Calcium Hypothesis and Transient Fusion Pore Formation

Although Katz and colleagues mainly used the neuromuscular junction and the giant synapse of the squid as model systems, employing technology that prevented the study of single subcellular organelles, their results were interpreted with most interesting and still valid conclusions:

In experiments where the role of Ca²⁺ in triggering secretion was tested, Katz and Miledi were able to block synaptic transmission in the giant synapse of the squid by applying a pulse of strong depolarization to the presynaptic membrane.Reference Katz and Miledi³ Rather high depolarization made the membrane terminal at least 130 mV positive with respect to outside, thus acting as an electric potential barrier preventing Ca²⁺ entry into the cell cytoplasm. As soon as the barrier was dropped, a large surge of secretion was detected as may be observed in panels 5/6 to 8 of Figure 2 (adapted from Ref. Reference Jones5, Figure 23).

Figure 2

Suppression of transmitter release during large ‘positive voltage step’ of presynaptic membrane potential. Taken with permission from Katz.Reference Katz and Miledi^3, Reference Katz⁴ Squid giant synapse treated with tetrodotoxin. Presaynaptic terminal loaded with tetraethylammonium ions. Blocks 1 to 8 display records obtained by increasing pulse intensity. In each block of records, the bottom trace shows the presynaptic voltage step, the middle record shows postsynaptic response, while the presynaptic current pulse is monitored in the top trace. Note the progressive suppression of ‘on’-response (increase in postsynaptic depolarization with a short delay after the current pulse application), and replacement by ‘off’-response (increase in postsynaptic depolarization at the end of the current pulse application), as presynaptic voltage is increased from 100 to about 200 mV (records 5/6 to 8).

This experiment provided compelling evidence to support the established current view according to which the entry of Ca²⁺ from the extracellular space is a prerequisite for the reactions that lead to the release of messenger molecules. Furthermore, the abrupt surge of secretion that followed the presynaptic depolarization also suggested that the vesicles had to be in a distinct physiological state to respond swiftly to the abrupt dissipation of the potential barrier and the ensuing increase in cytoplasmic calcium activity.

This issue is still relevant today, especially in the view of the function of the molecular machinery and the events leading to the presynaptic release. With the present knowledge of molecular structure of voltage-gated calcium channelsReference Jones⁵ which are orders of magnitude smaller than the much more complex and much larger molecular anatomy of the secretory vesicle,Reference Takamori, Holt, Stenius, Lemke, Grønborg, Riedel, Urlaub, Schenck, Brügger, Ringler, Müller, Rammner, Gräter, Hub, De Groot, Mieskes, Moriyama, Klingauf, Grubmüller, Heuser, Wieland and Jahn⁶ questions emerge of how the supramolecular structure of a vesicle can attain full and rapid release competence, and how the vesicle release can occur with such a minimal delay following the increase in cytosolic calcium activity after opening the voltage-gated calcium channels? It appears that the increased calcium activity triggers the vesicular apparatus to transit quickly from a state of no release to a state enabling the release surge of the transmitter, as described in the early experiments by B. Katz and colleagues. In particular, one needs to consider at least two scenarios of how vesicles release their cargo swiftly in response to the stimulus: (i) a stimulus triggers a chain of reactions that include the overcoming of the high energy barrier for membrane merger and the fusion pore formation, (ii) or the vesicle and plasma membranes are already fused before the stimulus delivery, whereby vesicles are release incompetent, unless the stimulus is delivered. In the latter scenario, which intuitively exhibits a much shorter delay to a stimulus versus the first scenario, where vesicles enter the chain of events leading to membrane merger, vesicles may be pre-fused before the actual stimulus delivery, yet their pre-existing fusion pores are too narrow to mediate the release of signalling molecules.

To address these intriguing questions experimentally it would be ideal to have a model system and techniques that enable the monitoring of transitions between the functional states of a single vesicle, ranging from the release incompetent to the release competent one. One such suitable model system is that of the pituitary cells (Figure 1, inset); and the fusion pore studies undertaken in these cells will be presented in the final part of this article.

Fusion pore exhibits complex properties

Since the advent of the electrophysiological patch-clamp technique in 1982Reference Neher and Marty⁷ it became possible to monitor time-dependent fusion of a single vesicle. This initiated a series of studies that addresses the classical view postulating that, upon stimulation, the fusion pore is formed, through which cargo molecules diffuse from the vesicle lumen into the cell exterior. Once formed, the fusion pore either closes (transient fusion), as already considered by B. Katz,Reference Katz⁴ to allow the vesicle to be reused in the next round of exocytosis (kiss-and-run exocytosis),Reference Ceccarelli, Hurlbut and Mauro⁸ or it fully widens leading to the complete merger of the vesicle membrane with the plasma membrane (full fusion exocytosis).Reference Heuser and Reese⁹ The patch-clamp membrane capacitance measurementsReference Neher and Marty⁷ revealed that the fusion pore can also fluctuate between an open and a closed state in the subsecond time domain (fusion pore flickering) before full fusion of the vesicleReference Fernandez, Neher and Gomperts¹⁰ or can retain the transient nature of opening and closing for several tens of minutes or even longer, sometimes periodically (‘the pulsing pore’)Reference Stenovec, Kreft, Poberaj, Betz and Zorec¹¹ as was observed in the anterior pituitary cellsReference Stenovec, Kreft, Poberaj, Betz and Zorec^11–Reference Jorgacevski, Stenovec, Kreft, Bajić, Rituper, Vardjan, Stojilkovic and Zorec¹³ in chromaffin cells,Reference Henkel, Meiri, Horstmann, Lindau and Almers¹⁴ and also in plant cells.Reference Thiel, Kreft and Zorec¹⁵ In anterior pituitary cells,Reference Stenovec, Kreft, Poberaj, Betz and Zorec^11–Reference Jorgacevski, Stenovec, Kreft, Bajić, Rituper, Vardjan, Stojilkovic and Zorec¹³ rhythmic fusion pore openings and closings were sometimes recorded for relatively long periods (>600 s), which indicates that the fusion pore itself may be considered as an energetically favourable structure.

It is important to note that electrophysiological membrane capacitance measurements, where the properties of the fusion pore can be studied directly,Reference Fernandez, Neher and Gomperts^10, Reference Stenovec, Kreft, Poberaj, Betz and Zorec^11–Reference Thiel, Kreft and Zorec¹⁵ have some limitations. For example, it is difficult to reconcile that the term ‘fusion pore closure’ actually reflects the complete fission of a vesicle from the plasma membrane. Due to recording noise limitations, extreme narrowing of the fusion pore diameter may be easily misinterpreted as the fusion pore closure.Reference Debus and Lindau¹⁶ This consideration is important if one aims to address the physiological relevance of transient repetitive fusion events, which can be observed in some cellular systems, and in particular in the pituitary cells.

Therefore, bearing these questions in our mind, we will first discuss spontaneous and stimulated release from a single peptidergic vesicle. Then we will address the nature of the vesicle cargo release in the light of an all-or-none event as proposed by Katz and colleagues. For this we will describe measurements obtained in pituitary lactotrophs at the single vesicle level, which clearly show that vesicle discharge may not be considered simply as an all-or-nothing event. Then we will ask how partial release from a single vesicle can be attained and, finally, we will consider stimulus-dependent modulation of vesicle exocytosis.

Differences in Spontaneous and Stimulated Exocytosis

In almost all secretory cell types, including neuronsReference Katz⁴ the basal cargo secretion, which is assumed to occur with low probability,Reference Murthy and Stevens¹⁷ can be detected in the absence of cell stimulation. In the past, the spontaneous exocytotic events were largely neglected because of their infrequent occurrence and the belief that they exhibit similar properties as the stimulated events.Reference Katz⁴ However, new methodological approaches revealed that spontaneous vesicle exocytosis differs from the stimulated one in many respects.

SNARE (soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptor) proteins were shown to mediate both spontaneous and stimulated exocytosis (reviewed in Ref. Reference Jahn and Scheller18); however, the deletions of SNARE proteins affect spontaneous and stimulated synaptic vesicle fusion to different extents.Reference Deitcher, Ueda, Stewart, Burgess, Kidokoro and Schwarz^19–Reference Washbourne, Thompson, Carta, Costa, Mathews, Lopez-Benditó, Molnár, Becher, Valenzuela, Partridge and Wilson²¹ Some results indicate that vesicles use different SNARE components when undergoing spontaneous and stimulated fusion.Reference Deák, Schoch, Liu, Südhof and Kavalali^22, Reference Hua, Raciborska, Trimble and Charlton²³ Furthermore, SNARE proteins synaptotagmin-1/-2 are thought to trigger the release of vesicle cargo by binding of Ca²⁺ upon stimulation.Reference Geppert, Goda, Hammer, Li, Rosahl, Stevens and Südhof^24–Reference Pang, Sun, Rizo, Maximov and Südhof²⁶ On the contrary, synaptotagmin-1/-2 were shown to restrict spontaneous release.Reference Pang, Sun, Rizo, Maximov and Südhof²⁶ Recently, synaptotagmin-12, a Ca²⁺-independent synaptic vesicle protein controlled by cAMP-dependent processes, has been identified as a possible selective modulator of spontaneous synaptic-vesicle exocytosis.Reference Maximov, Shin, Liu and Südhof²⁷ Exocytotic apparatus at rest differs from the stimulated one also in the requirement of distinct proteins involved in vesicle trafficking,Reference Wucherpfennig, Wilsch-Bräuninger and González-Gaitán²⁸ vesicle priming,Reference Virmani, Ertunc, Sara, Mozhayeva and Kavalali²⁹ and possibly vesicle recycling.Reference Sara, Virmani, Deák, Liu and Kavalali^30, Reference Groemer and Klingauf³¹ Recent studies have pointed out the role of lipids, i.e. cholesterol in balancing spontaneous and stimulated neurotransmitter release by hindering spontaneous and sustaining evoked exo-/endocytosis.Reference Wasser, Ertunc, Liu and Kavalali³² How sphingosine, a lipid molecule that activates the formation of the SNARE complex via synaptobrevin affects spontaneous exocytosis, remains to be determined.Reference Darios, Wasser, Shakirzyanova, Giniatullin, Goodman, Munoz-Bravo, Raingo, Jorgacevski, Kreft, Zorec, Rosa, Gandia, Gutiérrez, Binz, Giniatullin, Kavalali and Davletov³³ Spontaneous and stimulated exocytosis differ also in the kinetics of the vesicular content dischargeReference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² and in the size of the fusion pore,Reference Jorgacevski, Stenovec, Kreft, Bajić, Rituper, Vardjan, Stojilkovic and Zorec¹³ which will be discussed in the next sections.

Slow Spontaneous and Rapid Stimulated Peptide Release from Vesicles

The rateReference Stenovec, Kreft, Poberaj, Betz and Zorec^11, Reference Albillos, Dernick, Horstmann, Almers, Alvarez de Toledo and Lindau³⁴ and the amount of vesicle cargo releaseReference Angleson, Cochilla, Kilic, Nussinovitch and Betz³⁵ may be regulated at the level of an individual secretory vesicle after fusion pore establishment, which has been termed post-fusion regulation.Reference Rahamimoff and Fernandez³⁶ To study the kinetic properties of spontaneous and stimulated peptide release,Reference Stenovec, Kreft, Poberaj, Betz and Zorec¹¹ we have used lactotrophs, the prolactin secreting anterior pituitary cells (Figure 1, inset).Reference Walker and Farquhar³⁷ It has been shown that stimulated hormone discharge from a single vesicle can be 10–20 times faster than the discharge under resting conditions (Figure 3). Optical studies revealed that a fluorescently tagged peptide (ANP.Emd), similar in size to the hormone prolactin, is released from prolactin vesicles within seconds after stimulation. Simultaneously, vesicle loading with the styryl dye FM 4-64 was observed. On the contrary, the hormone release and the FM 4-64 loading was slow (∼3 min) in many spontaneously releasing vesicles, indicating that the fusion pore properties in resting and in stimulated conditions differ (Figure 3).Reference Stenovec, Kreft, Poberaj, Betz and Zorec¹¹

Figure 3

Rapid stimulated and slow spontaneous peptide hormone release from a single vesicle. Time-dependent fluorescent intensity changes of fluorescence peptide (ANP.emd) and styryl dye FM 4-64 indicate the release of peptides and loading of the extracellular dye into single vesicles in spontaneous and in stimulated conditions. The time-course of fluorescence intensity changes of the two fluorescent probes differ significantly; it is rather slow (minutes) in spontaneously releasing vesicles and much more rapid (seconds) in stimulated vesicles. Note that the time-course of loading and unloading is similar (synchronous) in spontaneously secreting vesicles whereas it is different in stimulated vesicles (stimulation was attained by exposing cells to a K⁺-enriched extracellular solution (100 mM K⁺)). Modified from Stenovec and coworkersReference Stenovec, Kreft, Poberaj, Betz and Zorec¹¹ with the permission of The Federation of American Societies for Experimental Biology.

Measurements of cell-attached membrane capacitance in resting lactotrophs revealed regular repetitive transient fusion pore openings (the phenomenon termed ‘the pulsing pore’), appearing in bursts with the duration up to 760 s.Reference Stenovec, Kreft, Poberaj, Betz and Zorec^11–Reference Jorgacevski, Stenovec, Kreft, Bajić, Rituper, Vardjan, Stojilkovic and Zorec¹³ The duration of a single transient event at rest was ∼50 ms (Figure 4)Reference Stenovec, Kreft, Poberaj, Betz and Zorec^11, Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² similar to the resting synapses of calyx of Held.Reference Sun, Wu and Wu³⁸ It has been proposed that the slow exchange of fluorescence probes through the fusion pore in resting lactotrophs (Figure 3) may be constrained kinetically by a long-lasting regular fusion pore gating.Reference Stenovec, Kreft, Poberaj, Betz and Zorec¹¹ Transient vesicle fusion can limit or prevent peptide hormone release because of the large molecular size of peptides and the consequent low diffusional mobility of peptide molecules.Reference Barg, Olofsson, Schriever-Abeln, Wendt, Gebre-Medhin, Renström. and Rorsman^39–Reference Obermüller, Lindqvist, Karanauskaite, Galvanovskis, Rorsman and Barg⁴¹ Similarly, fusion pore flickering can limit dopamine release from synaptic terminals in neurons.Reference Staal, Mosharov and Sulzer⁴² This indicates that despite the relatively high diffusional mobility of small chemical transmitters, fusion pore gating may also be involved in the regulation of small chemical transmitter release in synapses.Reference de Toledo, Fernández-Chacón and Fernández⁴³

Figure 4

Repetitive transient fusion pore openings in resting lactotophs. Fusion pore openings of a single vesicle in resting pituitary cells were recorded by the patch-clamp capacitance measurements. Inset shows expanded segments of the trace, showing individual transient fusion events. Note that changes in Cm (top trace, imaginary part of admittance) in the inset are not correlated with changes in the real part of admittance (lower trace), indicating the correct phase angle setting of the lock-in amplifier by the calibration pulse (**) at the end of the trace. Arrow indicates a fusion pore event with higher amplitude and a lower frequency of occurrence (appearing every ∼12 s), suggesting the presence of two vesicles in this cell-attached membrane. Note the pulsing behaviour of the fusion pore with fusion pore dwell-time of around 50 ms. Adapted from Stenovec and coworkersReference Stenovec, Kreft, Poberaj, Betz and Zorec¹¹ with the permission of The Federation of American Societies for Experimental Biology.

Interestingly, the long lasting repetitive transient fusion pore openings recorded in physiological experiments indicate that a vesicle is stably fused to the plasma membrane. Therefore, one may be able to see such a structure by electron microscopy even in resting conditions. Indeed, scanning electron microscopy studies in which the surface structure of the individual resting pituitary cells was monitored revealed tiny openings, which may represent structures related to the fusion pores observed in electrophysiological measurements (Figure 5).

Figure 5

Scanning electron micrograph of the pituitary lactotroph surface exhibits fusion-pore-like formations. Panel A shows a view of the whole cell with a diameter of 10 μm. Panel B is magnified view of the framed region in panel A. Several small openings in the cell surface (black), which may be related to the fusion pore-like structures, are noticeable.

Subnanometre Fusion Pore Diameters at Rest Expand upon Stimulation

The slower vesicle cargo release observed in resting lactotrophs (Figure 3) might also reflect a narrow fusion pore of spontaneously releasing vesicles. Vesicle fusion was monitored by confocal microscopy in resting and stimulated lactotrophs expressing synaptopHluorin (spH), a pH-sensitive green fluorescence protein that enables optical discrimination between unfused and fused vesicles.Reference Miesenböck, De Angelis and Rothman⁴⁴ Simultaneously, the permeation of FM 4-64 dye and HEPES molecules through the fusion pores was monitored.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² Fusion pore openings associated with a change in spH fluorescence, were observed in >50% of the spontaneous exocytotic events.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² In these events the fusion pore was permeable to protons, as indicated by spH fluorescence changes, but impermeable to FM 4-64 (left; molecular diameter = ∼0.9 nm) and HEPES (molecular diameter = ∼0.5 nm) molecules. Thus, the fusion pore diameter in many resting peptidergic vesicles is <0.5 nm, which is smaller than the size of peptide hormone prolactin stored in these vesicles (prolactin molecular diameter = ∼5.2 nm).Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² Membrane capacitance measurements confirmed subnanometre fusion pores in resting lactotrophsReference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec^12, Reference Spruce, Breckenridge, Lee and Almers⁴⁵, suggesting that exocytosis without release of vesicle cargo may occur before delivery of the stimulus (‘unproductive exocytosis’, meaning that vesicles are release incompetent).

Upon stimulation, >70% of exocytotic events exhibited a larger, FM 4-64–permeable pore (>0.9 nm) consistent with previous fusion pore permeation studies.Reference Barg, Olofsson, Schriever-Abeln, Wendt, Gebre-Medhin, Renström. and Rorsman^39, Reference Tsuboi and Rutter^40, Reference Takahashi, Kishimoto, Nemoto, Kadowaki and Kasai^46, Reference Fulop, Radabaugh and Smith⁴⁷ In capacitance measurements, stimulation decreased the fraction of events with lowest measurable fusion pore diameter of 3.2 nm from 25% to 2% reflecting much wider pore diameters upon stimulation and likely a massive vesicle cargo discharge in the majority of these events.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² Similarly, fusion pore expansion was observed in chromaffin cells upon stimulation, which likely improves the efficiency of release of small classical transmitters.Reference Fulop, Radabaugh and Smith^47, Reference Elhamdani, Palfrey and Artalejo⁴⁸ Moreover, in stimulated lactotrophs, the pore size can reversibly open to several nanometres in diameter, since quantum dots coupled to prolactin antibodies can be introduced into retrieving prolactin vesicles.Reference Vardjan, Jorgačevski, Stenovec, Kreft and Zorec⁴⁹

Stimulus Modulates Fusion Pore Kinetics

While transient fusion is accepted as a mode of vesicle cargo release, both in central neurons and neuroendocrine cells, the prevalence of this mechanism in comparison to full fusion is still under discussion.Reference LoGiudice and Matthews⁵⁰ In lactotrophs, transient vesicle exocytosis appears to be the predominant mode of exocytosis in spontaneous and in stimulated cells.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² Optical studies on lactotrophs expressing spH revealed that 65% of spontaneous events were transient, some of them were repetitive.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² In 35% of all events, spH fluorescence persisted after fusion for >100 s. On the contrary, the majority of stimulated events (>93%) were persistent.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² In persistent events the lateral diffusion of spH signal from the site of exocytosis into the plasma membrane was not detected,Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² suggesting that vesicle exocytosis occurred in a non-full-collapse mechanism.Reference Taraska, Perrais, Ohara-Imaizumi, Nagamatsu and Almers^51, Reference Thorn and Parker⁵² Since FM 4-64 loading was fourfold slower in persistent events than in transient events, spH signal likely persisted because of a rapidly flickering fusion poreReference Stenovec, Kreft, Poberaj, Betz and Zorec^11, Reference Staal, Mosharov and Sulzer⁴² and not because of a long open fusion pore lifetime.Reference Ohara-Imaizumi, Nakamichi, Tanaka, Katsuta, Ishida and Nagamatsu⁵³ This hypothesis was confirmed by capacitance measurements, which revealed that transient fusion pore openings with the burst duration of >100 s are the predominant mode of spontaneous and stimulated exocytosis in lactotrophs.Reference Stenovec, Kreft, Poberaj, Betz and Zorec^11, Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² Optical monitoring of fusion pore dynamics is limited by the characteristics of the spH construct. Thus, the effect of fusion pore closure is poorly reflected in these studies because of vesicle reacidification, which in comparison to the fusion pore closure observed as an off-step in capacitance measurements is rather slow (τ = 4–5 s for synaptic vesicles).Reference Atluri and Ryan⁵⁴ These results are in contrast to previous studies on chromaffin cells where increased cell stimulation shifted the mode of exocytosis from transient to full fusion, resulting in the modulation of the amount of cargo release from a single vesicle.Reference Fulop, Radabaugh and Smith^47, Reference Elhamdani, Azizi and Artalejo^55, Reference Harata, Aravanis and Tsien⁵⁶

Capacitance measurements also revealed that, in lactotrophs, transient vesicle fusion occurred four times more frequently after stimulation with a twofold longer fusion pore dwell-time and a wider pore diameter.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹² Therefore, a stimulus prolongs the effective opening of a fusion pore and expands its subnanometre diameter to enable hormone secretion without full fusion.Reference Vardjan, Stenovec, Jorgacevski, Kreft and Zorec¹²

Perspectives

In contrast to the views developed by Bernard Katz decades ago, where transient fusion was considered to be an event mediating complete discharge of the vesicle content, recent findings indicate that vesicle content may be emptied incompletely. A key role in partial vesicle discharge appears to occur at the post-fusion stage. Fusion pores are subject to physiological regulation and this affects the amount of vesicle cargo to be released from a single vesicle. Therefore, vesicles may exhibit different release competence. Current knowledge of mechanisms determining vesicle release competence include fusion pore diameter regulation as well as the fusion pore dwell-time determination.Reference Stenovec, Kreft, Poberaj, Betz and Zorec^11, Reference Rahamimoff and Fernandez³⁶ In lactotrophs, the experiments point to the existence of both mechanisms: release of vesicle cargo may be restrained kinetically and/or due to a narrow fusion pore. Under stimulation, a pre-formed fusion pore may retain the transient nature, but with a prolonged dwell-time, increased frequency of re-openings and an increased fusion pore diameter. All of these changes will facilitate the vesicle cargo release (Figure 6).

Figure 6

Fusion pore properties modulation before and after stimulation. The permeation of molecules through the fusion pore depends on 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 a faster, more effective release of peptides from vesicles in stimulated events. Modified from Stenovec and coworkersReference Stenovec, Kreft, Poberaj, Betz and Zorec¹¹ with the permission of The Federation of American Societies for Experimental Biology.

A key question to be answered in the future is related to the nature of the repetitive transient fusion events. Do they represent cycles of merger/fission of a single vesicle? Given that membrane merger involves high energy barrier, such a repetitive cycle is unlikely. Perhaps the transient fusion events represent fluctuations of an open fusion pore between the states where the pore is extremely narrow, with a neck formed by the fused vesicle and plasma membrane. A narrow fusion pore may structurally involve membrane domains with high local curvature, which may be attained by highly anisotropic molecules with negative curvatures.Reference Churchward, Rogasevskaia, Brandman, Khosravani, Nava, Atkinson and Coorssen⁵⁷ In support of such a distinct membrane structure adjacent to fused vesicles versus plasma membrane, it was reported that plasma membrane sites where prolactin vesicles engage into exocytosis are distinct from the plasma membrane areas devoid of docked prolactin vesicles.Reference Gonçalves, Stenovec, Chowdhury, Grilc, Kreft and Zorec⁵⁸ The basis of energetic stability of such special curved structures is to be addressed in the future as well. Finally, the role of SNARE proteins in these physiological states remains to be determined as well.