Membrane proteins exhibiting extra- and intracellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered bilayer lipid membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by way of a metal-chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19) possessing a cysteine residue in the N-terminal extremity for grafting onto gold and a C-terminal extremity modified by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed us to bind liposomes of variable compositions containing a 2% molar ratio of a chelating lipid, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] so-called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein. The formation of pep-tBLMs was characterized by surface plasmon resonance imaging (SPRi), and their continuity, fluidity, and homogeneity were demonstrated by fluorescence recovery after photobleaching (FRAP), with a diffusion coefficient of 2.5 × 10 cm/s, and atomic force microscopy (AFM). By using variable lipid compositions including phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol 4,5-bisphosphate (PIP), sphingomyelin (SM), phosphatidic acid (PA), and cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteoliposomes expressing the functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). We succeeded in reinserting CXCR4 in pep-tBLMs formed on P19-4H by the fusion of tethered proteoliposomes. AFM and FRAP characterization allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous, and continuous. The value of the diffusion coefficient determined in the presence of reinserted CXCR4 was 2 × 10 cm/s. Ligand binding assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin II), revealed that CXCR4 can be reinserted in pep-tBLMs with functional folding and orientation. This new approach represents a method of choice for investigating membrane protein reincorporation and a promising way of creating a new generation of membrane biochips adapted for screening agonists or antagonists of transmembrane proteins.
The dynamics of polyribosome abundance were studied in gravistimulated maize (Zea mays) stem pulvini. During the initial 15 min of gravistimulation, the amount of large polyribosomes transiently decreased. The transient decrease in polyribosome levels was accompanied by a transient decrease in polyribosome-associated mRNA. After 30 min of gravistimulation, the levels of polyribosomes and the amount of polyribosome-associated mRNA gradually increased over 24 h up to 3-to 4-fold of the initial value. Within 15 min of gravistimulation, total levels of transcripts coding for calreticulin and calmodulin were elevated 5-fold in maize pulvinus total RNA. Transcripts coding for calreticulin and calmodulin were recruited into polyribosomes within 15 min of gravistimulation. Over 4 h of gravistimulation, a gradual increase in the association of calreticulin and calmodulin transcripts with polyribosomes was seen predominantly in the lower one-half of the maize pulvinus; the association of transcripts for vacuolar invertase with polyribosomes did not change over this period. Our results suggest that within 15 min of gravistimulation, the translation of the majority of transcripts associated with polyribosomes decreased, resembling a general stress response. Recruitment of calreticulin and calmodulin transcripts into polyribosomes occurred predominantly in the lower pulvinus one-half during the first 4 h when the presentation time for gravistimulation in the maize pulvinus is not yet complete.The vector of the gravitational force provides a constant cue for the direction of plant growth. Changes in the orientation of a plant relative to the gravity vector result in positive or negative gravitropic growth of roots and shoots, respectively. The gravity vector is thought to be perceived through changes in tensegrity or the pressure exerted by statoliths (Sack, 1991; Kaufman et al., 1995;Yoder et al., 2001) or by the entire protoplast (Staves, 1997). Sedimentation of statoliths in starch-containing cells can occur within seconds to minutes of gravistimulation (Sack, 1991; Kaufman et al., 1995;Yoder et al., 2001). A cascade of coordinated biochemical events subsequently amplifies and distributes the signal through a responsive tissue, resulting in the redistribution of auxin between upper and lower sides of the gravistimulated organ and initiating the bending response (for review, see Kaufman et al., 1995; Lomax et al., 1995;Sinclair and Trewavas, 1997;Chen et al., 1999;Rosen et al., 1999). Although the gravitropic response of plants has been the subject of intensive research, our understanding of the signaling processes linking perception of gravity to differential growth is still limited.The stem pulvini of cereal grasses have previously been used as model systems for the investigation of gravitropic growth in a number of studies (Dayanandan and Kaufman, 1984; Kaufman et al., 1987 Kaufman et al., , 1995Winter et al., 1997; Collings et al., 1998;Perera et al., 1999Perera et al., , 2001 Johannes et al., 2001). The pulvini are d...
Individual cell addressing on an arrayed biochip is achieved. Gold layers are electrochemically functionalized by arraying antibodies using silicon‐made microcantilevers (see image). Because the spot size is close to a lymphocyte diameter, living blood cells are successfully bound and organized on the surfaces according to the micrometric features.
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