Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy

The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.

α-Synuclein kinetically regulates the nascent fusion pore dynamics

α-Synuclein (α-synFL) is central to the pathogenesis of Parkinson’s disease (PD), in which its nonfunctional oligomers accumulate and result in abnormal neurotransmission. The normal physiological function of this intrinsically disordered protein is still unclear. Although several previous studies demonstrated α-synFL’s role in various membrane fusion steps, they produced conflicting outcomes regarding vesicular secretion. Here, we assess α-synFL’s role in directly regulating individual exocytotic release events. We studied the micromillisecond dynamics of single recombinant fusion pores, the crucial kinetic intermediate of membrane fusion that tightly regulates the vesicular secretion in different cell types. α-SynFL accessed v-SNARE within the trans-SNARE complex to form an inhibitory complex. This activity was dependent on negatively charged phospholipids and resulted in decreased open probability of individual pores. The number of trans-SNARE complexes influenced α-synFL’s inhibitory action. Regulatory factors that arrest SNARE complexes in different assembly states differentially modulate α-synFL’s ability to alter fusion pore dynamics. α-SynFL regulates pore properties in the presence of Munc13-1 and Munc18, which stimulate α-SNAP/NSF–resistant SNARE complex formation. In the presence of synaptotagmin1(syt1), α-synFL contributes with apo-syt1 to act as a membrane fusion clamp, whereas Ca2+•syt1 triggered α-synFL–resistant SNARE complex formation that rendered α-synFL inactive in modulating pore properties. This study reveals a key role of α-synFL in controlling vesicular secretion.

Fusion pore expansion and contraction during catecholamine release from endocrine cells

ABSTRACTAmperometry recording reveals the exocytosis of catecholamine from individual vesicles as a sequential process, typically beginning slowly with a pre-spike foot, accelerating sharply to initiate a spike, reaching a peak, and then decaying. This complex sequence reflects the interplay between diffusion, flux through a fusion pore, and possibly dissociation from a vesicle’s densecore. In an effort to evaluate the impacts of these factors, a model was developed that combines diffusion with flux through a static pore. This model recapitulated the rapid phases of a spike, but generated relations between spike shape parameters that differed from experimental results. To explore the possibility of fusion pore dynamics, a transformation of amperometry current was introduced that yields fusion pore permeability divided by vesicle volume (g/V). Applying this transform to individual fusion events yielded a highly characteristic time course. g/V initially tracks the pre-spike foot and the start of the spike, increasing ∼15-fold to the peak. However, after the spike peaks, g/V unexpectedly declines and settles to a constant value that indicates the presence of a stable post-spike pore. g/V of the post-spike pore varies greatly between events, and has an average that is ∼3.5-fold below the peak value and ∼4.5-fold above the pre-spike value. The post-spike pore persists and g/V remains flat for tens of milliseconds, as long as catecholamine flux can be detected. Applying the g/V transform to rare events with two peaks revealed a stepwise increase in g/V during the second peak. The g/V transform offers an interpretation of amperometric current in terms of fusion pore dynamics and provides a new framework for analyzing the actions of proteins that alter spike shape. The stable post-spike pore conforms with predictions from lipid bilayer elasticity, and offers an explanation for previous reports of prolonged hormone retention within fusing vesicles.STATEMENT OF SIGNIFICANCEAmperometry recordings of catecholamine release from single vesicles reveal a complex waveform with distinct phases. The role of the fusion pore in this waveform is poorly understood. A model based on a static fusion pore fails to recapitulate important aspects of the waveform. A new transform of amperometric current introduced here renders fusion pore permeability in real time. This transform reveals rich dynamic behavior of the fusion pore as catecholamine leaves a vesicle. This analysis shows that fusion pore permeability rapidly increases and then decreases before settling into a stable post-spike configuration.