Floral to green: mating switches moth olfactory coding and preference

Mating induces profound physiological changes in a wide range of insects, leading to behavioural adjustments to match the internal state of the animal. Here, we show for the first time, to our knowledge, that a noctuid moth switches its olfactory response from food to egg-laying cues following mating. Unmated females of the cotton leafworm (Spodoptera littoralis) are strongly attracted to lilac flowers (Syringa vulgaris). After mating, attraction to floral odour is abolished and the females fly instead to green-leaf odour of the larval host plant cotton, Gossypium hirsutum. This behavioural switch is owing to a marked change in the olfactory representation of floral and green odours in the primary olfactory centre, the antennal lobe (AL). Calcium imaging, using authentic and synthetic odours, shows that the ensemble of AL glomeruli dedicated to either lilac or cotton odour is selectively up- and downregulated in response to mating. A clear-cut behavioural modulation as a function of mating is a useful substrate for studies of the neural mechanisms underlying behavioural decisions. Modulation of odour-driven behaviour through concerted regulation of odour maps contributes to our understanding of state-dependent choice and host shifts in insect herbivores

Charge-Dependent Translocation of the Trojan Peptide Penetratin across Lipid Membranes

We studied the interaction of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X(PG), by means of isothermal titration calorimetry. The work was aimed at getting a better understanding of factors that affect the peptide binding to lipid membranes and its permeation through the bilayer. The binding was well described by a surface partitioning equilibrium using an effective charge of the peptide of z(P) ≈ 5.1 ± 0.5. The peptide first binds to the outer surface of the vesicles, the effective binding capacity of which increases with X(PG). At X(PG) ≈ 0.5 and a molar ratio of bound peptide-to-lipid of ∼1/20 the membranes become permeable and penetratin binds also to the inner monolayer after internalization. The results were rationalized in terms of an “electroporation-like” mechanism, according to which the asymmetrical distribution of the peptide between the outer and inner surfaces of the charged bilayer causes a transmembrane electrical field, which alters the lateral and the curvature stress acting within the membrane. At a threshold value these effects induce internalization of penetratin presumably via inversely curved transient structures

Pfg NMR studies of lateral diffusion in oriented lipid bilayers

Abstract. The pfg-NMR diffusion technique is proposed to have an appreciable potential for future biophysical investigations in the field of membrane biology. Topics like transport of molecules both across and in the plane of the membrane can be successfully studied, as well as the formation of lipid domains and their intrinsic dynamics can be scrutinized. This short review will introduce the fundamental aspects of orientation dependent NMR interactions and the technique of macroscopically oriented bilayers for eliminating the unwanted effects of those interactions. The pfg-NMR technique will be briefly introduced and finally, some recent results illustrating the potential of the method are presented. Orientation dependent NMR interactions The NMR spectrum for a general spin system is determined by the spin Hamiltonian, H, which consists of a number of interaction terms, of which the following four terms are of interest: where H Z is the Zeeman term and the next three terms represent the static interactions. The static interactions have a common scaling term, (1/2)(3 cos 2 θ − 1), which is the second Legendre polynomial, P 2 (θ), where θ is an angle that relates the principle coordinate system of the specific interaction to the main magnetic field (B 0 ) -H CSA -The chemical shift anisotropy that represents the effect of induced magnetic fields due to orbital electronic motions, i.e. the chemical shift. Will change the chemical shift of the spectrum with a factor proportional to P 2 (θ). -H Q -The quadrupole interaction between the nuclear quadrupole moment and the surrounding electric field gradient. Will scale the quadrupolar coupling constant observed for nuclei with spin > 1/2 according to P 2 (θ). -H D -The dipolar interaction that represents the magnetic interaction between dipoles. Due to simultaneous coupling to several different dipoles this term will give rise to a linebroadening, which is proportional to P 2 (θ). For cos θ = 1/ √ 3, i.e. θ = 54.7 • , the scaling term (1/2)(3 cos 2 θ − 1) becomes zero and the static interactions "magically" disappear. This condition is generally hard to obtain since θ is distributed randomly in a non-oriented sample so one has to use special techniques in order to remove the static interactions. In the commonly used solid-state NMR technique, where magic angle spinning (MAS) is utilized, the sample is transferred into a rotor that can be spun at very high spinning rates. spinning of the sample causes all the static interactions to be projected onto the spinning axis of the rotor, which is then turned to the magic angle (54.7 • ) with respect to B 0 , thereby removing all the static interactions in the sample Preparation of macroscopically aligned lipid bilayers A number of methods has been employed to obtain macroscopically oriented lipid bilayers. The methods can roughly be divided according to weather a single bilayer or a stack of several bilayers is required. In the former case Langmuir-Blodgett films In our laboratory good results have been obtained by the following procedure. Lipids dissolved in a 1 : 4 mixture of methanol : 1-propanol are deposited onto thoroughly cleaned, but otherwise untreated, glass plates to a concentration of about 5-15 µg/mm 2 . The solvent is evaporated and the plates are placed into high vacuum for at least 4 hours to remove traces of solvent. The choice of solvent mixture gives a good adhesion to the glass surface and results in thin films covering the glass plates. The choice of solvent can be critical The sample tube is placed for several days in a humid atmosphere above the gel to L α phase transition temperature. During this time hydrated and oriented bilayers are formed. Hydration by humid atmosphere is generally preferred over the use of liquid water since addition of liquid water disrupts the bilayers and results in the formation of vesicular structures. This occurs especially when more than one glass plate is needed for obtaining a sufficient signal/noise ratio. If the glass plates are stacked before hydration, addition of liquid water will disrupt the bilayers as water is sucked in between the plates by capillary forces. Attempts to stack prehydrated plates often results in mechanical disruption of the lipid bilayers. G. Orädd and G. Lindblom / Pfg NMR studies of lateral diffusion in oriented lipid bilayers 193 • C. The spectrum consists of signals from all deuterons of the perdeuterated lipid and, due to the macroscopic orientation, is a sum of pairs of peaks with a frequency separation, ∆νq, governed by the averaged quadrupole coupling constant, ∆ν 0 q , and the orientation in which ∆ν 0 q is determined by the so-called order parameter of the C-D bond • of sample rotation/turn of the goniometer. The top right figure shows the spectrum at the magic angle (θ LD = 54.7 • ), where all the splittings have coalesced into the isotropic spectrum in which the two main peaks come from the choline headgroup and the acyl chain deuterons, respectively. This difference in isotropic chemical shift gives rise to the slight asymmetry in the innermost splittings seen in the left figure. 194 G. Orädd and G. Lindblom / Pfg NMR studies of lateral diffusion in oriented lipid bilayers Finally, after obtaining the desired water content (checked by weighing), the tube is sealed and the sample is left another day or two for final equilibration. This procedure results in that when the sample is watched between crossed polarizers along the bilayer normal, large dark areas are observed, since the sample is optically isotropic along the bilayer normal The pfg-NMR method for measuring lipid translational diffusion The NMR methods with pulsed magnetic field gradients provide some of the most attractive techniques for studies of molecular transport and the applicability of the NMR diffusion techniques has been growing fast due to many improvements of the NMR equipments for diffusion and microscopy The use of NMR for diffusion measurements rests on the ability to create transverse magnetization with a presession rate that is dependent on the local magnetic field. The details of this method are beyond the scope of this article and the interested reader is referred to previously published reviews Spins moving in an inhomogenous magnetic field will experience a varying precession rate and the refocussing of the magnetization will in general not be completed after the application of a refocussing pulse. This effect is enhanced by the application of magnetic field gradients during the dephasing/rephasing periods and the resulting attenuation of the echo amplitude [30] is utilized to obtain the self-diffusion coefficient, D, of the molecules. In this equation the summation goes over all diffusion components. A 0i is the echo amplitudes without applied gradients, γ is the gyromagnetic ratio, ∆ is the time interval between gradient pulses and δ and g are the duration and amplitude of the pulsed field gradients, respectively. The initial echo amplitude A 0i is determined by the longitudinal and transverse NMR relaxation times. Usually the second half of the spin echo is collected and Fourier transformed to obtain the spectrum in the frequency domain. In this case the amplitude at each frequency channel is governed by Eq. (2). In order to separate the bandshapes corresponding to differently diffusing species the use of the CORE method for global analysis of the entire data set has proven useful In the NMR experiment the translational diffusion is measured in the direction of the magnetic field gradient, which normally is directed parallel with B 0 . For a lipid membrane the observed diffusion co- For a bilayer oriented at the magic angle sin 2 θ LD = 2/3 and, since it is reasonable to assume that D ⊥ is orders of magnitude smaller than D L so that the second term in Eq. Our lab is equipped with a 10 mm 1 H probe for a 100 MHz NMR spectrometer with a maximum gradient strength of 3 T/m (routinely used for lipid diffusion measurements in the order of 1-10 µm 2 /s) and with a 5 mm dual 1 H/X probe for a 400 MHz system that is capable of giving gradient strengths up to 10 T/m (for slow diffusion of the order of 0.01-0.1 µm 2 /s and diffusion of isotopically enriched molecules, e.g. with 2 H, 31 P, 19 F and 13 C Applications Domains and rafts in lipid bilayers During the past decade the organization of lateral structures in biological membranes and their importance for biological activity have received increasing attention as the importance of domain formation and percolation phenomena to cellular processes have been recognized Since the translational motion of lipids will be affected by the presence of microdomains in the lipid bilayer, it is possible to use the pgf-NMR diffusion method to investigate such domains With this technique we have successfully investigated several potentially raft-forming systems in order to get an understanding of the physico-chemical mechanisms in the raft-forming process. Recently, we have extended the method to isotopically labelled molecules, which allows us to measure the diffusion of each individual lipid species in the raft mixture Lipid diffusion can also be analyzed in the context of solid, inpermeable obstacles, such as integral proteins and gel patches. In such systems the diffusion coefficient will be dependent on the total fraction of obstacles as well as the size and shape of the obstacles Anisotropic diffusion in oriented systems In the study of lipid diffusion the motion parallel to the bilayer normal is orders of magnitudes smaller than that within the bilayer plane. This is not the case for molecules that are partially dissolvable in both water and oil, since this feature allows the molecules to pass through both the water layers and the oily hydrocarbon core of the stacked bilayers. In this case the diffusion along the normal will be slowed down as compared to that along the layers and this anisotropy in the diffusion can be measured with pfg-NMR. For this application we use the ability of the imaging equipment on the 400 MHz spectrometer to produce pulsed field gradients in any direction, thereby allowing for diffusion measurements at any angle to the bilayer normal. With this method we have investigated water diffusion anisotropy in bilayers and found that the water diffuses at least two orders of magnitude slower along the normal than perpendicular to it Acknowledgemen

The Effect of Cholesterol on the Lateral Diffusion of Phospholipids in Oriented Bilayers

Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to ∼40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (l(o)) and the liquid disordered (l(d)) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the l(d) phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (E(A)) in the range of 28–40 kJ/mol. The l(o) phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The E(A) for this phase was significantly larger (55–65 kJ/mol) than for the l(d) phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the l(d) and l(o) regions in the bilayer on the timescale of the NMR experiment (100 ms). Thus, strong evidence has been obtained that fluid domains (with size of μm or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained E(A):s indicate that these systems are in the l(d) phase at all CHOL contents for temperatures above 25°C

Lipid Lateral Diffusion in Ordered and Disordered Phases in Raft Mixtures

Lipid lateral diffusion coefficients in the quarternary system of dioleoylphosphatidylcholine (DOPC), sphingomyelin, cholesterol, and water were determined by the pulsed field gradient NMR technique on macroscopically aligned bilayers. The molar ratio between dioleoylphosphatidylcholine and sphingomyelin was set to 1:1, the cholesterol content was varied between 0 and 45 mol %, the water content was 40 wt %, and the temperature was varied between 293 and 333 K. The diffusion coefficients were separated into fast and slow spectral components by using the CORE method for global analysis of correlated spectral data. A large two-phase region, tentatively assigned to the liquid disordered (l(d)) and the liquid ordered (l(o)) phases, was present in the phase diagram. The l(d) phase was enriched in dioleoylphosphatidylcholine and exhibited diffusion coefficients that were about three to five times larger than for the l(o) phase. Both the diffusion coefficients and the apparent activation energies for the quarternary systems were compatible with earlier reports on ternary phospholipid/cholesterol/water systems. However, in contrast to the latter ternary systems, the exchange of lipids between the l(o) and the l(d) phases was slow on the timescale for the diffusion experiment for the quarternary ones. This means that on the millisecond timescale fluid, ordered domains are floating around in a sea of faster diffusing lipids, assigned to consist of mainly dioleoylphosphatidylcholine

Effect of sterol structure on the bending rigidity of lipid membranes: A 2H NMR transverse relaxation study

AbstractThe effect of incorporation of 3–43 mol% sterol on the lipid order and bilayer rigidity has been investigated for model membranes of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. 2H NMR spectra and spin-lattice relaxation rates were measured for macroscopically aligned bilayers. The characteristics of spectra obtained at temperatures between 0–60 °C are interpreted in terms of a two-phase coexistence of the liquid disordered and the liquid ordered phases and the data is found to be in agreement with the phase diagram published by Vist and Davis (Biochemistry 29 (1990), pp. 451–464). The bending modulus of the bilayers was calculated from plots of relaxation rate vs. the square of the order parameter at 44 °C. Clear differences were obtained in the efficiency of the sterols to increase the stiffness of the bilayers. These differences are correlated to the ability of the sterols to induce the liquid ordered phase in binary as well as in ternary systems; the only exception being ergosterol, which was found to be unable to induce lo phases and also had a relatively weak effect on the bilayer stiffness in contrast to earlier reports