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NEUROENDOCRINOLOGY |
Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425
Address all correspondence and requests for reprints to: Dr. Fredric R. Boockfor, Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425. E-mail: boockfor{at}musc.edu
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Abstract |
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Introduction |
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To date, very little insight has been gained on the cellular and molecular basis for these episodes in individual GnRH-secreting cells. Recent efforts in our laboratory have been directed toward understanding the processes underlying this GnRH pulsatile function. Our development of a technique using FM1-43 fluorescent dye incorporation enabled the observation that exocytotic activity in individual GT1 cells occurs in pulses (12). In other studies we used a GnRH promoter-driven luciferase reporter activity to monitor GnRH gene expression (GnRH-GE) in single GT1 cells and found that GnRH-GE also occurs in episodes (13). The observation that both exocytosis and GnRH-GE are elaborated in a pulsatile fashion raised the intriguing possibility that a functional link existed between these two intermittent activities. Characterization of the temporal association of these episodic events in a single cell would be likely to provide some insight into the manner in which this pulsatile activity is initiated and/or propagated. In the present study we have developed a protocol that combines single cell analysis of exocytosis (by FM1-43 fluorescence) and GnRH-GE (by GnRH promoter-driven luciferase reporter expression) in the same living GT1-7 cells and used it to begin to define the relationship between these two cellular processes.
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Materials and Methods |
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Concurrent measurement of exocytosis and GnRH promoter activity in
single living cells
Concurrent measurement of these cellular functions was
accomplished by first microinjecting cells with the plasmid
pA3GnRH-LUC that contained the coding sequence of
the firefly luciferase placed under the control of 3 kb of the rat GnRH
promoter (provided by Dr. Margaret E. Wierman, University of Colorado
Health Sciences Center, Denver, CO), as reported previously from our
laboratory (13). These cells were kept in culture 24
h after microinjection and then loaded with the fluorescent dye FM1-43
(2 µM; Molecular Probes, Inc., Eugene, OR)
for 30 min at 37 C. The loading/perifusion medium consisted of high
glucose DMEM (without phenol red) supplemented with 10% FBS, 10
mM HEPES, 0.1 mM MEM sodium pyruvate solution,
4 mM glutamine, 1% penicillin/streptomycin, and 0.1
mM luciferin (substrate of the luciferase enzyme;
Sigma-Aldrich Corp., St. Louis, MO). Coverslips bearing
the cells were mounted in Sykes-Moore chambers (Bellco Glass, Inc.) and
then placed on the temperature-controlled stage of a fluorescence
microscope (Axiovert, Carl Zeiss, Jena, Germany) that was
fitted with a Carl Zeiss Fluar x40 oil immersion
objective (numerical aperture, 1.3). Microinjected cells were relocated
by their coordinates on the grid-etched coverslip and perifused
continuously (10 µl/min). At the beginning of each recording, a
brightfield image of the cells was captured for reference purposes.
Cells were then epiilluminated at 490 nm every 10 min to acquire the
FM1-43 fluorescent signal. Image acquisition was controlled by
Metafluor PC software (Universal Imaging Corp., West Chester, PA), and
fluorescent emission light (550 nm) was captured using a cooled CCD
camera (C-4880-80, Hamamatsu Photonics, Hamamatsu City, Japan).
Immediately after the fluorescent image was captured, photonic emission
from individual cells was collected over 10 min using a Hamamatsu VIM
Photon Counting Camera/Argus 20 Image Processor (Hamamatsu Photonics)
in series with Metafluor. Images of FM1-43 incorporation and photonic
emission were stored and later analyzed.
To investigate the influence of blocking transcriptional activity on the pulsatile dynamics of exocytosis, we treated cells microinjected with the pA3GnRH-LUC vector with actinomycin D (Sigma-Aldrich Corp.), which has been shown to prevent transcription by binding to single-stranded DNA (14). FM1-43 fluorescence and photonic emission from individual cells were recorded concurrently for 1–4 h as described above. The perifusion medium bathing the cells was then changed to medium containing 10 µM actinomycin D. A total exchange of the medium in the Sykes-Moore chamber took 4 min to complete (500 µl/min). Monitoring of fluorescence and photonic emission continued for as long as 10 h.
In studies designed to block exocytotic pulsatility, we first
microinjected cells with the pA3GnRH-LUC reporter
plasmid and 24 h later injected the cells with 1 mg/ml monoclonal
antibody raised against mouse SNAP-25 (SM81, Sternberger Monoclonals,
Inc., Lutherville, MD), an antibody shown to arrest exocytosis in
individual GT1-7 cells (12). Anti-SNAP-25 was coinjected
with 1.25% rhodamine dextran (10-kDa; 1:1; Sigma-Aldrich Corp.) for identification of cells microinjected with the
antibody. Immediately after injection of anti-SNAP-25/rhodamine
dextran, the chamber was placed on the fluorescence microscope, and the
exocytotic and gene expression activities were monitored as described
above. As control, the cells were coinjected with 1 mg/ml purified IgG1
that has the same isotype as that of anti-SNAP-25 (MOPC 21,
Sigma-Aldrich Corp.), and rhodamine dextran (1:1). Other
groups of GT1-7 cells were bathed with perifusion medium containing 12
µg/ml tannic acid. This nonintrusive substance has been shown to
eliminate the export of secretory material from the cell while still
allowing the initial fusion of secretory granules to the cell membrane
(15).
Data and statistical analyses
Profiles of photonic emission from individual cells were
analyzed as follows. Initially, the entire dataset was normalized to
the first six points (1 h) to ensure a comparable baseline for all
cells studied. Episodes of photonic emission were first resolved with
the pulse detector software PULSAR [developed by Drs. Merriam, Kozuch,
and Wachter, NICHHD (Bethesda, MD) and University of California
(Berkeley, CA)]. However, because this program yielded some
significant episodes that were questionable by visual inspection, we
added another criterion for pulse identification. Increases in photonic
emission were considered significant bursts of promoter activity if the
fluctuations were greater than 5% of the value at the beginning of the
episode plus 2 times the total signal-noise variation as reported by
Takasuka et al. (16). The latter parameter was
estimated as the square root of each value plus the
SD of the corresponding background values squared
[
(signal + (SD background)2)]. The
photonic emission signal was always greater than 3-fold the background
level. To avoid false positives, we did not consider single point
fluctuations as significant in any of the bioluminescence profiles
analyzed.
In parallel, fluorescence profiles were analyzed as described previously (12). Briefly, the data were transformed into the differential over time (dy/dt), representing the rate of dye uptake. This transformation yielded profiles with identifiable upstrokes and downstrokes that could be analyzed subsequently by the PULSAR program. Pulse amplitude was calculated from the original datasets as the difference between the areas under the curve defined by the three points following and preceding a pulse. Increases greater than 5% were considered significant deflections in FM1-43 incorporation. The signal to noise ratio was always higher than 2 at the beginning of each recording. As in photonic emission recordings, we did not include single point peaks as pulses in any of the FM1-43 profiles analyzed.
Comparison of data used one-way ANOVA to determine whether the data were parametric. This test was followed by two-tailed, unpaired t test, except for the comparison of frequency, amplitude, and duration of exocytotic pulses before and after treatment with actinomycin D, which used a Newman-Keuls multiple comparison test. Results were expressed as the mean ± SEM. 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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, A and B, are
micrographs of two adjoining GT1-7 cells that underwent dual monitoring
of GnRH promoter activity (photonic emission from luciferase reporter)
and exocytosis (endocytotic FM1-43 uptake after exocytotic events). As
revealed by the fluorescence image (Fig. 1A), the signal derived from
the FM1-43 dye incorporation was relatively strong, permitting us to
delineate precisely the boundaries of the cells. These regions of
interest were then exported to the bioluminescence image (Fig. 1B) to
identify the same cells for assessment of GnRH promoter activity. In
Fig. 1C, a representative profile of GnRH-GE (top) and
exocytotic (bottom) activities in a GT1-7 neuron under basal
conditions is provided. As shown, pulses or bursts of activity were
exhibited for both GnRH-GE and FM1-43 incorporation. GnRH-GE pulses
occurred with an average frequency of 0.53 ± 0.05 pulses/h
(n = 20 cells). This frequency was comparable to that observed
previously for GT1-1 cells when only photonic measurements were taken
(13). In addition, fluorescence measurements revealed
stepwise increases in FM1-43 uptake. This index, which reflects
endocytosis that immediately follows exocytosis, has been shown by our
laboratory (12) and others (17, 18, 19) to serve
as an excellent means of estimating exocytotic activity. On the
average, the frequency of exocytotic events (0.98 ± 0.06
pulses/h; n = 20 cells) was similar to that found previously for
individual GT1-7 cells in which single, and not dual, monitoring was
performed (12) as well as for entire GT1-7 cell
populations (7, 9, 10), and for hypothalamic GnRH neurons
(7).
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Effect of blockage of GnRH-GE episodes on exocytotic pulse
activity
To determine whether exocytotic pulse activity was dependent on
GnRH-GE, we blocked transcription with actinomycin D and assessed the
presence and dynamics of exocytotic pulses. Shown in Fig. 2A are the GnRH-GE and exocytotic
profiles in an individual cell after actinomycin D treatment. Analysis
revealed that this treatment induced a rapid decrease in GnRH-GE,
especially the frequency with which episodes occurred (0.77 ±
0.16 vs. 0.16 ± 0.05 pulses/h, before and after
exposure to actinomycin D, respectively; n = 10 cells). More
importantly, blockage of transcription did not abolish
exocytotic bursts in GT1-7 neurons, similar to that found in the entire
population of GT1–1 cells (20). Interestingly,
actinomycin D did reduce pulse frequency and duration in a
time-dependent manner (Fig. 2B). Exocytotic pulse frequency and
duration decreased only slightly during the first 6 h in cells
after actinomycin D treatment. This inhibitory effect became more
evident with prolonged treatment (>6 h). However, even though pulse
frequency and duration were reduced with time of treatment, pulse
amplitude was not influenced. Taken together, these results indicate
that GnRH-GE episodes do not account for the initiation or acute
modulation of exocytotic pulses.
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and found GnRH-GE episodes to occur at a frequency of
0.48 ± 0.04 episodes/h (n = 10 cells), a value comparable to
that observed in noninjected cells (Fig. 3A). Next, we injected anti-SNAP-25
antibody and, as anticipated, observed a cessation of exocytotic pulses
(Fig. 3B). Moreover, we also observed a strong reduction in the
frequency of GnRH-GE episodes (0.48 ± 0.04 vs.
0.21 ± 0.05 episodes/h in cells injected with control IgG and
anti-SNAP-25, respectively; n = 10 cells). This observation
strongly suggested that a functional link existed between the process
of granule fusion to the membrane and GnRH-GE pulse elaboration. At
this point it was difficult to determine whether these effects were
specific to this process of granule fusion or to other processes that
occurred later during exocytosis. To distinguish between these
possibilities, we treated the cells with tannic acid. Tannic acid
treatment has been shown to eliminate the export of secretory material
from the cell, but still allows the initial fusion of secretory
granules to the cell membrane (15). Treatment with this
agent did not modify the frequency of GnRH-GE pulses (0.54 ± 0.05
pulses/h; n = 15 cells), although their duration was slightly
increased (63.53 ± 3.32 vs. 73.09 ± 4.62 min in
control and treated conditions, respectively; P <
0.05) even though exocytotic pulsatility was abolished. The fact that
blockage of granule fusion to the cell membrane (via anti-SNAP-25
injection) abolished episodes of GnRH-GE, but blocking granule export
(via tannic acid) had no acute effect on this pulsatile activity,
indicated that the functional link identified exists between the
SNAP-25-mediated process of granule fusion and the process of
GnRH-GE.
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). As shown, we
found that larger pulses were more closely associated with GnRH-GE
episodes than smaller pulses. Moreover, we observed that as the
amplitude of exocytotic pulses reached a certain threshold (
1000
fluorescent units), the percentage of pulses associated with GnRH-GE
episodes rose rapidly, suggesting that this point represents the
minimum amplitude that an exocytotic pulse must display to be able to
trigger a GnRH-GE episode. We then divided the duration of the
recordings into 90-min bins to estimate the relative exocytotic pulse
frequency. Inasmuch as the average duration of exocytotic pulses was
27.50 ± 1.70 min, we chose this time interval to allow the
identification of multiple exocytotic pulses that might precede a
GnRH-GE episode (0–4 pulses/90 min). By using this analysis, we found
that the pulse amplitude threshold calculated above could be reached by
a large single exocytotic pulse (Fig. 4B) or by a combination of
numerous small exocytotic pulses immediately preceding a GnRH-GE
episode (Fig. 4C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further use of this technique in this investigation uncovered a strong temporal association between pulses of exocytosis and episodes of GnRH promoter activity in individual GT1-7 neurons. This observation invited speculation that a common intrinsic mechanism drives both cellular processes to maintain a precise equilibrium between release and biosynthesis. Indeed, in other cell types displaying massive exocytotic bursts, it has been demonstrated that transcriptional activation of genes coding for secretory products occurred immediately after the major exocytotic events, suggesting that exocytosis somehow modulates gene expression (22, 23). Moreover, in light of the observation that the state of occupation of cortical docking sites (i.e. active zones where secretory granules wait for fusion with the cell membrane) was inversely proportional to the transcriptional activity, it was suggested that some part of the cortical docking process plays an important role in regulating transcriptional turnover (22). In our study, in which a strategy of blocking one process while measuring the other was used, we were able to determine that a portion of the exocytotic process was needed to initiate GnRH promoter pulses. Indeed, our ability to inhibit GnRH-GE pulses by arresting the exocytotic pathway at a late postdocking stage with anti-SNAP-25 provides strong evidence that a docking site-related mechanism modulating transcriptional activity also operates in GnRH neurons. The importance of this latter stage of the exocytotic pathway is further suggested by the lack of influence of tannic acid treatment on GnRH-GE episodic activity. It has been shown by others that tannic acid treatment allows secretory granules to fuse to the cell membrane even though release is inhibited (15). In our studies the lack of effect of this drug on GnRH-GE episodes indicates that export of granule content is not critical in the GnRH-GE pulse initiation process. Regardless of the exact source of initiating signal, a communication mechanism between exocytosis and gene activation would probably involve components that arise during docking and/or granule fusion, such as changes in intracellular calcium concentration and/or the conformational changes in the cytoskeleton surrounding the cell membrane, both of which have been reported to mediate transcriptional activation (24, 25, 26). Two possible candidates that would subserve such a communicating role are calcium calmodulin-dependent protein kinases (for review, see Ref. 27) and the Rho family guanine triphosphatase proteins (for review, see Ref. 28). Specifically, in the first example it has been shown that increases in the calcium concentration that trigger fusion of granules to the cell membrane also activate calcium calmodulin-dependent protein kinases I, II, and IV. In turn, these components phosphorylate the cAMP response element-binding protein family of transcription factors (26). In the second case, Rho guanine triphosphatases, which have been implicated in the process of exocytosis by controlling cytoskeleton organization (29, 30, 31), activate transcription by up-regulating serum response factors (32, 33). Of course, these are just two examples of a growing number of intracellular factors and signal transduction cascades that are activated after cell membrane and cytoskeletal remodeling and that may be involved in exocytosis/gene expression coupling. Further investigation is required to define the common molecular signaling pathways linking these processes. In this regard we are presently pursuing this problem by identifying regulatory sites of the GnRH promoter involved in modulation of episodic GnRH-GE activity and determining their relationship with different signaling transduction pathways involved with exocytosis.
The rapid activation of biosynthesis by an exocytotic event may serve as a mechanism for the replenishment of products lost during exocytosis. Indeed, our results indicate that larger exocytotic pulses triggered GnRH-GE episodes in individual GT1-7 cells. This is not surprising in light of the findings of others that an increase in gene expression occurs when a majority of the cellular content is secreted during a single exocytotic burst (22, 23). Although these studies measured stimulated exocytotic events, the principle of hormone replenishment may still be comparable in our study, in which only basal, unstimulated exocytotic pulsatility is considered. Interestingly, our results further show that gene expression can be triggered not only by one larger episode, but also by several small exocytotic pulses, suggesting that an exocytotic threshold must be reached before a gene expression event can be triggered. Even though these pulses represent only small, basal fluctuations, it can be argued that either one larger pulse or several smaller exocytotic events may account for enough hormone loss in a relatively short period to trigger a compensating synthetic event. This would ensure that the cell does not become depleted over time. Our findings of the importance of overall pulse magnitude on the ability of one or a series of exocytotic events to trigger a GnRH-GE pulse are consistent with this hypothesis. Taken together, these results suggest that the process of exocytosis-dependent GnRH-GE activation serves as an important coordinating mechanism to ensure that GnRH neurons are maintained in a ready state able to respond to modulatory input necessary for changing reproductive function.
In summary, in the present study we have demonstrated that exocytosis is functionally coupled to GnRH-GE in GT1-7 cells. This finding raises the exciting possibility that similar mechanisms of control of gene expression may function in other secretory cells or with other genes associated with the secretory process in GnRH neurons. In the future, our approach of combined measurement of gene expression and exocytosis in the same cell may serve as a fundamental tool to address this and other basic issues of synthesis and secretion in a variety of endocrine cell types.
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Acknowledgments |
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Footnotes |
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Abbreviation: GnRH-GE, GnRH gene expression.
Received July 24, 2001.
Accepted for publication August 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12 and
G13 inhibit
Ca2+-dependent exocytosis through
Rho/Rho-associated kinase-dependent pathway. J Neurochem 75:708–717[CrossRef][Medline]
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