stimulated insulin secretion was
lowered by about two thirds; accordingly, mKHC expression in
Re-antisense treated cells was not significantly affected, and the
levels of both basal and glucose-stimulated insulin secretion were not
changed relative to those in untreated controls. Together with the
results of microtubule inhibitor studies performed by others (7, 8, 9),
which demonstrated that microtubule integrity is required for efficient
insulin secretion, these results support the hypothesis that kinesin
functions as a translocator that is responsible for transporting
insulin-containing vesicles from the cell interior to the cell surface
along microtubule tracks upon secretory stimulation. The findings from
these studies are in good agreement with the previous in
vitro studies from our laboratory, which demonstrated that the
ß-cell form of kinesin contains microtubule-stimulated ATPase
activity (13).
Partial depletion of the mKHC protein by an 84-h treatment with ATG-antisense oligo resulted in a 50% decrease in levels of basal insulin secretion. This indicated that the mKHC is essential for the persistent release of insulin from ß-cells. More interestingly, glucose-stimulated insulin secretion was decreased by approximately two thirds after ATG-antisense treatment of cultured ß-cells. This indicates that mKHC plays a critical role in the regulated secretory process. Upon glucose stimulation, ß-cells are triggered to secrete elevated amounts of insulin and need a mechanism for the synchronous intracellular transport of insulin granules required to meet the physiological requirement of bringing glucose levels back to normal. It is reasonable to conclude that a shortage of motor proteins, such as kinesin molecules, would severely affect glucose-stimulated insulin secretion.
Further studies are necessary to elucidate the details of the mechanism by which suppressed kinesin expression caused reduced insulin secretion and decreased cellular response to glucose. Recently, Lippincott-Schwartz et al. (50) and Feiguin et al. (29) have shown that kinesin also plays a role in pre-Golgi membrane traffic (i.e. endoplasmic reticulum membrane, Golgi stack, and trans Golgi network membrane) in fibroblasts and neurons, raising the possibility that suppression of kinesin expression in ß-cells would cause either a collapse of intracellular membrane systems or a total inhibition of insulin-containing vesicle production and insulin biosynthesis. To test this possibility, two studies were performed. First, the intracellular insulin content of cells receiving the different oligonucleotide treatments was assayed and compared to the amount of insulin in the untreated control cells. No significant differences in intracellular insulin content were found among cells in the various treatment groups, suggesting that depletion of KHC expression did not interfere with the balance of membrane traffic or the synthesis of insulin within mouse ß-cells. In addition, electron microscopic evidence demonstrated that treated cells were heavily granulated and contained well organized intramembranous organelles, including an intact Golgi apparatus. The reasons for the differences in the results reported here and the data presented by Feiguin et al. (29) and Lippincott-Schwartz et al. (50) are not clear at this time. However, it is possible that the decreased levels of kinesin were sufficient to maintain essential cellular functions, such as the maintenance of intracellular membrane systems, but were not capable of supporting a specialized cellular function such as secretion. This might partially explain why higher levels of the ATG-antisense oligo were toxic to cells. It is conceivable that concentrations of the ATG-antisense oligo above 2 µM may have reduced kinesin levels to a point where essential cellular functions could not be performed, resulting in cell death. Further studies will be necessary to resolve this inconsistency.
In conclusion, the cDNA encoding the mouse ß-cell kinesin heavy chain has been cloned and sequenced and an antisense strategy has been used to reduce the amount of KHC in cultured ß-cells. The results demonstrate that the ß-cell form of kinesin plays an essential and important role in both basal and glucose-stimulated insulin secretion. This study provides the first direct evidence that kinesin plays an important role in secretion from a nonneuronal mammalian cell type.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Sigma Xi Grant-in-Aid.
3 Sponsored by a Juvenile Diabetes Foundation Summer Research
Fellowship.
Received October 21, 1996.
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174 DNA: the effect of single base
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) or
treated for 84 h with 2 µM of either ATG-antisense
(ATG-as; 
) or Re-antisense (Re-as; 
) oligonucleotides in 5.5
mM glucose. Medium was collected at 36, 60, and 84 h
for measurement of insulin secretion. Data are presented as the
mean ± range. Two-way ANOVA was performed by comparing the level
of insulin secreted from untreated cells to the amounts secreted from
treated cells. ****, P < 0.0001 vs.
untreated.