The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < approximately 1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to approximately 1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (>/=+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections.
Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.
Lipid and protein lateral mobility is essential for biological function. Our theoretical understanding of this mobility can be traced to the seminal work of Saffman and Delbrück, who predicted a logarithmic dependence of the protein diffusion coefficient (i) on the inverse of the size of the protein and (ii) on the "membrane size" for membranes of finite size [Saffman P, Delbrück M (1975) Proc Natl Acad Sci USA 72:3111-3113]. Although the experimental proof of the first prediction is a matter of debate, the second has not previously been thought to be experimentally accessible. Here, we construct just such a geometrically confined membrane by forming lipid bilayer nanotubes of controlled radii connected to giant liposomes. We followed the diffusion of individual molecules in the tubular membrane using single particle tracking of quantum dots coupled to lipids or voltage-gated potassium channels KvAP, while changing the membrane tube radius from approximately 250 to 10 nm. We found that both lipid and protein diffusion was slower in tubular membranes with smaller radii. The protein diffusion coefficient decreased as much as 5-fold compared to diffusion on the effectively flat membrane of the giant liposomes. Both lipid and protein diffusion data are consistent with the predictions of a hydrodynamic theory that extends the work of Saffman and Delbrück to cylindrical geometries. This study therefore provides strong experimental support for the ubiquitous Saffman-Delbrück theory and elucidates the role of membrane geometry and size in regulating lateral diffusion.
An atomic force microscope (AFM) and a tunable infrared (IR) laser source have been combined in a single instrument (AFM-IR) capable of producing~200-nm spatial resolution IR spectra and absorption images. This new capability enables IR spectroscopic characterization of human stratum corneum at unprecendented levels. Samples of normal and delipidized stratum corneum were embedded, cross-sectioned and mounted on ZnSe prisms. A pulsed tunable IR laser source produces thermomechanical expansion upon absorption, which is detected through excitation of contact resonance modes in the AFM cantilever. In addition to reducing the total lipid content, the delipidization process damages the stratum corneum morphological structure. The delipidized stratum corneum shows substantially less long-chain CH 2 -stretching IR absorption band intensity than normal skin. AFM-IR images that compare absorbances at 2930/cm (lipid) and 3290/cm (keratin) suggest that regions of higher lipid concentration are located at the perimeter of corneocytes in the normal stratum corneum.
This article is available online at http://www.jlr.org complex and specifi c architecture of the inter-corneocyte lipid matrix ( 2 ) composed of a mixture of ceramides, cholesterol, and long-chain fatty acids. Ultrastructural studies, using electron microscopy and diffraction techniques, have revealed that these lipids are organized in stacked bilayers that are predominantly parallel to the skin surface ( 3-5 ). Part of the lipids is covalently bound to the corneocytes whereas another part represents the "free lipids" removable by solvent extraction.X-ray diffraction is an excellent technique for studying the spatial organization of the intercellular matrix because the lipid molecules are highly ordered and oriented in a crystalline structure. This technique has provided a number of important results with regard to the SC structure. In particular, small-angle X-ray scattering (SAXS) gives information on the inter-bilayer distance in the multi-lamellar lipid structures, whereas the wide-angle X-ray scattering (WAXS) data contain information about the in-plane crystalline arrangement of lipids (lattice type).Most of the SAXS studies of human SC fi nd refl ections corresponding to ف 4.5 nm and ف 6.5 nm inter-bilayer spacings ( 4, 6, 7 ). Some studies have also detected a refl ection corresponding to a spacing of 11-13.5 nm ( 6, 8 ), referred to as the "long periodicity phase" ( 6 ). These data indicate that the SC lipid matrix has a complex organization into multiple sublayers or even phase-separated domains with different lamellar repeat distances.Similar complexity is found at the molecular level using WAXS, where two sets of peaks are found, corresponding to orthorhombic (peaks at 0.41 and 0.37 nm) and hexagonal (peak at 0.41 nm) packing of the lipid chains. In addition, a fraction of lipids might exist in a fl uid or amorphous state, which would then contribute to the broad band corresponding to an average distance of 0.46 nm. Abstract Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the fi rst study where micro-focus X-ray scattering was used for assessing fi ne structural variations of the human skin barrier with micrometer resolution. We found that the scattering profi les were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profi les did not change as a function of depth in the SC, confi rming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that micro...
Single Particle Tracking (SPT) is a powerful technique for the analysis of the lateral diffusion of the lipid and protein components of biological membranes. In neurons, SPT allows the study of the real-time dynamics of receptors for neurotransmitters that diffuse continuously in and out synapses. In the simplest case where the membrane is flat and is parallel to the focal plane of the microscope the analysis of diffusion from SPT data is relatively straightforward. However, in most biological samples the membranes are curved, which complicates analysis and may lead to erroneous conclusions as for the mode of lateral diffusion. Here we considered the case of lateral diffusion in tubular membranes, such as axons, dendrites or the neck of dendritic spines. Monte Carlo simulations allowed us to evaluate the error in diffusion coefficient (D) calculation if the curvature is not taken into account. The underestimation is determined by the diameter of the tubular surface, the frequency of image acquisition and the degree of mobility itself. We found that projected trajectories give estimates that are 25 to 50% lower than the real D in case of 2D-SPT over the tubular surface. The use of 3D-SPT improved the measurements if the frequency of image acquisition was fast enough in relation to the mobility of the molecules and the diameter of the tube. Nevertheless, the calculation of D from the components of displacements in the axis of the tubular structure gave accurate estimate of D, free of geometrical artefacts. We show the application of this approach to analyze the diffusion of a lipid on model tubular membranes and of a membrane-bound GFP on neurites from cultured rat hippocampal neurons.
The lag-burst behavior in the action of phospholipase A(2) (PLA(2)) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T(m) of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA(2), evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA(2), involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form "mature" fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.
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