Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40–56 nm, upon increasing cholesterol content from 0 to 20%

The peridynamic model.

<p>(a) A small, randomly selected region in the distal membrane is represented as a collection of particles (small circles). Each particle represents a collection of lipid molecules, and is located at the center of a circular neighborhood (N_x). The motion of an arbitrary particle x (in yellow) at the center of N_x is influenced by the motion of every particle in N_x via bonds. If no forces apply to the membrane, the particles in N_x are considered to be in an undeformed state. The close-up shows vector ξ, representing the distance between bonded particles x and x’, where T is the force vector state that existed prior to the bond being broken. (b) As tension increases, the particles move apart from each other and the corresponding bonds stretch. At some critical value of stretch, the distance between the center particle (yellow dot) and some number of neighboring particles becomes too large, leading to broken bonds and disconnected particles (x', white dots). This corresponds to the rupture (pore) formation among membrane lipids.</p

Transition in rupture morphology with increasing shear moduli.

<p>(a-l) The peridynamic simulations of the lipid membrane which is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.g003" target="_blank">Fig 3</a> (j-l), but with gradually increased shear modulus. (a-d) The ruptures become rugged where G = 1 MPa. (e-h) The straight edges of the ruptures become more pronounced where G = 2.5 MPa and branches start to appear. (i-l) The ruptures appear as elongated finely branched structures where G = 7.5 MPa. These structures typically evolve into fractals(l). The color bar in (d) applies to all simulations shown in Fig 4, and is identical to the one in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.g003" target="_blank">Fig 3</a>. The number of pinning points in all simulations in this figure is 16, and the positions of the pinning sites are identical to the ones in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.g003" target="_blank">Fig 3J</a> and p (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.s004" target="_blank">S2 Movie</a>). The ratio of the diameter of the expanded membrane to the initial diameter (D/D₀) is shown below each snapshot of the simulations.</p

Floral and fractal biomembrane ruptures and corresponding peridynamic model simulations.

<p>(a-c) Confocal micrographs of a floral rupture occurring in the distal bilayer of a DLBM. Yellow arrow heads indicate threads of lipids between two layers, which are the pinned regions. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.s003" target="_blank">S1 Movie</a>) (d-f) Peridynamic simulations showing floral ruptures (G = 0 MPa). The ruptures nucleate at the pre-determined locations of pinned (fixed) particles, and then merge into one large floral pore. The pinning points (n = 6) are marked with red circles in (d). Black arrow heads show threads of points remain between two layers which correspond to the pinned regions, similar to (c). (g-i) Confocal micrographs showing small circular pores opening and progressing in the distal bilayer. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.s004" target="_blank">S2 Movie</a>) (j-l) Peridynamic simulations showing circular pores opening over time. Shear modulus G is 0 MPa as in (a-f), with the number of pinning points increased (n = 17). (m-o) Confocal micrographs of fractal ruptures occurring in the distal bilayer. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.s005" target="_blank">S3 Movie</a>) (p-r) Peridynamic simulations showing fractal ruptures (G = 5 MPa). The number and location of pinning points are the same as in (g-l). The color bar in f applies to all simulations in Fig 3 and shows the amount of material point damage (%) where 100% damage corresponds to a complete breaking of all bonds associated with the material point. The scale bar in (e) applies to all simulations in Fig 3. The ratio of the diameter of the expanded membrane to the initial diameter (D/D₀) is shown below each snapshot of the simulations.</p

The experiment.

<p>(a) Confocal laser scanning micrograph of a spreading double lipid bilayer membrane(DLBM), top view. (b) Schematic drawing of the DLBM in (a), side view. DLBM consists of a distal (upper, red color) bilayer and the proximal (lower, blue color) bilayer. The spreading edge of the double bilayer performs a 'tank-tread' motion. (c) Micrograph of a ruptured membrane. (d) Schematic drawing showing a rupture in the distal membrane. Upon rupturing, the lipid material migrates towards the edges onto the substrate.</p

Fractal dimension analysis of ruptures in actual membranes and simulations.

<p>(a-c) Binary images showing the contour of the fractal ruptures in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.g003" target="_blank">Fig 3O and 3R</a> (G = 5) and 4l (G = 7.5). (c) Plots showing the fractal dimension (D) of rupture patterns in (a),(b) and (c). The slope of the red line shows the fractal dimension of the pattern in panel a, D = 1.63, the pattern in panel (b), D = 1.70, and the pattern in c, D = 1.56. The circular rim forming around the expanding membrane in the simulations has been removed manually with image processing software. All fractal dimensions have been calculated by using the reticular cell counting (box counting) method. The plots show the relation between the number of occupied boxes (y-axes) and the box size. The fractals in biological membranes (not shown) feature D values around 1.7[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165947#pone.0165947.ref001" target="_blank">1</a>]). The analysis from the simulations show that both slope and D are similar to the experimental values.</p

A Heating-Superfusion Platform Technology for the Investigation of Protein Function in Single Cells

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s

Nanopatterning of Mobile Lipid Monolayers on Electron-Beam-Sculpted Teflon AF Surfaces

Direct electron-beam lithography is used to fabricate nanostructured Teflon AF surfaces, which are utilized to pattern surface-supported monolayer phospholipid films with 50 nm lateral feature size. In comparison with unexposed Teflon AF coatings, e-beam-irradiated areas show reduced surface tension and surface potential. For phospholipid monolayer spreading experiments, these areas can be designed to function as barriers that enclose unexposed areas of nanometer dimensions and confine the lipid film within. We show that the effectiveness of the barrier is defined by pattern geometry and radiation dose. This surface preparation technique represents an efficient, yet simple, nanopatterning strategy supporting studies of lipid monolayer behavior in ultraconfined spaces. The generated structures are useful for imaging studies of biomimetic membranes and other specialized surface applications requiring spatially controlled formation of self-assembled, molecularly thin films on optically transparent patterned polymer surfaces with very low autofluorescence

Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy

We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger

Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy

We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger