Degranulation enhances presynaptic membrane packing, which protects NK cells from perforin-mediated autolysis

Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule–cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell–mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid “shields” against self-destruction.

Shortened tethering filaments stabilize presynaptic vesicles in support of elevated release probability during LTP in rat hippocampus

Long-term potentiation (LTP) is a cellular mechanism of learning and memory that results in a sustained increase in the probability of vesicular release of neurotransmitter. However, previous work in hippocampal area CA1 of the adult rat revealed that the total number of vesicles per synapse decreases following LTP, seemingly inconsistent with the elevated release probability. Here, electron-microscopic tomography (EMT) was used to assess whether changes in vesicle density or structure of vesicle tethering filaments at the active zone might explain the enhanced release probability following LTP. The spatial relationship of vesicles to the active zone varies with functional status. Tightly docked vesicles contact the presynaptic membrane, have partially formed SNARE complexes, and are primed for release of neurotransmitter upon the next action potential. Loosely docked vesicles are located within 8 nm of the presynaptic membrane where SNARE complexes begin to form. Nondocked vesicles comprise recycling and reserve pools. Vesicles are tethered to the active zone via filaments composed of molecules engaged in docking and release processes. The density of tightly docked vesicles was increased 2 h following LTP compared to control stimulation, whereas the densities of loosely docked or nondocked vesicles congregating within 45 nm above the active zones were unchanged. The tethering filaments on all vesicles were shorter and their attachment sites shifted closer to the active zone. These findings suggest that tethering filaments stabilize more vesicles in the primed state. Such changes would facilitate the long-lasting increase in release probability following LTP.

SNARE Complex Alters the Interactions of the Ca2+ sensor Synaptotagmin 1 with Lipid Bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca2+ sensor Synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca2+ binding. Prior to fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which binds the C2B domain of Syt1. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. The MD simulations showed that the C2AB tandem of Syt1 can either bridge SV and PM or immerse into PM, and that the latter configuration is more favorable energetically. Surprisingly, C2 domains did not cooperate in penetrating into PM, but instead mutually hindered the lipid penetration. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the Syt1-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains of Syt1 and promotes the immersion of both domains into the PM bilayer. Subsequently, we simulated this pre-fusion protein complex between lipid bilayers imitating PM and SV and found that the immersion of Syt1 into the PM bilayer within this complex promotes PM curvature and leads to lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.Statement of SignificanceNeuronal transmitters are packed in synaptic vesicles (SVs) and released by fusion of SVs with the presynaptic membrane (PM). SVs are attached to PM by the SNARE protein complex, and fusion is triggered by the Ca2+ sensor Synaptotagmin 1 (Syt1). Although Syt1 and SNARE proteins have been extensively studied, it is not yet fully understood how the interactions of Syt1 with lipids and the SNARE complex induce fusion. To address this fundamental problem, we took advantage of Anton2 supercomputer, a unique computational environment, which enables simulating the dynamics of molecular systems at a scale of microseconds. Our simulations produced a dynamic all-atom model of the prefusion protein-lipid complex and demonstrated in silico how the Syt1-SNARE complex triggers fusion.

Direct imaging of rapid tethering of synaptic vesicles accompanying exocytosis at a fast central synapse

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.

Synaptic Vesicles Having Large Contact Areas with the Presynaptic Membrane are Preferentially Hemifused at Active Zones of Frog Neuromuscular Junctions Fixed during Synaptic Activity

Synaptic vesicles dock on the presynaptic plasma membrane of axon terminals and become ready to fuse with the presynaptic membrane or primed. Fusion of the vesicle membrane and presynaptic membrane results in the formation of a pore between the membranes, through which the vesicle’s neurotransmitter is released into the synaptic cleft. A recent electron tomography study on frog neuromuscular junctions fixed at rest showed that there is no discernible gap between or merging of the membrane of docked synaptic vesicles with the presynaptic membrane, however, the extent of the contact area between the membrane of docked synaptic vesicles and the presynaptic membrane varies 10-fold with a normal distribution. The study also showed that when the neuromuscular junctions are fixed during repetitive electrical nerve stimulation, the portion of large contact areas in the distribution is reduced compared to the portion of small contact areas, suggesting that docked synaptic vesicles with the largest contact areas are greatly primed to fuse with the membrane. Furthermore, the finding of several hemifused synaptic vesicles among the docked vesicles was briefly reported. Here, the spatial relationship of 81 synaptic vesicles with the presynaptic membrane at active zones of the neuromuscular junctions fixed during stimulation is described in detail. For the most of the vesicles, the combined thickness of each of their contact sites was not different from the sum of the membrane thicknesses of the vesicle membrane and presynaptic membrane, similar to the docked vesicles at active zones of the resting neuromuscular junctions. However, the combined membrane thickness of a small portion of the vesicles was considerably less than the sum of the membrane thicknesses, indicating that the membranes at their contact sites were fixed in a state of hemifusion. Moreover, the hemifused vesicles were found to have large contact areas with the presynaptic membrane. These findings support the recently proposed hypothesis that, at frog neuromuscular junctions, docked synaptic vesicles with the largest contact areas are most primed for fusion with the presynaptic membrane, and that hemifusion is a fusion intermediate step of the vesicle membrane with the presynaptic membrane for synaptic transmission.

The Benefits of Botulinum Neurotoxin Treatment in a Multitude of Medical Conditions

Botulinum neurotoxins (BoNTs) are responsible for botulism in humans and vertebrates, being one of the six most catastrophic potential bioterrorism agents. This are ~150 kDa proteins, assembled as a ~50 kDa light chain (LC) and a ~100 kDa heavy chain (HC). The LC acts like a zinc metalloproteinase that cleaves three proteins in neurons, members of the SNARE (Soluble N-ethylmaleimide sensitive fusion attachment protein receptors) family: VAMP (vesicle-associated membrane protein) / synaptobrevin, SNAP-25 (synaptosomal-associated protein 25) and syntaxin. After cleavage of any of this proteins, neurotransmission is blocked and flaccid paralysis of the muscle is installed. This extraordinary restricted tropism for the cholinergic presynaptic membrane makes this drug unique regarding its toxicity, pharmacological and therapeutic use. Taking into consideration the potential of this substance, this paper aims to summarize the most relevant data regarding the mechanism of actions and its main clinical applications, in order to improve medical practice. Therefore, we presented the mechanism of action in order to understand its usage in different pathologies, such as dystonias, spasticity, nephrologic and urologic conditions, cosmetic use, depression, gastroenterologic and proctologic diseases, dermatologic conditions, pathologies specific to plastic surgery and also the role of BoNT therapy in pain management. It is well documented in the literature that important discoveries have been made through recent experimental and clinical studies. Even so, there is still much to learn about the therapeutic action of this drug in terms of molecular and pathophysiological mechanisms, in order to benefit of the whole healing potential of this amazing toxin.