Lipid species dependent vesicles clustering caused by alpha-synuclein as revealed by single-vesicle imaging with total internal reflection fluorescence microscopy

Single-molecule methods have been applied to study the mechanisms of many bio-physical systems that occur on the nanometer scale. To probe the dynamics of the such systems including vesicle docking, tethering, fusion, trafficking, protein-membrane interactions, etc., and to obtain reproducible experimental data; proper methodology and framework are crucial. Here, we address this need by developing a protocol for immobilization of vesicles composed of synthetic lipids and measurement using total internal reflection fluorescence (TIRF) microscopy. Furthermore, we demonstrate applications including vesicle clustering mediated by proteins such as alphaSynuclein (αSyn) and the influence of external ions by using TIRF microscopy. Moreover, we use this method to quantify the dependence of lipid composition and charge on vesicle clustering mediated by αSyn which is based on the methods previously reported.


Introduction
The series of events with synaptic vesicles in association with proteins are common in synaptic nerve terminals. For example synaptic vesicles (SV) transportion, fusion, and recycling processes facilitate the realese of neurotransmitters (Esposito et al. 2011). These overall process take place in many steps such as formation of cluster, docking, teathering, exocytosis and endocytosis cycle. This is however possible only after the mediation of cytosolic proteins (e.g alpha-Synuclein (αSyn)) (Murphy et al. 2000), membrane bound proteins e.g. soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) (Bonifacino and Glick 2004) and other accessory proteins and ions. Specifically, αSyn w/o SNARE or ions plays a critical role for regulation in clustering (Cai et al. 2020;Diao et al. 2013), docking (Lai et al. 2014), teathering (Cai et al. 2019), fusion , and endo/exocytosis (Schlaugher et al. 2017) by stabilizing the vesicles (Kaur and Lee 2021).
αSyn is a functional protein found predominantly in cytosol of neuron with primarily high abundance in presynaptic terminal (Stefanis 2012) and is associated with both physiology as well as pathology. Whereas physiological role involves with synaptic vesicles recycling by interaction with the lipid membrane (Lautenschlager et al. 2017); the pathological role is relevant in several synucleinopathies such as dementia. Remarkably, its aggregation into the Lewy bodies become the hallmark of Parkinson's disease (PD) (Spillantini et al. 1997). Indeed, the structural variation  or mutation (Polymerpous et al. 1997) or post-translational modifications (Wu et al. 2020;Bu et al. 2017) are causative for the functioning of αSyn from physiological function to the toxic condition. It is comprised of 140 amino acids (~14 kD) and known to be an intrinsically disordered protein. The structural basis shows that it can be divided into three main regions namely: N-terminus, non-amyloid-beta component (NAB) and C terminus contributing distinct structural and dynamical properties for the physiochemical regulation relevant to the content of amino acid sequences (Fusco et al. 2014). Of many functionalities of αSyn such as docking, tethering, fusion, endo/exo-cytosis are influenced by lipid species binding as mentioned earlier. Upon membrane binding αSyn adopts the structural transition (alpha helix) (Pfefferkorn et al. 2011). Studies have shown that αSyn has high tendency of binding only with the anionic lipids like phosphatidylserine (PS) (Middleton and Rhoades 2010), and preferential to the small and highly curved membrane . However, needs specific combination of polyunsaturated chain which provides the loose packing (Kubo et al. 2005). Other studies have shown that presence of zwitterionic phospholipid particularly phosphatidylethonalamine (PE) cause the elevated binding (Jo et al. 2000), whereas Jianjun Pan et al (Pan et al. 2018) shows the inhibitory effect of PE in membrane remodeling. So, the distincint role of specific lipid's head and chains for αSyn binding remain elusive.
Synaptic vesicles of size ~40 nm contain significant amount of phospholipids with 12 mole% PS, 23 mole% PE, and several other lipids (Cholesterol, Spingomyline, Phosphatidylinositol, hexylceramide, ceramide etc.) covering ~50% surface and is asymmetric in terms of lipid distribution (Takamuri et al. 2006). Every lipids in a synaptic vesicles have specific role in association with the αSyn binding (Shvadchok et al. 2011).
Biophysical studies of mimic synaptic vesicles  or membrane mediated by αSyn in vitro (for detail refer to review Candace M Pfefferkorn et al (Pfefferkorn et al. 2012)) have been studied by using different kind of model vesicles and bilayer using variable lipids (Pan et al. 2018). So having insight of how lipid species and their head or tail are affected by αSyn binding is important to understand the functionalities of synaptic vesicles recycling and pathogenicity caused by this protein.
Several techniques have been employed to study the membrane association of αSyn such as Nuclear magnetic resonance spectroscopy (NMR) (Eliezer et al. 2001), atomic force microscopy (AFM) and electron paramagnetic resonance spectroscopy (EPR) (Pan et al. 2018), computer simulation (West et al. 2016), neutron refloctmetry (NR) (Hellstrand et al. 2013), electron microscopy (EM) Madine et al. 2009) etc. Here we study the interaction of αSyn and membrane for lipid spicificity at the single-molecule level Ferreon et al. 2009;Gong et al. 2016) which overcomes the limitation of ensemble average (Deniz et al. 2007) by using total internal reflection fluorescence microscopy (TIRFM or TIRF) Hu et al. 2017;Du et al. 2021). TIRF is often known as evanescent wave or field microscopy providing a high contrast imaging of dynamics at or at the proximity of cellular membrane (Mattheyses et al. 2010). The physical basis of this technique is that it utilizes the evanescent field as an excitation field which decays exponentially with distance when the incoming bean passes through the medium with high refractive index (usually glass) to low refractive index (buffer or sample) at an angle above the critical angle (Fish 2009). This technique is often compared with epifluorescence microscopy. Unlike epifluorescence microscopy that fits for bulk imaging, it is capable to provide the information about the process that happen within ~100 nm (Steyer and Almers 2001). So, utilizing this technique for selective excitation using two solid state lasers (green (532 nm)-Nd:YAG and red (640 nm)-HeNe) has been particularly useful to study the vesicles docking as induced by protein (Cai et al. 2019). Moreover, it has been established as a powerful technique to probe several modern processes in cell biology (Mattheyses et al. 2010). For instance, peptide-lipid interaction (Fox et al. 2009), actin nucleation and elongation (Jiang and Huang 2017), vesicles clustering as mediated by αSyn and regulated by calcium ions (Cai et al. 2020), translocation of signaling molecule (Tengholm et al. 2003), and docking and priming of vesicles (Becherer et al. 2007) have been successfully studied using TIRF. Figure 1 shows the schematic of measurement by prism based TIRF.
Small unilamellar vesicles (SUV) provides the excellent platform to monitor the several biological phenomenon in vitro Aryal et al. 2020;Pan et al. 2017;Khadka et al. 2018;Khadka et al. 2021). To monitor and quantify the lipid species dependent clustering synaptic mimicking SUV and effectiveness of the technique; in this paper, we report a protocol of small unilamellar vesicles immobilization followed by interaction with free vesicles (Diao et al. 2009) as mediated by synaptic protein αSyn as measured by TIRF. Specifically, vesicles of size ~45 nm with DiD labeled vesicles (DiIC18(5); DiD-vesicles onward) are immobilized and vesicles with DiI labelled (DiIC18(3); DiI-vesicles onward) are floating. The floating vesicles depending upon the lipid species it contains in the presence or absence of divalent cations show the distinct quantitative clustering in the presence of αSyn. Clustering as mediated by αSyn and the influence of divalent cations (Cai et al. 2018) particularly the concentration dependent non-linear role of Ca 2+ has been already studied by Diao's group (Cai et al. 2020). By following the similar protocol, it may be relevant to study the influence of lipid species such as PS, PE in clustering of vesicles as mediated by αSyn and modulated by another divalent cation (Mg 2+ ). Understanding the influence of particular lipid species on the protein induced process in a single vesicles level will provide the kinetic and dynamic insight into the mechanism of action in molecular level in cell membranes (Man et al. 2021). The method described here is particularly designed for those who wish to perform the imaging and analysis of protein mediated action in singlemolecule level, but could be applied to study the other macromolecule-macromolecule interactions and endocytosis/exocytosis, budding, fusion/fission process etc. Similar assay has been already discussed to study the conformational dynamics of αSyn during the interaction with membrane (Ma et al. 2019) and SNARE (Sun et al. 2019) for its functional relevance. Table 1. Table 2. Table 3.

Research
Design-Based on the initial hypothesis that lipids species influence the αSyn binding (particularly PS, Lyso-PC and PE), while the role of Ca 2+ has been studied (Diao et al. 2009), Mg 2+ will change the ionic strength, suppressing the binding of αSyn at low concentration as a competitive binding. Acknowledging this idea, we experimentally validate the distinction of αSyn binding as caused by PE and PS, since both are presented in the synaptic vesicles. Furthermore, it has been reported that whereas phosphatidylcholine (PC) lipid plays little role in binding, PE has relatively higher; this study can be extended to understand the actual role of PE for αSyn interaction in a single molecule level. The experiment covers several tests for a given αSyn binding by identifying the optimized working condition satisfying the physiological relevance. Since experimental evidence shows the presence of anionic lipid enhance the binding of αSyn with vesicles, the project is being initialized with the extreme content of PS (Rhoades et al. 2006;Diao et al. 2009) for example using 30 mole% on both DiD and DiI-vesicles and or reproducing the earlier reported results from our lab on the effect of different concentrations of calcium ions. Below, we outline the methodology and preliminary results.

Surface Cleaning:
The pre-drilled quartz slides and the glass coverslips obtained from vendors must first be thoroughly cleaned. Here, we have described our standard cleaning procedure, but others have used slightly different protocol (Du et al. 2021;Lamichhane et al. 2010). Scrub and thoroughly rinse slides with ethanol (100%) and then with Milli-Q water. Since the presence of impurities on the surface increase background fluorescence, scrubbing is required for removing debris from previous experiments. Place slides and fresh coverslips in the clean and dry Coplin staining jars separately. Rinse with Milli-Q water. Sonicate with acetone for 20 min to remove any remaining organic material and non-specifically interacting debris. Again rinse with Milli-Q water three times. Fill jars with 1 M KOH. Sonicate for 20 min followed by rinsing with Milli-Q water three times. Fill jars with methanol and sonicate for 20 min. Burn one side of each quartz slide for at least two mins using propane torch, then rinse both slides and cover slips with fresh methanol, three times.

Silanization:
The next step is silanization which is achieved by aminosilanization with amino silane ((3-(2-aminoethylamino) propyl) trimethoxysilane (APTS). In aminosilanization reaction, methanol is used as a solvent and acetic acid as a catalyst (Chandradoss et al. 2014). Mix 100 mL methanol, 5 mL acetic acid and 1 mL APTS reagent in a clean, dry beaker. Pour into the jars and make sure it fully covers slides and coverslips. Incubate for 10 min. Sonicate for 1 min and again incubate for 10 min. Rinse slides and coverslips with methanol, and then at least three times with Milli-Q water. Completely dry the slides and coverslips with blowing air. Note that, amino silane solution should be freshly prepared.

PEGylation:
Polyethylene glycol (PEG) is commonly used for surface passivation to prevent non-specific interactions with protein, lipids (Ha and Joo 2002; or vesicles ). This is achieved by adding 100 μL of freshly prepared reaction solution (120 mg of mPEG, 4 mg of biotin-PEG, and 700 μL of 0.1 M sodium bicarbonate solution) onto the imaging surface of the slide. Each slide was covered by one clean coverslip to be a set, which was incubated overnight and then rinsed with Milli-Q water, dried, used immediately or stored at 20 °C for further use.

Microfluidic Chamber Assembly:
Prepare 4-8 mm wide and ~150 μm deep flow channels. For this place a quartz slide on a flat surface with the PEGylated side facing up. Make a channel on the PEGylated surface by putting double-sided tape over the quartz slide in such a way that the holes are positioned at the center of the channel. Place a coverslip facing the PEGylated surface at the top of taped quartz slide. Remove extra tape by using a razor blade. Seal the chamber by pressing the coverslip over the area where double-sided tapes are placed using properly mixed Epoxy (5 min) and wait at least 20 min for epoxy to set.

Vesicles Preparation:
SUV were prepared by using extrusion method discussed elsewhere (Aryal et al. 2020). Briefly; Mix the lipids in appropriate ratio (Table 3) from stock kept at −20 °C in a glass tube. For each consecutive lipid pipetation, syring should be cleaned at least 5 times. Vacuum the mixture for 4 hours to remove the organic solvents (chloroform/ethanol). Rehydrate the obtained dry film with HEPES (25 mM HEPES, 100 mM NaCl, pH 7.4 (with NaOH)) buffer. Vertex and sonicate the suspension until the film is fully dissolved. Repeat 7 cycles of freezing and thawing by submerging into the water at 50 °C and introducing into the dry ice (−79 °C) alternatively to obtain the polydispersed unilamellar vesicles solution. Finally, obtain the monodispersed vesicles (size ~50 nm) by extrusing the solution (21 times) through 50 nm polycarbonate membrane. Use the obtained vesicles immediately or keep at −80 °C for future use. Note that; chloroform is carcinogenic agent, lipid mixing is advised to be conducted under fume hood. Vesicles preparation steps are summarized in Figure 2.

Vesicles Immobilization and Slide Preparation:
For the measurement by using TIRF, inject 40 μL (2 shots of 20 μL) of 0.1 mg/mL NeutrAvidin (Thermo Scientific, 0.1 mg/mL in 10 mM Tris-HCL, pH 7.5, 50 mM NaCl) per channel and incubate for 15 min. This will create a binding surface for biotin labeled samples (DiD-vesicles). Inject the 100 μL of DiD-vesicles with appropriate dilution and incubate for 30 min. This is basis framework for many types of experiments, but for us we are interested in vesicle clustering and the interaction with αSyn. Inject 50 μL of αSyn (appropriate concentration) and incubate for 30 min. Inject 100 μL DiI-vesicles with appropriate dilution and incubate for 30 min. The channel without αSyn is considered as control channel. Note, in each consecutive steps, HEPES buffer exchange is required at least 5 times (200 μL each time) to remove unbound vesicles/protein. Thus, prepared slides were imaged using TIRF under identical intensity.
DiD-vesicles which contain Biotin-PE moiety bounds to the NeutrAdivin and hence get immobilized. Since optimized number of DiD-vesicles provides the homogenous distribution, it can be visualized using the red laser under TIRF. After the confirmation of DiD-vesicles coverage, protein was injected for affected channel whereas only buffer was injected in control channel. Control and affected channel should be prepared within a slide leaving the consecutive channel without any sample to save it if there is any tape to tape leakage, and for better batch to batch comparison. Replication was also done within same slide for the comparison. Since αSyn as definite binding ability, the optimal DiI-vesicle concentration depends upon protein. The imaging sequence is tabulated in Table 4.
After the experiment, the slides can be reused. For recycling, soak the slide in acetone overnight to soften any residue of epoxy or tape from the previous experiment. Use a razor blade to scrape off residue. Rinse the slides several times with Milli-Q water and dry them.

Optimization of Working Condition:
There are many variables to consider to obtain the consistent and reproducible results and optimization relies on fixing many of the variables to be as uniform as possible. Beyond the laser alignment one must optimize surface density of bound substrate vesicles (DiD-vesicles) e.g., full coverage, homogenous and isotropic distribution in channel, number (ratio) of DiD and DiI-vesicles for proper concentration of αSyn (pM to μM) binding (Diao et al. 2012), choice of background (high or low), time of incubation (e.g. few minute to many hours), temperature (about 0 °C or room temperature (RT) or 37 °C), DiD or DiI-vesicles as substrate, mole% of DiD and DiI fluorescent in a given composition and any surface imperfections on the quartz surface of oil-prism interface which results the formation of diffraction patterns in the images which in turn make them useless. It is to be noted that, the vesicles count differs from channel to channel, sample to sample and batch to batch, so direct comparison of the count may not be possible.

Buffer Preparation:
PEG buffer: This is prepared freshly for immediate use. It contains 0.1 M sodium bicarbonate (pH 8.5) HEPES buffer: This can be used for up to 1 month if stored at 4 °C. It contains 25 mM HEPES and100 mM NaCl (pH 7.4 with NaOH) T50 buffer: This can be stored at 4 °C to be used for a month. It contains 10 mM Tris-HCl and 50 mM NaCl (pH 8.0 with NaOH)

TIRF Imaging-
The sample is mounted in the following sequence for the imaging: DI water drop at the top of objective, sample coverslip down in contact with DI water, immersion oil on the surface of quartz slide, and finally the prism at the top of oil. The prism is held in place with frame and screw. The photograph of TIRF system used for this experiment is as shown in Figure 3.
For reproducibility, at least 10 different randomly selected sites were scanned for each sample flow channel and at least 3 channel a day were scanned. The experiment was conducted at least three different days with freshly prepared vesicles.

Image
Processing and Quantification-Fluorescence signal or images as well as real time recording can be taken and analyzed using separate software, but we do both using the custom-built software obtained from Dr. Taekjip Ha's group (John's Hopkins University, USA). Statistical results and plots are typically obtained in external program, such as Excel, MATLAB, Python, Mathematica etc. Protein induced changes are analyzed by using Student t-test. If p < 0.05, the change is considered as statistically significant. (Table 4) was implemented in the imaging process: 2.2.6. Imaging Troubleshoot-Here are some issues experienced and solutions associated with the imaging by TIRF and samples during the experiments (Table 5). For more details, please refer to our previous publication (Diao et al. 2012). Figure 4 shows the representative images of clustering of vesicles as an effect of αSyn or ion binding under the excitation of green laser. We quantify the observation by measuring the number of DiI-vesicles as a result of αSyn binding as a function of lipid head charge and ion concentration. As the measurement is done with various kind of samples in the optimization process with different αSyn concentrations, we present the representative data here. Figure  5A shows the change in clustering count of DiI-vesicles on the top of DiD-vesicles (both contains 12 mol% PS) as the effect of αSyn. Where-as an enhanced count can be observed for same set of vesicles with the calcium ion as compared to both 'without αSyn' and 'with αSyn' as shown in Figure 5B. Experiments were also performed in high background. Figure   5C and D show the significant increased count as a result of αSyn binding in 30 mol% PS containing DiI-vesicles and the 12 mol% PS DiI-vesicles respectively, where, 30 mol% PS containing DiD-vesicle was substrate. Furthermore, No PS DiI-vesicles (on 30 mol% PS DiD-vesicle substrate) shows less enhanced count even in the presence of αSyn (Fig. 5E). However, NO PS DiI-vesicles on 12 mol% PS DiD-vesicles shows the pretty much similar count as background (Fig. 5F). With this preliminary results, it can be said that; αSyn shows the strong binding affinity with the lipids having charged head specifically PS, and highly enhanced in the presence of higher concentration of calcium ion is in good agreement with the previous finding (Cai et al. 2020).

Summary
Surface passivation and immobilization strategy and measurement by TRIF has been widely accepted technique to study the several biophysical interactions in a single molecule level (Joo and Ha 2012a;Lamichhane et al. 2010). This technique and protocol can be generalized in different ways including to study the interacting behaviour of protein and lipid in real time and to probe the complex system of proteins and lipids relevant to physiological condition by using multi-color fluorophore/excitation. Furthermore, TIRF coupled with other highresolution techniques and single level spectroscopies to probe molecular mechanisms and dynamics in the qualitative as well as quantitative manner. Here, we have described the detailed protocol in quantification of the role of lipid's charge or ion on clustering behavior caused by αSyn by using TIRF aiming to extend our study in αSyn's binding with several other lipids that are physiologically relevant for synaptic vesicles. Since this assay has been already proven to be useful in elucidating the conformational dynamics of protein in protein-membrane interaction and RNA/DNA-protein interaction in conjugation with single-molecule FRET (Forster Resonance Energy Transfer), it can be applied to study other protein-lipids, protein-protein, and protein-ion interactions. One immediate and potential application of this protocol could be to distinguish between the forces such as charges versus hydrophobicity and imperfection that govern the protein interaction with lipids species. Furthermore, this protocol could be extended in the in vitro biophysical studies of protein reconstituted system of vesicles (e.g. segment of SNARE) for their relevancy of docking, fusion etc with the membrane. Photograph of the different parts of the TIRF system in the lab. Representative images of DiI-vesicle clustering before and after protein and or ion addition under the excitation of green laser. Statistic results on DiI-vesicle clustering count induced by αSyn and or ion (mean ± SEM) for: A) 12% PS containing DiI-vesicles on 12% PS containing DiD-vesicles (2 μM αSyn; p<0.001); B) 12% PS DiI-vesicles with 10 mM Ca 2+ ion on 12% PS DiD-vesicles (5 μM αSyn; p<0.001 with respect to both controls i.e. 'without αSyn' and 'with αSyn'); C) 30% PS DiI-vesicles on 30% PS DiD-vesicles (20 nM αSyn; p<0.001); D) 12% PS DiI-vesicles on 30% PS DiD-vesicles (20 nM αSyn; p<0.001); E) No PS DiI-vesicles on 30% PS DiD-vesicles (20 nM αSyn; p<0.001); F) No PS DiI-vesicles on 12% PS DiD-vesicles (2 μM αSyn; difference is statistically insignificant). All the incubation steps and imaging were conducted at room temperature. The significance was calculated by Student's t-test.

Action Reason
Focus Initial focusing is preferred with green laser because of low photobleaching effect.
DiD-vesicles with red laser To confirm the background/substrate vesicles if they are distributed homogenously.
DiD-vesicles with green laser To check if there are vesicles in background that can be probed by green (ideally zero).
DiI-vesicles w/o protein or ions with green laser To examine the clustering/tethering of DiI-vesicles on top of DiD-vesicles driven or not by protein or ions.
Whole sample set with red laser To identify if DiD-vesicles are still there as a substrate; confirmatory test to assure the DiI-vesicles tethering is on the top of DiD-vesicles (but not due to in-specific binding)

Issue Reason Solution
Parabolic shaped photobleaching covering at reasonable field of view with high intensity Improper focus with high intensity, high concentration of vesicles Play with focus knob gently. There are photobleaching in either direction, but there is short window where focus can be made, and lower the intensity, if not fixed change the vesicles concentration.
Bleeding of vesicles in irregular pattern throughout the region Tunneling of vesicles inside the tape; causing the unusual maximum intensity while playing with X-Y knob At this point the actual beam might be hitting off-center the objective which mislead with pattern or number in the screen. Reposition the beam is required.
False center Sample can be seen around the actual center as if it is focused but goes away while moving the position Identify the proper center by playing with X-Y knob which gives the high intensity number.
False focus Sample can be visualized even if the beam is at the other edge of aperture showing the fuzzy focus Realign the beam by taking the slide out in a such a way that initial beam (without slide and prism) will be at the inner side of microscope's aperture.
Unstable imaging Unlikely to focus the sample properly Need to wash a sample properly with HEPES.
Bad sample Whiteness in tape caused by channel to channel sample leakage Whiteness at the edge of tape may be ok, but if the tape separating the flow channel turn out to be completely white, it is likely caused by the tape to tape leakage of NeutrAvidin or sample itself. Skip conjugate channel to load the sample or prepare a new slide.