Journal: bioRxiv

Article Title: A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

doi: 10.1101/2025.04.17.649308

Figure Lengend Snippet: a , Structure of 14:0-6:0 NBD-PC. b , Scheme of BSA cuvette assay. Fatty acid-free BSA is added to a suspension of vesicles in a cuvette. Short-chain NBD-labeled lipids in the outer leaflet of the vesicles partition into the aqueous phase, where they bind to BSA. As the fluorescence quantum yield of the NBD fluorophore is ∼2-fold lower when NBD-PC is bound to BSA versus located in the membrane, a drop in fluorescence is observed. In vesicles with a functioning scramblase, inner-leaflet lipids translocate outward and are captured by BSA, resulting in a greater drop in fluorescence compared with protein-free liposomes. This assay enables real-time measurement of lipid translocation and extraction kinetics. c , Time-course analysis of cuvette BSA assay on symmetrically 14:0-6:0 NBD-PC labeled, protein-free liposomes, expressed as normalized fluorescence intensity relative to the initial value, following the addition of excess BSA. The desorption process is monitored by measuring the decrease in fluorescence over time, which reflects the transfer of NBD-PC from the liposomal membrane to BSA in solution. d , Kinetic analysis of NBD-PC desorption from protein-free liposomes. Fluorescence decay traces were fitted to a monoexponential decay function (for example, dashed line in panel c), yielding a rate constant of k = 0.07 s⁻¹, corresponding to the desorption kinetics of the 14:0-6:0 NBD-PC probe ( n = 11). e , Fluorescence intensity as a function of NBD-PC concentration in an aqueous environment, used to determine its solubility threshold (0.75 µM) as the point at which monomeric NBD-PC self-associates, resulting in a change in fluorescence quantum efficiency. This point was determined as the intersection of two lines corresponding to linear fits of the two portions of the graph. f , Symmetrically labeled liposomes were immobilized and just after initial TIRF microscopy image was taken (t=0 min), buffer flow was started. For each vesicle, lipid fluorescence signals were normalized to their respective initial intensities, Representative traces show the time-dependent loss of 14:0-6:0 NBD-PC fluorescence from individual symmetrically labeled vesicles. g , The fluorescence decay traces from (f) were fitted to a monoexponential decay function. The desorption rate constant ( γ ) was determined for each individual vesicle (dots), yielding an average desorption rate of 0.0124 ± 0.0045 s⁻¹ (mean ± S.D.).

Article Snippet: Image acquisition was performed using an inverted TIRF microscope (IX83, Olympus, Hamburg, Germany) with a 100x oil-immersion objective (1.5 NA; Olympus), an Orca-flash 4.0 sCMOS camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), and a quad band emission filter cube (DAPI/FITC/CY3/CY5).

Techniques: Suspension, Labeling, Fluorescence, Membrane, Liposomes, Translocation Assay, Extraction, Concentration Assay, Solubility, Microscopy

Journal: bioRxiv

Article Title: A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

doi: 10.1101/2025.04.17.649308

Figure Lengend Snippet: a , Illustration of the sample chamber, with tubing for flushing, depicted in the context of a TIRF microscope objective lens. b , Illustration of a single vesicle, containing an Alexa647-labeled VDAC1 dimer and a trace quantity of fluorescent lipid (14:0-6:0 NBD-PC), immobilized on a polymer-passivated (PLL-g-PEG/PLL-g-PEG-biotin) glass coverslip via biotin-neutravidin tethering. c , Representative TIRF images of immobilized vesicles visualized in the lipid channel (NBD) and protein channel (Alexa647). The lipid signal serves as a read-out of scramblase activity, while the protein signal estimates the number of proteins per vesicle. d , Theoretical fluorescence traces for individual immobilized liposomes with and without scramblase activity according to the protocol described in panel e. e , Schematic presentation of the assay steps. After vesicle immobilization, continuous buffer flow (wavy line) in the imaging chamber removes 14:0-6:0 NBD-lipids that readily partition from the vesicle outer leaflet into the buffer (phase I). At this juncture (turning point) vesicles contain 14:0-6:0 NBD-lipids only in the inner leaflet. Next, in phase II of the sequence, these remaining 14:0-6:0 NBD-lipids are flipped to the outer leaflet in scramblase-containing vesicles where they become accessible to buffer extraction. Note that these phases partially overlap. Vesicles are depicted to illustrate the outcome for liposomes with a scramblase (right half) or without a scramblase (left half).

Article Snippet: Image acquisition was performed using an inverted TIRF microscope (IX83, Olympus, Hamburg, Germany) with a 100x oil-immersion objective (1.5 NA; Olympus), an Orca-flash 4.0 sCMOS camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), and a quad band emission filter cube (DAPI/FITC/CY3/CY5).

Techniques: Microscopy, Labeling, Polymer, Activity Assay, Fluorescence, Liposomes, Imaging, Sequencing, Extraction

Journal: bioRxiv

Article Title: A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

doi: 10.1101/2025.04.17.649308

Figure Lengend Snippet: Verification of the single vesicle scramblase assay platform. a-c , Liposomes labeled symmetrically or asymmetrically with the indicated NBD-lipids were immobilized as in . TIRF microscopy imaging was started simultaneously with buffer flow (t=0 min). For each vesicle, lipid fluorescence intensity was normalized to the corresponding initial intensity, and individual traces were combined into a plot showing the mean ± SD (the number of averaged individual liposome traces is indicated at the top right of the graphs). a , Symmetrically labeled liposomes with head-labeled N -NBD-PE in both leaflets. b , Asymmetrically labeled liposomes with 14:0-6:0 NBD-PC in the outer leaflet. c , Symmetrically labeled liposomes with 14:0-6:0 NBD-PC in both leaflets. d , 14:0-6:0 NBD-PC-containing proteoliposomes reconstituted with non-crosslinked Alexa647-labeled VDAC1 were imaged by TIRF microscopy alongside protein-free liposomes to determine reconstitution efficiency. After vesicle immobilization, buffer flow was used to remove outer-leaflet NBD lipids. This was followed by sodium dithionite (SDT) addition to selectively bleach inner-leaflet NBD lipids in VDAC1-containing vesicles. Alamethicin (ALA) was then added to permeabilize all vesicles, confirming complete bleaching. Exemplary fluorescence traces are shown for a vesicle containing supposedly one protein (orange) and a protein-free liposome (yellow). The control without SDT accounts for photobleaching effects (grey). e , NBD fluorescence intensity of individual vesicles reconstituted with the indicated amounts non-crosslinked VDAC1 after dithionite addition, with the initial protein signal indicated by a colormap (scale on the right; the protein colormap corresponds to the initial Alexa647 fluorescence of the vesicles as this fluorophore is also bleached on SDT treatment). The control sample indicated in the first column was not subjected to SDT treatment. f , Comparison of vesicle protein occupancy determined by dithionite single vesicle assay (black), single vesicle protein signal (red) and bulk dithionite assay in cuvette (grey). The x-axis represents the amount of protein used per reconstitution.

Article Snippet: Image acquisition was performed using an inverted TIRF microscope (IX83, Olympus, Hamburg, Germany) with a 100x oil-immersion objective (1.5 NA; Olympus), an Orca-flash 4.0 sCMOS camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), and a quad band emission filter cube (DAPI/FITC/CY3/CY5).

Techniques: Liposomes, Labeling, Microscopy, Imaging, Fluorescence, Control, Comparison, Single-vesicle Assay

Journal: bioRxiv

Article Title: A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

doi: 10.1101/2025.04.17.649308

Figure Lengend Snippet: a , Size-exclusion chromatography elution profile of EGS-crosslinked Alexa647-labeled VDAC1 (top panel) and an Alexa647 fluorescence image of an SDS-PAGE gel of collected fractions (bottom panel). Fraction 8, highly enriched in VDAC1 dimers (pCL, purified dimer) was used for reconstitution into NBD-PC liposomes to yield pCL proteoliposomes. b, Bulk cuvette-based sodium dithionite (SDT) and c , BSA assays comparing proteoliposomes with pCL VDAC1 dimers with mock-reconstituted liposomes. d , Dynamic light scattering intensity distribution, revealing an average vesicle diameter of 203 nm (solid line). The lipid fluorescence intensity distribution histogram of the sample obtained via TIRF microscopy, normalized by DLS measurements, with vesicles categorized as protein-containing (red) or empty (grey). e , Correlation analysis of protein signal intensity versus lipid signal intensity, assessing the dependence of protein occupancy on vesicle diameter. f , Values of the scrambling rate constant (alpha, see ), derived from single-vesicle traces fitted to the three-compartment kinetic model, plotted against initial protein fluorescence signal of the vesicles. Data points are color-coded by vesicle diameter, based on lipid signal normalized via DLS.

Article Snippet: Image acquisition was performed using an inverted TIRF microscope (IX83, Olympus, Hamburg, Germany) with a 100x oil-immersion objective (1.5 NA; Olympus), an Orca-flash 4.0 sCMOS camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), and a quad band emission filter cube (DAPI/FITC/CY3/CY5).

Techniques: Size-exclusion Chromatography, Labeling, Fluorescence, SDS Page, Purification, Liposomes, Microscopy, Derivative Assay

Journal: bioRxiv

Article Title: A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

doi: 10.1101/2025.04.17.649308

Figure Lengend Snippet: 14:0-6:0 NBD-PC proteoliposomes containing either non-crosslinked (NCL) or purified crosslinked (pCL) Alexa647-labeled VDAC1 dimers, as well as protein-free NBD-PC liposomes were immobilized and subjected to TIRF microscopy as illustrated in . Imaging was started just before buffer flow (t=0 min). Panels show time-resolved comparison of lipid signal for ( a,d ) protein-free liposomes, ( b,e ) NCL-VDAC1 proteoliposomes, and ( c, f ) pCL-VDAC1 dimer proteoliposomes. For each individual liposome, lipid fluorescence intensity is shown as a normalized line plot relative to the initial fluorescence. Protein occupancy in each liposome is color-coded based on Alexa647 signal intensity, as indicated by the colormap on the right. d - f , Fluorescence traces from vesicles with similar protein occupancy were grouped and plotted in increasing order of protein contents, with the protein signal and intensity grouping indicated by the colormap on the right. g , Schematic representation of the three-compartment model used for data fitting, illustrating the inner leaflet (L in ), outer leaflet (L out ) and extracted pool (L ex ), along with the respective rate constants. h , Representative NBD-fluorescence intensity traces (solid lines) fitted using the three-compartment model (dashed lines) for three single vesicles exhibiting two-step Alexa647 photobleaching events (panels on the right), indicative of the presence of a single pCL-VDAC1 dimer in the vesicles. i , Scrambling rate for active vesicles with a single pCL-VDAC1 dimer. Symbols are color-coded to indicate number of lipids per vesicle according to the color map in panel j. j , Number of transported lipids per second for individual vesicles with a single pCL-VDAC1 dimer, color-coded to indicate number of lipids per vesicle according to the color map on the right with histogram illustrating the distribution of values. i - j The colormap represents the total number of lipids for each vesicle, deduced from initial NBD fluorescence intensity (see ).

Article Snippet: Image acquisition was performed using an inverted TIRF microscope (IX83, Olympus, Hamburg, Germany) with a 100x oil-immersion objective (1.5 NA; Olympus), an Orca-flash 4.0 sCMOS camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), and a quad band emission filter cube (DAPI/FITC/CY3/CY5).

Techniques: Purification, Labeling, Liposomes, Microscopy, Imaging, Comparison, Fluorescence

Journal: bioRxiv

Article Title: A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

doi: 10.1101/2025.04.17.649308

Figure Lengend Snippet: 14:0-6:0 NBD-PC proteoliposomes containing pCL Alexa647-labeled VDAC1 were immobilized and subjected to photobleaching step analysis using TIRF microscopy. a , Initial protein fluorescence signal of each vesicle is plotted against the total number of detected fluorophores per vesicle, determined by discrete photobleaching steps. This analysis provides insight into the fluorophore occupancy within individual proteoliposomes. Note that the fitting success is reduced with increasing fluorophore number. b , Final photobleaching step intensity, categorized by fluorophore count per vesicle, represents single-fluorophore emission, ranging from ∼250–550 A.U.

Article Snippet: Image acquisition was performed using an inverted TIRF microscope (IX83, Olympus, Hamburg, Germany) with a 100x oil-immersion objective (1.5 NA; Olympus), an Orca-flash 4.0 sCMOS camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), and a quad band emission filter cube (DAPI/FITC/CY3/CY5).

Techniques: Labeling, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

doi: 10.1101/2025.04.17.649308

Figure Lengend Snippet: 14:0-6:0 NBD-PC proteoliposomes containing pCL Alexa647-labeled VDAC1 were immobilized and subjected to TIRF microscopy. Imaging was started just before buffer flow (t=0 min). For each individual liposome, lipid fluorescence intensity is shown as a normalized line plot relative to the initial fluorescence. The scramblase kinetic parameter α was determined using a three-compartment model (fit shown as dashed line) and is displayed in the lower-left corner. The Inset shows the corresponding photobleaching step analysis for the protein fluorophores.

Article Snippet: Image acquisition was performed using an inverted TIRF microscope (IX83, Olympus, Hamburg, Germany) with a 100x oil-immersion objective (1.5 NA; Olympus), an Orca-flash 4.0 sCMOS camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), and a quad band emission filter cube (DAPI/FITC/CY3/CY5).

Techniques: Labeling, Microscopy, Imaging, Fluorescence