Characterization of GFP-MAL expression and incorporation in rafts

During myelin formation, membrane-associated proteins have to be sorted and transported in specified membrane regions such as compact and non-compact myelin membranes. One protein that may be involved in such a process is the Myelin and Lymphocyte protein MAL (VIP17/ MVP17). MAL was identified as a novel myelin membrane component expressed by oligodendrocytes and Schwann cells. Since MAL has been shown to be important in the apical sorting machinery of polarized cells, we have started to investigate the possible functional role of MAL in sorting myelin membrane-associated molecules. In this study, we have generated cDNA constructs with green fluorescent protein (GFP) either at the N- or C-terminus of MAL. Transfection experiments showed that GFP-MAL expression resembles that of normal MAL, whereas the MAL-GFP fusion construct was not properly transported within the cell. Furthermore, we could demonstrate that GFP-MAL is enriched in detergent insoluble glycolipid-enriched microdomains as already seen for untagged MAL. As a prerequisite for the generation of transgenic mice expressing GFP-MAL under the control of its own regulatory elements, we have generated a cDNA construct with an 8-kb MAL promotor fragment fused to GFP-MAL. Transfection experiments of the Oli-neu oligodendrocyte cell line showed that GFP-MAL was expressed, but only in cells, which were stimulated for differentiation with cAMP. In summary, the results confirm that the fusion protein GFP-MAL is incorporated into detergent-insoluble complexes and the 8-kb MAL promotor fragment is sufficient to be activated in oligodendrocytes.     

Neural Membrane Signaling Platforms

Throughout much of the history of biology, the cell membrane was functionally defined as a semi-permeable barrier separating aqueous compartments, and an anchoring site for proteins. Little attention was devoted to its possible regulatory role in intracellular molecular processes and neuron electrical signaling. This article reviews the history of membrane studies and the current state of the art. Emphasis is placed on natural and artificial membrane studies of electric field effects on molecular organization, especially as these may relate to impulse propagation in neurons. Implications of these studies for new designs in artificial intelligence are briefly examined.     

Optimized time‐gated generalized polarization imaging of Laurdan and di‐4‐ANEPPDHQ for membrane order image contrast enhancement

The membrane dyes Laurdan and di‐4‐ANEPPDHQ can be used to image membrane order due to a spectral blue‐shift in the fluorescence emission between the liquid‐ordered and liquid‐disordered phases. These images typically take the form of a normalized intensity ratio image known as a generalized polarization (GP) plot. Here, we exploit the known excited state photophysics and time‐resolved data acquisition via time‐correlated single‐photon counting (TCSPC) to demonstrate GP contrast enhancement for these two probes of 7 and 31%, respectively. This improvement in image contrast enhancement will be invaluable when studying the role of lipid rafts in fixed and live cell systems. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

Mechanical properties of Al2O3 and Al2O3/Al with Gyroid structure obtained by stereolithographic additive manufacturing and melt infiltration

To obtain both plasticity and toughness of the material at the same time, various manufacturing techniques of ceramic-metal composites and structures have been studied. In this work, a bio-inspired Al₂O₃ ceramic scaffold with Gyroid structure was designed and prepared by stereolithographic (SL) additive manufacturing, then the Al₂O₃/Al ceramic-metal hybrid structure was prepared by infiltrating molten Al into the Al₂O₃ ceramic structure. The performances of the Al₂O₃ ceramic scaffold and the Al₂O₃/Al ceramic-metal hybrid structure were compared and analyzed by a quasi-static compression experiment. The quasi-static compressive strength of the pristine Al₂O₃ scaffold was 14.36 MPa, while that of the Al₂O₃/Al ceramic-metal hybrid structure was up to 89.06 MPa. Moreover, the plasticity of the Al₂O₃/Al ceramic-metal hybrid structure was much higher than that of the Al₂O₃ scaffold. During compression, the Al₂O₃/Al ceramic-metal hybrid structure had excellent energy absorption, reaching up to 2569.16 KJ/m³, 15 times that of the Al₂O₃ scaffold. Therefore, this method can obtain materials with excellent ductility and toughness.     

Lipid Droplets and Their Autophagic Turnover via the Raft-Like Vacuolar Microdomains

Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs’ basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways.     

梳型丙烯酰胺共聚物在水溶液中的缔合结构

采用荧光探针研究了丙烯酰胺(AM)/4-乙烯苄基辛烷基酚聚氧乙烯(10)醚(VBPOE)/2-丙烯酰胺基-2-甲基丙磺酸钠(NaAMPS)梳型共聚物(PAVA)在水溶液中的疏水缔合微结构。结果发现,在纯水和盐水溶液中,随聚合物浓度的增大,疏水微区的数量和非极性明显增加,但当PAVA浓度高于0.20 g/dL时,疏水微区的非极性趋于恒定,而且0.5 g/dLNaCl的加入对I1/I3值(I1/I3的变化可以表征疏水微区是否形成以及监测疏水缔合微区的变化)的影响极小,这证实了分子链在盐水中的构象仍然较伸展,使得PAVA在盐水中也能形成连续的缔合结构,从而赋予聚合物良好的抗盐和增粘性能。随着盐浓度的增加,缔合微结构的非极性变化不大,但其紧密度和数量增加,Ie/Im值持续增大。     

Tuned phase behavior of weakly interacting polystyrene-block-poly(n-pentyl methacrylate) by selective solvent

We investigated, via small angle X-ray scattering, depolarized light scattering, rheometry, and transmission electron microscopy, the phase behavior of the mixture of a symmetric polystyrene-block-poly(n-pentyl methacrylate) copolymer (PS-b-PnPMA) showing the closed-loop phase behavior and excellent baroplasticity, and dodecanol, a PnPMA-selective solvent. We found that the addition of a selective solvent is simple, but very effective to obtain various microdomains including hexagonally packed cylinders and gyroids. Also, with increasing temperature, the mixtures showed multiple ordered-to-ordered transitions (OOTs) in addition to upper ordered-to-disordered transition (UODT). The first observation of gyroid microdomains in PS-b-PnPMA is very important, although they have been widely reported in many block copolymers, for instance, PS-block-polyisoprene copolymer (PS-b-PI) and PS-block-poly(d,l-lactide) copolymer (PS-b-PLA). Since the gyroid microdomains of PS-b-PnPMA show excellent baroplasticity, external pressure instead of temperature could easily change the microdomains.     

Analysis of membrane microdomain-associated proteins in the insular cortex of post-mortem human brain

Membrane microdomains (MM) are membrane rafts within the cell membrane enriched in cholesterol and glycosphingolipids that have been implicated in the trafficking and sorting of membrane proteins, secretory and endocytotic pathways, and signal transduction. To date, MM have not been characterised in the human brain. We reason that by identifying MM in the normal human cortex, we may better understand the molecular mechanisms of human brain dysfunction. To characterize the protein composition of MM in the human brain, we have carried out a comprehensive proteomic analysis of detergent resistant membranes (DRMs) associated proteins derived from human postmortem insular cortex using 1-DE separation prior to LC coupled to MS/MS or GeLC-MS/MS. Eighty five proteins were identified including 57 unique to human brain cortex DRMs (by comparison with DRM proteins reported in other cell types). High levels of signal transduction, cell adhesion, cell transport and cell trafficking proteins were identified including synaptic proteins such as synapsin II and synaptic vesicle membrane protein, mitochondrial proteins such as ATPase subunits and metabolic enzymes such as malate dehydrogenase. This data will facilitate our understanding of protein expression changes within membranes in candidate brain regions in human brain diseases such as schizophrenia, bipolar disorder and other psychiatric and neurodegenerative disorders.     

Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm)” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal.     

改造细胞质膜促进途径酶组装及N-乙酰氨基葡萄糖合成

功能膜微域（FMMs）可以作为内源性空间支架进行途径酶组装,适度地提高FMMs在质膜中的占比能够明显提高产物合成量,然而对FMMs进行改造后,细胞生长和产物的合成均受到抑制。在课题组前期研究的基础上,对细胞质膜进行理性改造以缓解FMMs过度改造对细胞造成的影响。首先通过过表达PlsX,PlsY和PlsC改造细胞质膜,之后,以枯草芽孢杆菌合成N-乙酰氨基葡萄糖（GlcNAc）为例,发现质膜改造的菌株中GlcNAc产量达到（5.18±0.16） g/L,与对照菌株相比产量提升了41.9%,同时,细胞质膜改造也明显降低FMMs过度修饰对菌株生长产生的不利影响。