ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2009             Vol. 52, [t. 2: 95–106
Mehanizmi eksocitoze/Mechanisms of Exocytosis
Nina Vardjan,2, Matjaž StenoVec1,2, Jernej Jorgačevski2, Marko kreft2,1 and Robert Zorec2,1,* 
1Celica Biomedical Center, LCI, Tehnološki park 24, 1000 Ljubljana, Slovenia
2LN-MCP, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 
1000 Ljubljana, Slovenia
Correspondence*: Robert Zorec, Ph. D., Professor, Email: robert.zorec@mf.uni-lj.si, University of 
Ljubljana, Faculty of Medicine Institute of Pathophysiology, LN-MCP, Zaloška 4, 1000 Ljubljana, 
Slovenia, Tel.: +386 1 543 70 20, Fax: +386 1 543 70 36
Abstract: Vesicles are cellular organelles, in which signaling molecules (neurotransmitters 
or hormones) are stored and are essential for the function of neurons and endocrine cells in sup-
porting the communication between tissues and organs. Upon stimulation the signaling molecules 
stored inside vesicles are released from cells by exocytosis. This fundamental biological process 
consists of membrane fusion between the vesicles and the plasma membrane, leading to the for-
mation of an aqueous channel – the fusion pore – through which signaling molecules exit into the 
extracellular space or blood stream. The vesicle cargo discharge initially requires the delivery of 
vesicles to the plasma membrane, where vesicles dock and get primed for fusion with the plasma 
membrane. Classical view holds that stimulation initiates the fusion pore formation and vesicle 
cargo discharge in an all-or-none fashion. Once formed the fusion pore may close (transient, 
“kiss-and-run” exocytosis) or expand, leading to the full collapse of the vesicle membrane into 
the plasma membrane (full fusion exocytosis). However, recent studies indicate that exocytosis 
may not be as simple. Here we highlight the novel findings which indicate that transient fusion 
pore is subject to regulations, which affect the release competence of a single vesicle. Our recent 
studies have shown that in pituitary lactotrophs vesicle release of peptide signaling molecules 
involves modulation of fusion pore kinetics and fusion pore conductance.
Keywords: exocytosis, vesicle, fusion pore, transient/full fusion, pituitary lactotrophs, 
peptide hormones
Izvleček: Mešički so celični organeli, v katerih so shranjene signalne molekule (živčni prena-
šalci, hormoni), ki so nujno potrebne za delovanje živčnih in endokrinih celic, saj omogočajo ko-
munikacjo med tkivi in organi. Po stimulaciji se signalne molekule izločijo iz mešičkov s pomočjo 
eksocitoze. Eksocitoza je temeljni biološki proces, pri katerem pride do zlitja membrane mešička in 
plazemske membrane, pri čemer nastane kanal – fuzijska pora, skozi katerega se izločijo signalne 
molekule v zunajcelični prostor ali krvni obtok. Pred eksocitozo mešički potujejo v neposredno 
bližino plazemske membrane, kjer se vsidrajo in pripravijo za zlitje s plazemsko membrano. V 
naslednjih fazah naj bi stimulacija sprožila nastanek fuzijske pore in popolno sprostitev vsebine 
mešička skozi fuzijsko poro. Fuzijska pora se lahko po odprtju zapre (t.i. prehodna, angl. »kiss-
and-run« eksocitoza) ali pa razširi, kar vodi do popolnega zlitja membrane mešička s plazemsko 
membrano (t.i. popolna, angl. »full fusion« eksocitoza). Nedavne raziskave so pokazale, da proces 
eksocitoze ni tako preprost. V preglednem članku se bomo osredotočili na najnovejše raziskave, ki 
kažejo, da je prehodna eksocitoza lahko uravnavana, kar vpliva na zmožnost izločanja signalnih 
molekul iz posameznega mešička. Naše nedavne raziskave so pokazale, da v laktotrofih iz hipofize, 
sproščanje peptidnih signalnih molekul iz mešičkov vključuje tako modulacijo kinetike fuzijske 
pore kot tudi uravnavanje prevodnosti (premera) fuzijske pore.
Ključne besede: eksocitoza, mešiček, fuzijska pora, prehodna/popolna fuzija, hipofizni 
laktotrofi, peptidni hormoni
96 Acta Biologica Slovenica, 52 (2), 2009
Introduction
Exocytosis is a fundamental cellular process 
used by eukaryotic cells to secrete different bio-
logical compounds. The basic machinery required 
for exocytosis has been well conserved throughout 
evolution from yeast to man. However, the precise 
molecular mechanisms underlying the process of 
exocytosis are still poorly understood and therefore 
extensively studied in different cell systems. 
The secretory process consists of few different 
stages. First vesicles packed with the secretory 
compounds are transported to the plasma mem-
brane. Then the vesicles are tethered or docked 
to the appropriate sites at the plasma membrane 
and prepared for fusion (priming). In the last step 
the fusion of vesicle with the plasma membrane 
(exocytosis) occurs and the vesicle content is 
released through the fusion pore (Fig. 1 and 2) 
and/or the vesicle membrane components are 
incorporated into the plasma membrane.
Exocytosis occurs in almost all cells in the 
form of constitutive exocytosis, which serves 
to release the components of the extracellular 
matrix, or just to deliver and incorporate newly 
synthesized membrane lipids and proteins into 
the plasma membrane (i.e. constitutive exocy-
tosis). In many cells an alternative secretory 
pathway exists, where an extracellular stimulus 
is required to trigger exocytosis, therefore al-
lowing a controlled release of peptide hormones 
and neurotransmitters. The stimulus typically 
triggers an increase in intracellular Ca2+ activity
([Ca2+]i), which activates vesicle fusion with the 
plasma membrane (i.e. regulated or Ca2+-triggered 
exocytosis; Fig. 2). 
In recent years new techniques have allowed 
direct measurements of elementary exocytotic 
events in real time in neurons and neuroendo-
crine cells. By measuring membrane capacitance 
(Cm) one can reveal changes in cell surface area, 
which are reflecting vesicle fusion and fission 
(Neher & Marty 1982, LiNdau & Neher 1988). 
Detection of vesicle fusion with amperometry is 
based on the indirect electrochemical detection 
of released molecules with a suitable oxidation 
potential (WightMaN & al.1991, chow & al. 
1992). Furthermore optical imaging with dif-
ferent fluorescence markers can provide a direct 
readout of vesicle fusion and recycling (BetZ & 
Figure 1: Scanning electron micrograph of the pituitary 
lactotroph surface exhibits fusion-pore-like formations. 
(A) Panel shows a view of the whole cell with a diameter 
of 10 µm. (B) Panel is a magnified view of the framed 
region in panel A. Several small openings in the cell 
surface (black) are seen, which may be related to the 
fusion pore-like structures.
BeWick 1992, MieseNBöck & al. 1998, Shaner 
& al. 2005). These multidisciplinary approaches 
led to the discovery that the rate (aLBiLLos & al. 
1997, StenoVec & al. 2004) and the amount of 
vesicle cargo release (angleSon & al. 1999) are 
controlled by the stimulus before the vesicle fu-
sion, and more importantly, also after the fusion 
pore formation (i.e. post-fusion regulation of a 
release, rahaMiMoff & ferNaNdez 1997). 
In this article we focus on the mechanisms of 
regulated exocytosis. We first present an overview 
97N. Vardjan, M. Stenovec, J. Jorgačevski, M. Kreft, R. Zorec: Mehanizmi eksocitoze
Figure 2: Exocytosis involves fusion of the vesicle 
membrane with the plasma membrane.
Membrane fusion leads to the formation of the fusion 
pore – an aqueous channel connecting the vesicle lumen 
with the extracellular space. Constitutive exocytosis 
does not require a stimulus to occur, whereas regulated 
exocytosis is triggered by a stimulus, such as an increase 
in the activity in cytosolic Ca2+. Endocytosis is a process 
of plasma membrane retrieval, often balancing exocy-
tosis to keep the surface area of a cell constant over a 
longer period of time.
Models of exocytosis
One of the first models of regulated vesicle 
exocytosis, introduced by del Castillo and Katz, 
predicts that in neurons the vesicles fuse transiently 
with the membrane and that the vesicle cargo is 
released in an all-or-none fashion (katz 1969). 
In the early 1970s, the first systematic studies on 
vesicle recycling were performed on frog neu-
romuscular junction by electron microscopy in the 
presence of the marker horseradish peroxidase. 
The studies were performed independently by 
two research groups, interestingly, their results 
yielded different interpretations (ceccareLLi & 
al. 1973, heuser & reeSe 1973).
Heuser and Reese discovered that vesicle 
fusion is followed by full collapse of the vesicle 
membrane into the plasma membrane (i.e. full 
fusion exocytosis) emptying the entire vesicle 
content to the extracellular space (all-or-nothing 
release). The retrieval of vesicle membrane, which 
is necessary for maintaining the cell size and 
integrity, was proposed to occur at the location 
distal to the fusion site (heuser & reeSe 1973). 
On the other hand, Ceccarelli and his co-workers 
proposed that vesicle fusion involves the opening 
of a small fusion pore, followed by its fast closure 
at the same site of fusion, without full pore dilation 
and vesicle membrane collapse into the plasma 
membrane (ceccareLLi & al. 1973). The model 
they proposed was similar to Katz’s initial model, 
that vesicles fuse with the plasma membrane only 
Figure 3: Models of exocytosis. Schematic drawing of exocytosis modes. (A) Full fusion of a vesicle after the 
initial formation of a narrow fusion pore. After the fusion pore widening the vesicle membrane is fully incorporated 
into the plasma membrane. (B) An example of transient fusion (a “kiss-and-run” event), where a vesicle forms a 
transient fusion pore with the plasma membrane and releases only part of its content. After the fusion pore closure, 
the vesicle lumen is reloaded and vesicle reused.
of the present knowledge on single vesicle fusion 
events in lactotrophs, neuroendocrine cells of 
anterior pituitary, which secrete peptide hormone 
prolactin. We then discuss mechanisms of post-
fusion regulation of vesicle cargo release from 
neurons and neuroendocrine cells.
98 Acta Biologica Slovenica, 52 (2), 2009
transiently (i.e. transient or “kiss-and-run” exocy-
tosis as proposed by fesce & al. (1994)). Further 
studies have revealed that the transient opening of 
the fusion pore may lead only to the partial release 
of vesicle cargo (StenoVec & al. 2004, reviewed 
in harata & al. 2006) and that once opened, the 
fusion pore can change its dimensions dynamically 
and even close and reopen repetitively (fusion pore 
flickering, the pulsing fusion pore, ferNaNdez & 
al.1984, StenoVec & al. 2004, Vardjan & al. 2007). 
More than 30 years after, these two models (Fig. 
3) have been still intensively debated (reviewed 
in he & al. 2006, Logiudice & MatthewS 2006, 
sMith & al. 2008). 
Stimulus increases the rate of cargo 
release from a single vesicle in 
lactotrophs
Figure 4 shows immunolabelled prolactin 
hormone accumulation at the surface of the plasma 
membrane following cell stimulation. Recently, it 
has been observed that in lactotrophs stimulated 
hormone discharge from a single vesicle is up to 
20 times faster than spontaneous peptide hormone 
discharge (StenoVec & al. 2004). Time-lapse con-
focal imaging was performed on cells expressing 
fluorescently tagged peptide ANP.emd (han & al. 
1999) in their vesicle lumen and in the presence 
of the extracellularly added fluorescent styryl dye 
FM 4-64 (BetZ & BeWick 1992). In lactotrophs, 
FM-membrane dye stains not only the plasma 
membrane when in the extracellular medium, but 
also the matrix of individual prolactin vesicles 
(angleSon & al. 1999, StenoVec & al. 2005), 
upon exposure to the extracellular solution. The 
studies were carried out in resting conditions 
and following stimulation by exposing cells to a 
high potassium-containing solution. When exo-
cytotic cargo release occurred, the fluorescence 
intensity of the ANP.emd probe decreased at the 
vesicle site and the vesicle was loaded through 
the same fusion pore with the FM 4-64. In resting 
lactotrophs, in 50% of spontaneously releasing 
vesicles, the peptide hormone release and the FM 
4-64 loading were slow (~3 min). However, high 
potassium stimulation triggered hormone release 
and FM 4-64 loading within seconds, indicating 
that in lactotrophs stimulation increases the rate 
of vesicle cargo release, very likely at the stage 
when the fusion pore is already formed (post-fusion 
regulation of a release; rahaMiMoff & ferNaNdez 
1997, StenoVec & al. 2004). 
Figure 4: (A) Confocal image of secreted rat prolactin (rPRL) visualized on the surface of the plasma membrane 
by anti-rPRL antibody and fluorescent secondary antibody (red). The cell was exposed to a solution containing 
100 mM K+, to depolarize the membrane and stimulate the cells prior to the immunocytochemical reaction. (B) 
Transmitted light image with DIC contrast of the cell displayed in A. Bar: 5 µm.
99N. Vardjan, M. Stenovec, J. Jorgačevski, M. Kreft, R. Zorec: Mehanizmi eksocitoze
Mechanisms of post-fusion regulation of 
vesicle cargo release in lactotrophs
Recent studies have shown that fusion pores 
are subject to regulations, which affect the release 
competence of a single vesicle (StenoVec & al. 
2004, reviewed in harata & al. 2006). In the next 
two chapters we will discuss two mechanisms 
that control the rate of vesicle cargo release in 
lactotrophs: (I) fusion pore kinetics and (II) fusion 
pore diameter (Fig. 5).
I. Fusion pore kinetics
It has been proposed that transient, “kiss-
and-run”, mode of vesicle fusion can limit or 
completely prevent peptide hormone release 
because of the relatively large size of peptide 
molecules and the consequent low diffusion 
mobility of these molecules (Barg & al. 2002, 
tsuBoi & rutter 2003, oBerMüller & al. 2005). 
To determine, if transient mode of vesicle fusion 
is slowing the release of vesicle cargo in resting 
lactotrophs, Cm measurements of single vesicle 
fusion events were performed by cell-attached 
patch-clamp technique (neher & Marty 1982). 
A stepwise increase in Cm (on-step) indicates full 
fusion exocytosis. If the on-step is followed by a 
downward, off-step in Cm, of a similar amplitude 
to the preceding on-step, the fusion of a vesicle 
with the plasma membrane is transient (transient 
Figure 5: Mechanisms of vesicle cargo discharge modulation.
The permeation of molecules through the fusion pore depends on the fusion pore conductance (G; diameter) and/
or fusion pore kinetics. A wider fusion pore (higher G) and/or faster frequency of fusion pore openings with longer 
fusion pore dwell-times (faster kinetic of fusion pore openings) leads to increased release of peptides from a single 
vesicle in stimulated conditions.
or “kiss-and-run” exocytosis) (neher & Marty 
1982, ferNaNdez & al. 1984, scepek & LiNdau 
1993, LoLLike & al. 1995).
The majority of exocytotic events (> 99%) ob-
served by Cm measurements in resting lactotrophs 
were transient fusion events (i.e. transient fusion 
pore openings). The occurrence of non-reversing 
steps, representing full vesicle fusion (neher & 
Marty 1982), was very low (~0.4% of all events). 
The fusion pore open-state duration of a single 
transient event was at rest ~50 ms (StenoVec & 
al. 2004, Vardjan & al. 2007), similar as in rest-
ing synapses of calyx of Held (suN & al. 2002). 
These transient events appeared repetitively in 
bursts lasting for as long as 760 s (“the pulsing 
pore”; StenoVec & al. 2004, Vardjan & al. 2007), 
some of them were more complex with multiple 
amplitude on- and off-steps representing repetitive 
compound vesicle-to-vesicle-to-plasma membrane 
fusion events (Vardjan & al. 2009). Regular 
repetitive fusion pore openings and slow, but 
synchronous, loading and unloading of fluorescent 
probes in lactotrophs, likely indicate that in rest-
ing lactotrophs the slow probe exchange through 
the fusion pore may be constrained kinetically by 
regular fusion pore openings (fusion pore flicker-
ing, also termed fusion pore gating; StenoVec & al. 
2004). Amperometric studies on neurons revealed 
that fusion pore flickering can limit the release 
of dopamine from synaptic terminals (Staal & 
al. 2004). This indicates that flickering transient 
100 Acta Biologica Slovenica, 52 (2), 2009
fusion pores may also be involved in the regula-
tion of small molecular weight transmitter release 
in synapses despite the relatively fast diffusion 
mobility of small chemical molecules.
Transient and full fusion exocytosis were 
reported to coexist in some systems and it has 
been suggested that switching between the tran-
sient to full fusion mode due to cell stimulation 
can result in the modulation of the amount of 
cargo release from a single vesicle (reviewed 
in harata & al. 2006). Interestingly, we have 
observed by Cm measurements that transient 
fusion pore openings with estimated mean burst 
duration of >100 s were the predominant mode 
of exocytosis not only in resting but also in high 
potassium stimulated lactotrophs (StenoVec & al. 
2004, Vardjan & al. 2007). Full vesicle fusion 
occurred with practically the same relatively low 
incidence before and after stimulation (Vardjan 
& al. 2007). However, transient events occurred 
fourfold more frequently in stimulated compared 
to resting lactotrophs. Moreover, the fusion pore 
dwell time was twofold longer after stimulation. 
Stimulus thus prolongs the effective open time of 
the transient pores, facilitating hormone secretion 
without full fusion (Vardjan & al. 2007). More 
frequent transient events with prolonged fusion 
pore open state dwell-time were observed also 
in lactotrophs after hypotonic stimulation (jor-
gacevski & al. 2008). These results are in contrast 
to previous studies in chromaffin cells where low 
levels of stimulation triggered “kiss-and-run” 
exocytosis, whereas with stronger stimulation the 
predominant mode of exocytosis was full fusion 
(eLhaMdaNi & al. 2001, fuLop & al. 2005). 
II. Fusion pore diameter 
Recently, it has been shown that the fusion pore 
diameter can be the limiting factor preventing the 
permeation of molecules through the fusion pores 
(Barg & al. 2002, takahashi & al. 2002, tsuBoi 
& rutter 2003, fuLop & al. 2005). Therefore, the 
release of vesicle cargo from vesicles undergoing 
transient, “kiss-and-run”, exocytosis may depend 
also on the diameter of the fusion pore. We used 
electrophysiological and optical methods to meas-
ure the size of the effective fusion pore diameter 
in exocytic vesicles in lactotrophs before and after 
stimulation (Vardjan & al. 2007). 
The effective diameter of a fusion pore can 
be calculated from the fusion pore conductance 
(Gp; spruce & al. 1990, LoLLike & LiNdau 1999). 
Cell-attached patch-clamp technique can be used 
to monitor the time course of the Gp by model-
ling a vesicle fusing with the cell membrane as 
a conductance (the fusion pore) in series with a 
capacitor (the vesicle membrane) (ziMMerBerg & 
al. 1987). We have measured the Gp as described in 
LoLLike & LiNdau (1999) and then we calculated 
the effective fusion pore diameters from the Gp 
values (described in spruce & al. 1990). At rest, 
the Gp values in transient events ranged from 8 
to 200 pS (the average 53 pS), corresponding to 
fusion pore diameters of 0.4 nm to 2.0 nm. After 
stimulation, the Gp values of transient events 
increased to an average measurable Gp of 81 pS. 
The maximal measurable Gp value after stimula-
tion was ~530 pS (i.e. 3.2 nm), indicating that 
the diameter of fusion pore in the majority of 
stimulated events had effective pore diameters 
>3.0 nm. In >98% stimulated events the Gp values 
increased after stimulation to an unmeasurable final 
conductance, representing fusion pore expansion, 
and then decreased again to measurable values, 
indicating fusion pore narrowing/closing. This 
is consistent with previous results observed in 
different cell systems, where fusion pores enlarge 
their diameters several folds and close completely 
afterwards (ferNaNdez & al. 1984, MoNck & al. 
1990, spruce & al. 1990, MeLikyaN & al. 1995, 
LariNa & al. 2007). Events with the measurable 
size of the fusion pore were more frequent in rest-
ing than in stimulated conditions (25% vs. 2%), 
indicating that the stimulus increases the size of 
the transient fusion pore (Vardjan & al. 2007). 
To confirm our observations, we performed 
optical fusion pore studies. Permeation of FM 
4-64 (molecular diameter ~ 0.9 nm) and HEPES 
(molecular diameter ~ 0.5 nm) through spon-
taneously forming fusion pores was studied in 
lactotroph vesicles expressing synaptopHluorin 
(spH; Vardjan & al. 2007). spH is a pH-dependent 
protein consisting of the vesicle membrane-
targeted protein VAMP2 (synaptobrevin-2) with 
a pH-sensitive enhanced green fluorescent protein 
(superecliptic pHluorin) fused to its luminal side 
(MieseNBöck & al. 1998). At the acidic pH of 
resting vesicles, spH fluorescence is quenched by 
protons because of the H+-ATPase activity. After 
101N. Vardjan, M. Stenovec, J. Jorgačevski, M. Kreft, R. Zorec: Mehanizmi eksocitoze
fusion with the plasma membrane, the vesicle 
interior becomes accessible from the relatively 
alkaline extracellular pH environment, allowing 
the protons to escape. The fluorescence intensity 
of spH increases rapidly and remains elevated 
until the pore closes and the vesicle is reacidified 
(MieseNBöck & al. 1998). 
Confocal imaging showed that half of the 
spontaneous exocytotic events exhibited fusion 
pore openings associated with an increase in spH 
fluorescence, indicating permeation of protons, 
however the pores were impermeable to FM 4-64 
and HEPES molecules. Together with the results 
on Gp measurements these findings indicate an 
open fusion pore diameter in resting peptidergic 
vesicles of <0.5 nm. This is much narrower than 
the size of neuropeptides stored in these vesicles 
(prolactin molecular diameter = ~5.2 nm; Vardjan 
& al. 2007), indicating that a narrow open fusion 
pore (lower conductance - Gp) prevents or slows 
down the release process in resting lactotrophs. 
Probes used in the earlier fusion pore permeability 
studies in resting secretory cells (FM-dyes, horse-
radish peroxidase, antibodies) were of relatively 
large size >0.9 nm (MaLgaroLi & al. 1995, ryan 
& al. 1997, Sara & al. 2005), leading to the 
underestimation of fusion pore diameter and the 
extent of spontaneous fusion.
In stimulated lactotrophs, >70% of exocytotic 
events exhibited a larger, FM 4-64–permeable pore 
(>0.9 nm) consistent with previous fusion pore 
permeation studies (Barg & al. 2002, takahashi 
& al. 2002, tsuBoi & rutter 2003, fuLop & al. 
2005) and our Gp measurements (Vardjan & 
al. 2007, Jorgacevski & al. 2008). Stimulation-
induced fusion pore widening is consistent with a 
facilitated vesicle cargo discharge in the majority 
of stimulated fusion events recorded (Vardjan & 
al. 2007). Similarly, stimulation led to the fusion 
pore expansion in chromaffin cells, therefore 
increasing the efficiency of release of small clas-
sical transmitters (eLhaMdaNi & al. 2001, fuLop 
& al. 2005). 
Summary
Transient fusion was considered to be an event 
mediating complete discharge of the vesicle content 
(katz 1969), however recent findings indicate that 
vesicle content may be emptied incompletely in 
transient fusion events. Partial vesicle discharge 
appears to occur at the post-fusion stage. Tran-
sient fusion pores are subject to physiological 
regulation which affects the amount of vesicle 
cargo discharge from a single vesicle. Current 
knowledge of regulatory mechanisms at the 
post-fusion stage, involving fusion pore diameter 
and/or fusion pore open-time modulation, is still 
limited (rahaMiMoff & ferNaNdez 1997, Vardjan 
& al. 2007). However, in lactotrophs the transient 
“kiss-and-run” mode of exocytosis is a robust 
physiological phenomenon and thus represents a 
model for further studies towards the unravelling 
the nature of the exocytotic fusion machinery. 
Based on the current results the release of vesicle 
cargo may be restrained kinetically and/or due 
to a narrow fusion pore. Under stimulation, the 
pre-formed fusion pore may retain the transient 
nature, but with a longer dwell-time, increased 
frequency of re-openings and a wider effective 
fusion pore diameter. All of these changes will 
facilitate the vesicle cargo release (Fig. 5; Vardjan 
& al. 2007). Regulation of fusion pore dynamics is 
important, as any modulation of release processes 
could have an impact on specific physiological 
functions of cells.
Membrane fusion involves high energy bar-
rier, therefore it is unlikely that transient fusion 
events represent cycles of fusion/fission of a single 
vesicle. Transient fusion events may represent 
fluctuations of an open fusion pore between states 
where the pore is extremely narrow. A narrow fu-
sion pore is likely composed of molecules with 
highly negative curvatures (churchWard & al. 
2008). It was reported recently that the structure 
of plasma membrane sites where prolactin vesicles 
undergo exocytosis are distinct from the plasma 
membrane areas devoid of docked prolactin 
vesicles (gonçalVeS & al. 2008). In the future the 
determination of the structure of the fusion pore 
and of the cellular mechanisms underlying the 
nature of the repetitive transient fusion events will 
be critical for a better understanding of exocytosis 
of peptidergic vesicles.
102 Acta Biologica Slovenica, 52 (2), 2009
Povzetek 
Dolgo je veljalo, da se pri procesu prehodne 
eksocitoze vsebina mešička popolnoma izloči 
skozi fuzijsko poro (katz 1969), vendar pa so 
novejše raziskave pokazale, da se lahko vsebina 
mešička pri prehodni eksocitozi sprosti le delno. 
Delno izločanje vsebine mešička je najverjetneje 
uravnavano po zlitju mešička s plazemsko mem-
brano oz. na stopnji že oblikovane fuzijske pore. 
Fuzijska pora je torej prehodna struktura, ki je po 
samem nastanku lahko fiziološko uravnavana, kar 
vpliva na sproščanje količine vsebine mešička. 
Dosedanje znanje o postfuzijskem uravnavanju 
izločanja vsebine mešička na ravni modulacije 
premera fuzijske pore in časa odprtja fuzijske pore 
je pomanjkljivo (rahaMiMoff & ferNaNdez 1997, 
Vardjan & al. 2007). V laktotrofih je prehodna 
eksocitoza robusten fiziološki fenomen, zato lahko 
celice laktotrofov uporabljamo kot model za študij 
prehodne eksocitoze, kar bi v prihodnje pripomoglo 
k razjasnitvi osnovnih mehanizmov eksocitoze. Na 
podlagi dosedanjih rezultatov je izločanje vsebine 
mešička lahko ovirano kinetično in/ali zaradi ozke 
fuzijske pore. Po stimulaciji lahko fuzijska pora 
ohrani svojo prehodno naravo odpiranja, vendar 
pa je prehodna fuzijska pora po stimulaciji dlje 
časa odprta, se pogosteje odpira in zapira in ima 
večji premer. Vse te spremembe pospešijo izločanje 
vsebine mešička (Fig. 5; Vardjan & al. 2007). 
Uravnavanje dinamike izločanja vsebine mešička 
skozi fuzijsko poro je fiziološko zelo pomembno, 
saj ima lahko vsaka modulacija izločanja vpliv na 
specifične fiziološke funkcije celic. 
Fuzija membran je energijsko neugoden 
proces, zato prehodna narava odpiranja fuzijske 
pore najverjetneje ni posledica ponavljajoče fuzije/
fisije posameznega mešička. Prehodno odpiranje 
fuzijske pore je bolj verjetno posledica fluktuacije 
odprte fuzijske pore med stanji, ko je fuzijska pora 
skrajno ozka in stanji, ko se le-ta prehodno bolj od-
pre. Ozka fuzijska pora je zelo verjetno sestavljena 
iz molekul, ki imajo močno negativno ukrivljenost 
(churchWard & al. 2008). Nedavno je bilo po-
kazano, da se struktura plazemske membrane na 
področjih, kjer poteka eksocitoza prolaktinskih 
mešičkov razlikuje od plazemske membrane, kjer 
ni pripetih prolaktinskih mešičkov (gonçalVeS 
& al. 2008). V prihodnje bo določitev strukture 
fuzijske pore in pa razjasnitev celičnih mehaniz-
mov, ki uravnavajo prehodno odpiranje fuzijske 
pore, kritično pripomoglo k boljšemu razumevanju 
mehanizmov eksocitoze peptidnih hormonov.
Acknowledgements
This work was supported by grants P3 310 
381, Z3 7476 from the Ministry of Higher Educa-
tion, Sciences and Technology of the Republic of 
Slovenia, NS36665 from the National Institutes 
of Health, EC grant #QLG3 2001-2004, EC 
support DECG, CLG3-CT-2001-02004, and EC 
GROWBETA QLG1-CT-2001-02233.
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aLBiLLos A., G. derNick, H. horStMann, W. alMerS, G. aLvarez de toLedo & M. LiNdau 1997: The 
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angleSon J., A. cochiLLa, G. kiLic, I. Nussinovitch & W. Betz 1999: Regulation of dense core re-
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440–446.
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2002: Delay between fusion pore opening and peptide release from large dense-core vesicles in 
neuroendocrine cells. Neuron. 33: 287–299.
BetZ W. & G. BeWick 1992: Optical analysis of synaptic vesicle recycling at the frog neuromuscular 
junction. Science 255: 200–203.
ceccareLLi B., W. hurLBut & a. Mauro 1973: Turnover of transmitter and synaptic vesicles at the 
frog neuromuscular junction. J. Cell. Biol. 57: 499–524.
chow r., l. Von rüden & e. neher 1992: Delay in vesicle fusion revealed by electrochemical moni-
toring of single secretory events in adrenal chromaffin cells. Nature 356: 60–63.
105N. Vardjan, M. Stenovec, J. Jorgačevski, M. Kreft, R. Zorec: Mehanizmi eksocitoze
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and calcium entry in calf chromaffin cells. Neuron 31: 819–830.
ferNaNdez J., e. Neher & B. goMperts 1984: Capacitance measurements reveal stepwise fusion events 
in degranulating mast cells. Nature 312: 453–455.
fesce r., f. grohovaz, f. vaLtorta & J. MeLdoLesi 1994: Neurotransmitter release: fusion or ’kiss-
and-run’? Trends Cell Biol. 4: 1–4.
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adrenal chromaffin cells. J. Neurosci. 25: 7324–7332.
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haN W., y. Ng, d. axeLrod & e. LevitaN 1999: Neuropeptide release by efficient recruitment of 
diffusing cytoplasmic secretory vesicles. Proc. Natl. Acad. Sci. U.S.A. 96: 14577–14582.
harata N., a. aravaNis & r. tsieN 2006: Kiss-and-run and full-collapse fusion as modes of exo-
endocytosis in neurosecretion. J. Neurochem. 97: 1546–1570.
he L., x. Wu, r. MohaN & L. Wu 2006: Two modes of fusion pore opening revealed by cell-attached 
recordings at a synapse. Nature 444: 102–105.
heuser J. & t. reese 1973: Evidence for recycling of synaptic vesicle membrane during transmitter 
release at the frog neuromuscular junction. J. Cell Biol. 57: 315–344.
Jorgacevski J., M. steNovec, M. kreft, a. BaJic, B. rituper, N. vardJaN, s. stoJiLkovic & r. zorec 
2008: Hypotonicity and Peptide Discharge from a Single Vesicle. Am. J. Physiol. Cell Physiol. 
295: C624–631.
katz B. 1969: The release of neural transmitter substances. Liverpool University Press, Liverpool.
LariNa o., p. Bhat, J. pickett, B. LauNikoNis, a. shah, W. kruger, J. edWardsoN & p. thorN 2007: 
Dynamic regulation of the large exocytotic fusion pore in pancreatic acinar cells. Mol. Biol. Cell 
18: 3502–3511.
LiNdau M. & e. Neher 1988: Patch-clamp techniques for time-resolved capacitance measurements 
in single cells. Pflugers Arch 411: 137–146.
Logiudice L. & g. MattheWs 2006: The synaptic vesicle cycle: is kissing overrated? Neuron 51: 
676–677.
LoLLike k., N. Borregaard & M. LiNdau 1995: The exocytotic fusion pore of small granules has a 
conductance similar to an ion channel. J. Cell Biol. 129: 99–104.
LoLLike k. & M. LiNdau 1999: Membrane capacitance techniques to monitor granule exocytosis in 
neutrophils. J. Immunol. Methods 232: 111–120.
MaLgaroLi a., a. tiNg, B. WeNdLaNd, a. BergaMaschi, a. viLLa, r. tsieN & r. scheLLer 1995: 
Presynaptic component of long-term potentiation visualized at individual hippocampal synapses. 
Science 268: 1624–1628.
MeLikyaN g., W. NiLes, v. ratiNov, M. karhaNek, J. ziMMerBerg & f. coheN 1995: Comparison of 
transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar 
membranes. J. Gen. Physiol.106: 803–819.
MieseNBöck g., d. de aNgeLis & J. rothMaN 1998: Visualizing secretion and synaptic transmission 
with pH-sensitive green fluorescent proteins. Nature 394: 192–195.
MoNck J., g. aLvarez de toLedo & J. ferNaNdez 1990: Tension in secretory granule membranes 
causes extensive membrane transfer through the exocytotic fusion pore. Proc. Natl. Acad. Sci. 
USA 87: 7804–7808.
neher e. & a. Marty 1982: Discrete changes of cell membrane capacitance observed under condi-
tions of enhanced secretion in bovine adrenal chromaffin cells. Proc. Natl. Acad. Sci. USA 79: 
6712–6716.
oBerMüLLer s., a. LiNdqvist, J. karaNauskaite, J. gaLvaNovskis, p. rorsMaN & s. Barg 2005: 
Selective nucleotide-release from dense-core granules in insulin-secreting cells. J. Cell Sci. 118: 
4271–4282.
106 Acta Biologica Slovenica, 52 (2), 2009
rahaMiMoff r. & J. ferNaNdez 1997: Pre- and postfusion regulation of transmitter release. Neuron 
18: 17–27.
ryaN t., h. reuter & s. sMith 1997: Optical detection of a quantal presynaptic membrane turnover. 
Nature 388: 478–482.
sara y., t. virMaNi, f. deák, x. Liu & e. kavaLaLi 2005: An isolated pool of vesicles recycles at rest 
and drives spontaneous neurotransmission. Neuron 45: 563–573.
scepek s. & M. LiNdau 1993: Focal exocytosis by eosinophils-compound exocytosis and cumulative 
fusion. EMBO J. 12: 1811–1817.
shaNer N., p. steiNBach & r. tsieN 2005: A guide to choosing fluorescent proteins. Nat. Methods 
2: 905–909.
sMith s., r. reNdeN & h. voN gersdorff 2008: Synaptic vesicle endocytosis: fast and slow modes 
of membrane retrieval. Trends Neurosci. 31: 559–568.
spruce a., L. BreckeNridge, a. Lee & W. aLMers 1990: Properties of the fusion pore that forms during 
exocytosis of a mast cell secretory vesicle. Neuron 4: 643–654.
staaL r., e. Mosharov & d. suLzer 2004: Dopamine neurons release transmitter via a flickering 
fusion pore. Nat. Neurosci. 7: 341–346.
steNovec M., M. kreft, i. poBeraJ, W. Betz & r. zorec 2004: Slow spontaneous secretion from single 
large dense-core vesicles monitored in neuroendocrine cells. FASEB J. 18: 1270–1272.
steNovec M., i. poBeraJ, M. kreft & r. zorec 2005: Concentration-dependent staining of lactotroph 
vesicles by FM 4–64. Biophys. J. 88: 2607–2613.
suN J., x. Wu & L. Wu 2002: Single and multiple vesicle fusion induce different rates of endocytosis 
at a central synapse. Nature 417: 555–559.
takahashi N., t. kishiMoto, t. NeMoto, t. kadoWaki & h. kasai 2002: Fusion pore dynamics and 
insulin granule exocytosis in the pancreatic islet. Science 297: 1349–1352.
tsuBoi t. & g. rutter 2003: Multiple forms of »kiss-and-run« exocytosis revealed by evanescent 
wave microscopy. Curr. Biol. 13: 563–567.
vardJaN N., J. Jorgacevski, M. steNovec, M. kreft & r. zorec 2009: Compound exocytosis in 
pituitary cells. Ann. N. Y. Acad. Sci. 1152: 63–75.
vardJaN N., M. steNovec, J. Jorgacevski, M. kreft & r. zorec 2007: Subnanometer fusion pores 
in spontaneous exocytosis of peptidergic vesicles. J. Neurosci. 27: 4737–4746.
WightMaN r., J. JaNkoWski, r. keNNedy, k. kaWagoe, t. schroeder, d. LeszczyszyN, J. Near, e. J. 
diLiBerto & o. viveros 1991: Temporally resolved catecholamine spikes correspond to single 
vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. USA 88: 10754–10758.
ziMMerBerg J., M. curraN, f. coheN & M. BrodWick 1987: Simultaneous electrical and optical me-
asurements show that membrane fusion precedes secretory granule swelling during exocytosis of 
beige mouse mast cells. Proc. Natl.Acad. Sci. USA 84: 1585–1589.