| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 4, 2004 as doi:10.1096/fj.03-1397fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
STENOVEC*,1
,1
,2
1Celica Biomedical Sciences Center, Ljubljana, Slovenia;
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Medical School, University of Ljubljana, Ljubljana, Slovenia;
Department of Physics, Faculty of Mathematics and Physics, Ljubljana, Slovenia; and
Department of Physiology & Biophysics, University of Colorado Health Sciences Center, Denver, Colorado, USA
2Correspondence: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical School, University of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia. E-mail: robert.zorec{at}mf.uni-lj.si
SPECIFIC AIMS
Stimulated secretion in synapses is thought to be associated with rapid release of vesicle content by "complete" exocytosis. A stimulus may also result in vesicle content discharge via "kiss-and-run" exocytosis, where vesicles docked to the surface membrane transiently form a channel to the extracellular space via a fusion pore. In synaptic vesicle exocytosis, the actual amount of neurotransmitter released from each vesicle is unlikely to be affected by fusion pore closure on the time scale of kiss-and-run, because the transmitter diffuses rapidly, whereas in large dense-core vesicles the transient mode of exocytosis may limit or prevent vesicle peptide discharge.
In resting synapses and neuroendocrine cells, vesicle cargo appears to be released, but the mechanism of vesicle discharge is not known. The aim of this study was to measure vesicle cargo discharge from a single vesicle in basal and stimulated conditions.
PRINCIPAL FINDINGS
We studied basal secretion of prolactin from rat pituitary lactotrophs. We used confocal microscopy to study the discharge of preloaded green fluorescent probe (proatrial natriuretic peptide fused with the emerald green fluorescence protein, or ANP.emd) and simultaneous vesicle loading by an extracellular FM 4-64 red fluorescent probe. We monitored the properties of stationary vesicles positioned within 2 µm of the plasma membrane by measuring time-dependent fluorescence intensity changes of the labeled vesicles. A decrease in ANP.emd fluorescence is consistent with the vesicle content discharge, although it may be due to a drift of the vesicle out of focus. To rule out that a decrease in green fluorescence intensity is due to vesicle movement out of focus, we added a styryl dye (FM 4-64) into the bath, which stains the membrane and matrix of individual vesicles, when a fusion pore forms between the docked vesicle and the plasma membrane. The properties of hormone discharge from single stimulated vesicles were studied first.
1. Dissimilar kinetics of fluorescence loading and unloading in stimulated vesicles
We stimulated lactotrophs by exposure to 100 mM extracellular KCl, which resulted in a rapid increase in FM 4-64 fluorescence intensity of vesicles with a concomitant loss of the ANP.emd probe (Fig. 1
). Of 99 stimulated vesicles in 6 cells, 20 apparently docked, initially green vesicles turned red within 90 s. Measurements of the time required for a 20 to 80% change of fluorescent signals revealed that dimming of ANP.emd was 4.5 ± 0.6 s (n=20), 2- to 3-fold slower than uptake of FM 4-64 (1.9±0.2 s; n=20; 4 µM Fig. 1C
). Assuming that FM 4-64 and the ANP.emd molecules are transported into and out of the vesicle through an open fusion pore, respectively, the difference between the time course of FM 4-64 loading and ANP.emd loss is consistent with the view that larger molecules diffuse more slowly because of their lower mobility.
|
2. Slow synchronous kinetics of probe transport in spontaneously secreting vesicles
Next we examined vesicle properties at rest. In the absence of stimulation, when vesicles were observed for 90 s as the stimulated ones, we found that none of the 824 apparently docked green vesicles turned red. Therefore, we monitored resting vesicles for several minutes. If exocytotic release of prolactin occurs at rest, then the intensity of the ANP.emd probe should decrease and the vesicle should be loaded through the same fusion pore by the FM 4-64 dye, as observed (Fig. 2
). To rule out the possibility that a decrease in fluorescence intensity was due to a movement of the vesicle out of focus, we recorded stacks of images in the presence of FM 4-64. Figure 2B
shows the time course of spontaneous release from two vesicles recorded every 2 min. Properties of these spontaneous exocytotic events were studied at a higher temporal resolution, recording stacks of images every 30 s. Of 412 vesicles studied in 15 cells, 36 were found to undergo spontaneous release in 15 min. In 50% of these vesicles (18 of 36) we saw the spontaneous exchange of the ANP.emd for the FM 4-64 in two consecutive images. In the rest of the vesicles studied, spontaneous uptake of FM 4-64 was very slow. Figure 2C
shows slow uptake of the FM 4-64 observed in 18 of 36 vesicles. In 14 of these vesicles, the slow FM 4-64 uptake was paralleled by slow ANP.emd loss. The time course of the slow spontaneous release was studied by measuring the time required for a 20 to 80% change of the fluorescence signal (Fig. 2C
). The average time for FM 4-64 fluorescence increase was 180 ± 22 s (n=18) and 216 ± 31 s (n=14) for ANP.emd intensity dimming, not significantly different from each other (Fig. 2D
). These results show that spontaneous discharge of hormone occurs from single vesicles and that in 50% of these vesicles it proceeds with a slow time course. It is unlikely that prolonged vesicle attachment to the plasma membrane is a property of constitutive fusion; hence, the events observed likely represent spontaneous peptide discharge from vesicles that enter regulated fusion. In contrast to the dissimilar kinetics of FM 4-64 loading and ANP.emd discharge in stimulated vesicles, loading and unloading of both probes in resting vesicles was synchronous. The relatively slow time course of FM 4-64 loading and ANP.emd loss in resting vesicles may be accounted for by a narrower opening than the open fusion pore in stimulated vesicles. However, assuming that the binding and unbinding of the two probes with the vesicle matrix is unaltered in the two physiological states, the synchronous transport of the probes used cannot simply be assigned to a constantly open fusion pore. If diffusion in the lumen of the open fusion pore determined the exchange of fluorescent probes in resting vesicles, the rates of each probe should be proportional to the respective probe mobility, which was not observed. Hence, the results (Fig. 2D
) suggest a synchronization mechanism for the fusion pore probe exchange at rest. Transport of probes through the fusion pore may be affected by fusion pore flickers or gating. Therefore, we considered a model of synchronized transport of probes with different mobilities through a gated fusion pore (Fig. 3
).
|
|
3. Modeling transport through the fusion pore by considering pore flickering
To interpret the transport of fluorescent probes through the fusion pore, we considered that the binding and unbinding properties of fluorescence probes are unaltered under the different physiological conditions studied. Three processes were considered: 1) diffusion of hormone from vesicular matrix with volume V1 into the vesicular aqueous space with volume V2; 2) diffusion of hormone between the vesicular aqueous space and the extracellular medium through the fusion pore with gates, and 3) fusion pore gating. In other cells it was found that the discharge of peptide fluorescent probes from a stimulated single vesicle occurs in the submillisecond range. Therefore, the discharge of ANP.emd in stimulated lactotroph vesicles (Fig. 1C
) likely represents the ANP.emd discharge from the vesicle matrix into the vesicular aqueous space V2 with a time constant of 
2 = 4.5 s; the FM 4-64 dye is loaded with a time constant of 1 = 1.9 s (Fig. 1C
). Once in the aqueous milieu of the vesicle, the diffusion of probes out into the extracellular space is in the submillisecond range when the fusion pore gates are open. Synchronization of transport of substances of different degrees of mobility through the fusion pore can be attained in such a model if the dwell time of the fusion pore is short enough to allow the aqueous diffusion of substances out of the aqueous vesicle space and the gating is sufficiently slow; i.e., the period (Fig. 3)
between fusion pore openings occurs within the time required for nonaqueous diffusion of hormone from the matrix. We found that the model describes well the time course of fluorescence changes of vesicle release and accounts for the different kinetics of FM 4-64 loading and green dye loss in stimulated vesicles (Fig. 1C
).
4. Fusion pore flickers
Fusion pore flickers have been measured in cells before, but no evidence of this process is known for peptide-secreting lactotrophs. To test the hypothesis that the fusion pore in lactotrophs exhibits pore flickers, we used the cell-attached patch-clamp technique to monitor discrete changes in membrane capacitance (Cm) that reflect unitary fusion and fission events. Of 30 patches with a total recording time of 4.3 h (435 s average time), we recorded spontaneous discrete "on" and "off" steps in Cm in 14 patches with 1 to 5 fF amplitude (as reported), which agree well with the size of prolactin-containing vesicles in lactotrophs. In six (43%) patches, which exhibited Cm steps we observed that an "on" step in Cm was followed by an "off" step of similar amplitude within 50 ms, representing a transient fusion event. Moreover, these events appeared at regular intervals for as long as 760 s (average pulsing duration 170±78 s, n=10). The results show that fusion pores in resting lactotrophs exhibit significant fusion pore flickers.
CONCLUSIONS AND SIGNIFICANCE
Our results clearly show that the properties of elementary vesicle hormone discharge differ under stimulated and basal conditions. Distinct modes of vesicle hormone discharge reported here might be relevant for other fields in neurobiology and cell biology of secretion. The view advanced by Bernard Katz some decades ago—that, at rest, the vesicle discharge of hormones and neurotransmitters is similar to that occurring after stimulation—needs to be extended. In addition to the classical paradigm that secretory capacity of a cell is determined by controlling the probability of occurrence of elementary exocytotic events, one will have to consider activity modulation of elementary exocytotic events.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1397fje; doi: 10.1096/fj.03-1397fje
1 M.S. and M.K. contributed equally to this work. 
This article has been cited by other articles:
|
|
 |
|
  A. E. Gonzalez-Iglesias, T. Murano, S. Li, M. Tomic, and S. S. Stojilkovic Dopamine Inhibits Basal Prolactin Release in Pituitary Lactotrophs through Pertussis Toxin-Sensitive and -Insensitive Signaling Pathways Endocrinology, April 1, 2008; 149(4): 1470 - 1479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Vardjan, M. Stenovec, J. Jorgacevski, M. Kreft, and R. Zorec Elementary properties of spontaneous fusion of peptidergic vesicles: fusion pore gating J. Physiol., December 15, 2007; 585(3): 655 - 661. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Pangrsic, M. Potokar, M. Stenovec, M. Kreft, E. Fabbretti, A. Nistri, E. Pryazhnikov, L. Khiroug, R. Giniatullin, and R. Zorec Exocytotic Release of ATP from Cultured Astrocytes J. Biol. Chem., September 28, 2007; 282(39): 28749 - 28758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Vardjan, M. Stenovec, J. Jorgacevski, M. Kreft, and R. Zorec Subnanometer Fusion Pores in Spontaneous Exocytosis of Peptidergic Vesicles J. Neurosci., April 25, 2007; 27(17): 4737 - 4746. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. KREFT, M. STENOVEC, and R. ZOREC Focus-Drift Correction in Time-Lapse Confocal Imaging Ann. N.Y. Acad. Sci., June 1, 2005; 1048(1): 321 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Stenovec, I. Poberaj, M. Kreft, and R. Zorec Concentration-Dependent Staining of Lactotroph Vesicles by FM 4-64 Biophys. J., April 1, 2005; 88(4): 2607 - 2613. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
