Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behavior — it has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid–lipid and protein–lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein–lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.     

Low Copy Numbers of DC‐SIGN in Cell Membrane Microdomains: Implications for Structure and Function

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC‐SIGN, a C‐type lectin, in membrane microdomains. DC‐SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC‐SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1 µm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3 T3 cells contains only 4–8 molecules of DC‐SIGN, consistent with our preliminary super‐resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (～50 nm) pathogen, dengue virus, leading to infection of host cells.      

Recent advances in understanding the molecular mechanism of chloroplast photorelocation movement

Plants are photosynthetic organisms that have evolved unique systems to adapt fluctuating environmental light conditions. In addition to well-known movement responses such as phototropism, stomatal opening, and nastic leaf movements, chloroplast photorelocation movement is one of the essential cellular responses to optimize photosynthetic ability and avoid photodamage. For these adaptations, chloroplasts accumulate at the areas of cells illuminated with low light (called accumulation response), while they scatter from the area illuminated with strong light (called avoidance response). Plant-specific photoreceptors (phototropin, phytochrome, and/or neochrome) mediate these dynamic directional movements in response to incident light position and intensity. Several factors involved in the mechanisms underlying the processes from light perception to actin-based movements have also been identified through molecular genetic approach. This review aims to discuss recent findings in the field relating to how chloroplasts move at molecular levels. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Pattern of rise in subplasma membrane Ca concentration determines type of fusing insulin granules in pancreatic β cells

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca²⁺ concentration ([Ca²⁺]_PM) with the Ca²⁺ indicator Fura Red in rat β cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca²⁺]_PM caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca²⁺]_PM induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca²⁺]_PM rise directly determines the types of fusing granules.     

The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: A physico-chemical study with lecithin phospholipids

Nonsteroidal anti-inflammatory drugs (NSAIDs) represent non-specific inhibitors of the cycloxygenase pathway of inflammation, and therefore an understanding of the interaction process of the drugs with membrane phospholipids is of high relevance. We have studied the interaction of the NSAIDs with phospholipid membranes made from dimyristoylphosphatidylcholine (DMPC) by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). FTIR data obtained via attenuated total reflectance (ATR) show that the interaction between DMPC and NSAIDs is limited to a strong interaction of the drugs with the phosphate region of the lipid head group. The FTIR transmission data furthermore are indicative of a strong effect of the drugs on the hydrocarbon chains inducing a reduction of the chain-chain interactions, i.e., a fluidization effect. Parallel to this, from the DSC data beside the decrease of T_m a reduction of the peak height of the melting endotherm connected with its broadening is observed, but leaving the overall phase transition enthalpy constant. Additionally, phase separation is observed, inducing the formation of a NSAID-rich and a NSAID-poor phase. This is especially pronounced for Diclofenac. Despite the strong influence of the drugs on the acyl chain moiety, FRET data do not reveal any evidence for drug incorporation into the lipid matrix, and ITC measurements performed do not exhibit any heat production due to drug binding. This implies that the interaction process is governed by only entropic reactions at the lipid/water interface.     

Amyloidogenic Propensities and Conformational Properties of ProIAPP and IAPP in the Presence of Lipid Bilayer Membranes

Human islet amyloid polypeptide (hIAPP), which is considered the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in β-cells of the pancreas from its precursor pro-islet amyloid polypeptide (proIAPP), which may be important in early intracellular amyloid formation as well. We compare the amyloidogenic propensities and conformational properties of proIAPP and hIAPP in the presence of negatively charged lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. Circular dichroism studies verify the initial secondary structures of proIAPP and hIAPP to be predominantly unordered with small amounts of ordered secondary structure elements, and exhibit minor differences between these two peptides only. Using attenuated total reflection-Fourier transform infrared spectroscopy and thioflavin T fluorescence spectroscopy, as well as atomic force microscopy, we show that in the presence of negatively charged membranes, proIAPP exhibits a much higher amyloidogenic propensity than in bulk solvent. Compared to hIAPP, it is still much less amyloidogenic, however. Although differences in the secondary structures of the aggregated species of hIAPP and proIAPP at the lipid interface are small, they are reflected in morphological changes. Unlike hIAPP, proIAPP forms essentially oligomeric-like structures at the lipid interface. Besides the interaction with anionic membranes [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) + x1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]], interaction with zwitterionic homogeneous (DOPC) and heterogeneous (1,2-dipalmitoyl-sn-glycero-3-phosphocholine:DOPC:cholesterol 1:2:1 model raft mixture) membranes has also been studied. Both peptides do not aggregate significantly at DOPC bilayers. In the presence of the model raft membrane, hIAPP aggregates markedly as well. Conversely, proIAPP clusters into less ordered structures and to a minor extent at raft membranes only. The addition of proIAPP to hIAPP retards the hIAPP fibrillation process also in the presence of negatively charged lipid bilayers. In excess proIAPP, increased aggregation levels are finally observed, however, which could be attributed to seed-induced cofibrillation of proIAPP.     

A Rudimentary labellar speculum in Cymbidium lowianum (Rchb.f.) Rchb.f. and Cymbidium devonianum Paxton (Orchidaceae)

BACKGROUND AND AIMS: The labellar 'hairs' of some Cymbidium spp. are said to be thin-walled and to contain 'plasma', oil and sugars and it has long been speculated that they may function as food-hairs. However, the present authors' preliminary studies showed that certain atypical papillae may have a different role and, by reflecting light, function as a speculum. The purpose of the paper is to test this hypothesis. METHODS: Light microscopy, scanning electron microscopy, transmission electron microscopy, histochemistry and ultraviolet photography were used to investigate the structure, food content and light-reflecting properties of these papillae. KEY RESULTS AND CONCLUSIONS: The labellum of Cymbidium lowianum (Rchb.f.) Rchb.f. is densely clothed with obconical to conical papillae with wide bases and pointed tips. However, on either side of the median axis of the lip occur silvery patches comprising papillae with truncated tips and it is thought that these reflect light and thereby attract insect pollinators. Similar patches are also found in Cymbidium devonianum Paxton, and in both species, they are set against a reddish background, which, since bees cannot perceive this colour, probably appears dark to the insect thus enhancing the visual impact of the light-reflecting patches. In Cymbidium tigrinum Parish ex Hook. and Cymbidium mastersii Griff. ex Lindl., however, the labellum is mainly white and no light-reflecting patches were observed. Instead, unlike C. lowianum and C. devonianum, these species are highly fragrant and the attraction of insects probably depends to a greater extent on olfactory cues. In C. lowianum both types of papillae contain protein, starch and lipid bodies but only protein is seemingly present at elevated concentrations. However, lipoidal material also occurs upon the surface of the labellum and it is possible that this may be gathered by insects as reported for C. iridifolium A. Cunn (syn. C. madidum Lindl.). The labellar papillae of C. lowianum, thus, have the potential to function as food-hairs, although direct evidence for this is lacking.     

偏振光激发的单分子荧光纵向成像研究

本文从单分子的水平上,详细分析并模拟了不同偏振光下的单个荧光分子的成像,指出荧光成像强度的差别是由分子的纵向位置及跃迁偶极矩的取向共同决定的。给出了确定荧光分子偶极矩取向的方法,并在此基础上给出了重构分子间纵向间隔的公式。     

同时应用近场光学显微镜的透射和反射模式研究单个细胞的光学性质

通常认为．在近场光学显微技术的光收集模式中,观察透光性好的样品时采用透射模式．研究不透明样品时采用反射模式。本文同时采用透射和反射两种模式观察透明性较好的PCI2细胞和淋巴细胞样品．初步研究单个细胞的反射、吸收、透射和荧光等光学性质,以促进组织光学和激光生物医学等领域的进一步发展。细胞光学的时代就要到来。     

A Transmembrane Polar Interaction Is Involved in the Functional Regulation of Integrin αLβ2

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of α and β subunits. Each subunit contains a single α-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of αβ TM packing. The leukocyte integrin αLβ2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of αLβ2 TMs is consistent with that of the integrin αIIbβ3 TMs. However, molecular dynamics simulations of αLβ2 TMs in lipids predicted a polar interaction involving the side chains of αL Ser1071 and β2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled αLβ2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of αLβ2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of β2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated αLβ2, αMβ2, and αXβ2 in 293T transfectants. We also show that the expression of mutant β2 Thr686Gly in β2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1α treatment as compared to wild-type β2-expressing cells. These two TM polar residues are totally conserved in other members of the β2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar interactions within the low dielectric environment of the membrane interior and demonstrate its importance in the regulation of αLβ2 function.