Implications of lipid microdomains for membrane curvature, budding and fission.

Recent studies have highlighted the importance of monolayer and bilayer curvature for the budding and fission of biological membranes. Other lines of research, addressing the structure of planar biological membranes, have revealed the existence of cholesterol-based membrane microdomains. Here, we comment on the significance of microdomains for curved membranes, with special emphasis on budding and fission.     

Association of GM3 with Zap-70 induced by T cell activation in plasma membrane microdomains: GM3 as a marker of microdomains in human lymphocytes.

Recent evidence demonstrated that T cell activation leads to the redistribution of membrane and intracellular kinase-rich raft microdomains at the site of TCR engagement. In this investigation we demonstrated by high performance thin layer chromatography, gas chromatographic, and mass spectrometric analyses that GM3 is the main ganglioside constituent of these microdomains in human lymphocytes. Then we analyzed GM3 distribution and its interaction with the phosphorylation protein Zap-70. Human T lymphocytes were stimulated with anti-CD3 and anti-CD28. Immunofluorescence microscopy analysis revealed a clustered GM3 distribution over the cell surface and an intracellular localization resembling specific cytoplasmic compartment(s). Scanning confocal microscopy showed that T cell activation induced a significant association between GM3 and Zap-70, as revealed by nearly complete colocalization areas; very few colocalization areas were detected in unstimulated cells. Coimmunoprecipitation experiments revealed that GM3 was immunoprecipitated by anti-Zap-70 only after co-stimulation through CD3 and CD28 as detected by both thin layer chromatography and immunoblotting. Therefore, T cell activation does not promote a redistribution of glycosphingolipid-enriched microdomains but induces Zap-70 translocation in selective membrane domains in which Zap-70 may interact with GM3. These findings suggest that GM3 is a component of a multimolecular signaling complex involved in T cell activation.     

Microelectrode measurement of cell membrane potential in isolated hepatocytes attached to collagen

Isolated spherical rat hepatocytes attached to collagen-coated cover slips generate a mean membrane potential (Em) of -78 +/- 9 mV as measured with high-resistance microelectrodes. The recordings were biphasic and were stable for upto 20 minutes. The correlation between external potassium concentration and Em was not linear. Several potassium-channel blockers did not effect the membrane potential. Addition of ouabain added to the incubation solution slowly depolarized the cells. The results indicate a high potassium permeability of the isolated spherical hepatocytes attached to collagen.     

Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy

The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.     

Annexins in cell membrane dynamics. Ca(2+)-regulated association of lipid microdomains

The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca²⁺ concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca²⁺ sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca²⁺ concentrations. Annexin VI participates in the formation of a reversible, membrane–cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191–35195). Annexin II promotes the Ca²⁺-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca²⁺ flux.     

A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment

The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, does not become GPI anchored. We now show that this null sequence for GPI attachment can be converted to a strong GPI signal by mutating a pair of residues (valine-glutamate) in the LDLR sequence at a position corresponding to the normal cleavage/attachment site, to serine-glycine, as found in the DAF sequence. A single mutation (converting valine at the anchor addition site to serine, the normal acceptor for GPI addition in DAF) was insufficient to produce GPI anchoring, as was mutation of the valine-glutamate pair to serine-phenylalanine (a bulky residue). These results suggest that a pair of small residues (presumably flanking the cleavage point) is required for GPI attachment. By introducing the sequence serine-glycine (comprising a cleavage-attachment site for GPI addition) at different positions in the LDLR sequence of the fusion protein, HLD, we show that optimal GPI attachment requires a processing site positioned 10-12 residues NH2-terminal to the hydrophobic domain, the efficiency anchor attachment dropping off sharply as the cleavage site is moved beyond these limits. These data suggest that the GPI signal consists solely of a hydrophobic domain combined with a processing site composed of a pair of small residues, positioned 10-12 residues NH2-terminal to the hydrophobic domain. No other structural motifs appear necessary.     

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus

In most bacteria, the tubulin‐like GTPase FtsZ forms an annulus at midcell (the Z‐ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z‐ring assembly and early FtsZ‐directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C‐terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane‐anchored FtsZ in the regulation of cell wall hydrolysis.     

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.     

Vascular cell responses to a hybrid transforming growth factor-beta molecule.

Functional biological assays were performed using a hybrid molecule of Transforming Growth Factor-Beta (TGF-5 beta) where nine amino acids near the cleavage site of TGF-beta 1 were substituted with nine amino acids located in the identical position of TGF-beta 2. Bovine aortic endothelial and smooth muscle cells as well as rat epididymal fat pad microvascular endothelia were studied in three distinct bioassays examining proliferation, migration and angiogenesis. The data suggested TGF-5 beta elicited results that do not differ significantly from the TGF-beta 1 isoform, while TGF-beta 2 expressed unique characteristics. We have also shown that these amino acid substitutions to TGF-beta 1 do not, in fact, alter the biological functions of the growth factor.     

Adenosine deaminase in cell transformation. Biophysical manifestation of membrane dynamics

Cell transformation is associated with a dramatic collapse of a graphic fingerprint characteristic of normal cells, as measured by phase fluorimetry. This is demonstrated on adenosine deaminase (ADA, EC 3.5.4.4), an established malignancy marker. ADA activity is known to decrease markedly in chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus. The high affinity between the catalytic small subunit ADA (SS-ADA) and its membranal complexing protein (ADCP) (which abounds on the plasma membrane of CEF) allowed the hybridization of fluorescent labeled SS-ADA with native ADCP on CEF. Multifrequency differential phase fluorimetry responded remarkably to the state of this hybrid membrane protein. The transformation process is shown to have led to increased membrane fluidity and rotational mobility of ADCP as well as to its reduced availability to SS-ADA binding. The hypothesis of protein vertical sinking into the lipid core of the membrane is now given support by our spectroscopic data. Additional models are considered. A regulatory role is thus suggested for the complexing protein, which may also account for (a) reduced ADA activity in transformed cells and (b) detachment, exclusive to normal cells, upon addition of SS-ADA in excess.     

Perturbation of membrane microdomains reduces mitogenic signaling and increases susceptibility to apoptosis after T cell receptor stimulation

Acid sphingomyelinase-deficient (asmase-/-) mice generated by gene targeting abundantly store sphingomyelin in the reticuloendothelial system of liver, spleen, bone marrow, and in brain. Liver cells of asmase-/- mice accumulate sphingomyelin and glycosphingolipids in purified lipid bilayers of microsomes, Golgi, and the plasma membrane, but cholesterol is depleted in the plasma membrane. Detergent-insoluble glycolipid-enriched membrane microdomains (GEM) can be isolated from hepatocytes, embryonic fibroblasts, and splenocytes of wild-type, but not of asmase-/- mice, by sucrose gradient density centrifugation. Lck and other Src-family kinases are reduced in isopycnic fractions of asmase-/- splenocytes compared to GEM-containing fractions of wild-type cells. The proliferation of asmase-/- T lymphocytes is reduced, whereas their susceptibility to Fas-induced apoptosis is increased after T cell receptor (TCR) stimulation. TNF receptor I signaling remains unimpaired. The perturbation of GEM impairs tyrosine phosphorylation and, consequently, mitogenic signaling of the TCR. Reduced MAPK activity-dependent FLICE-like inhibitory protein (FLIP) expression in asmase-/- T lymphocytes increases their sensitivity towards Fas-mediated apoptosis.     

Analysis of the signal for attachment of a glycophospholipid membrane anchor

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophospholipid (GPI) membrane anchor. To define this signal we deleted portions of the DAF COOH terminus and expressed the mutant cDNAs it CV1 origin-deficient SV-40 cells. Our results show that the COOH-terminal hydrophobic domain (17 residues) is absolutely required for GPI anchor attachment. However, when fused to the COOH terminus of a secreted protein this hydrophobic domain is insufficient to direct attachment of a GPI anchor. Additional specific information located within the adjacent 20 residues appears to be necessary. We speculate that by analogy with signal sequences for membrane translocation, GPI anchor attachment requires both a COOH-terminal hydrophobic domain (the GPI signal) as well as a suitable cleavage/attachment site located NH2 terminal to the signal.     

Partitioning of myelin basic protein into membrane microdomains in a spontaneously demyelinating mouse model for multiple sclerosis.

We have characterized the lipid rafts in myelin from a spontaneously demyelinating mouse line (ND4), and from control mice (CD1 background), as a function of age and severity of disease. Myelin was isolated from the brains of CD1 and ND4 mice at various ages, and cold lysed with 1.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate). The lysate was separated by low-speed centrifugation into supernatant and pellet fractions, which were characterized by Western blotting for myelin basic protein (MBP) isoforms and their post-translationally modified variants. We found that, with maturation and with disease progression, there was a specific redistribution of the 14-21.5 kDa MBP isoforms (classic exon-II-containing vs exon-II-lacking) and phosphorylated forms into the supernatant and pellet. Further fractionation of the supernatant to yield detergent-resistant membranes (DRMs), representing coalesced lipid rafts, showed these to be highly enriched in exon-II-lacking MBP isoforms, and deficient in methylated MBP variants, in mice of both genotypes. The DRMs from the ND4 mice appeared to be enriched in MBP phosphorylated by MAP kinase at Thr95 (murine 18.5 kDa numbering). These studies indicate that different splice isoforms and post-translationally modified charge variants of MBP are targeted to different microdomains in the myelin membrane, implying multifunctionality of this protein family in myelin maintenance.     

Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor.

The pattern of solubilization of nine kidney microvillar ectoenzymes by a range of detergents distinguished two classes of membrane proteins: those released from the membrane by bacterial phosphatidylinositol-specific phospholipase C and those not so released. The latter group of transmembrane proteins were solubilized efficiently (greater than 80%) by all the detergents examined. In contrast, proteins released by phosphatidylinositol-specific phospholipase C were solubilized effectively only by octyl glucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate and sodium deoxycholate. Octyl glucoside solubilized the amphipathic forms of the ectoenzymes examined, suggesting that this may be a useful detergent in the purification of glycosyl-phosphatidylinositol-anchored ectoenzymes.     

Cell-free processing of nascent proteins destined to be linked to the plasma membrane by a phosphatidylinositol-glycan anchor.

Certain proteins are anchored to the outer plasma membrane by a phosphatidylinositol-glycan (PI-G) linker. Nascent forms of PI-G anchored proteins contain both NH2- and COOH-terminal signal peptides. The function and structural requirements of the COOH-terminal signal peptide as discussed and some studies on the cell-free processing of a nascent protein to its mature PI-G tailed form are presented.     

Direct binding of integrin alphavbeta3 to FGF1 plays a role in FGF1 signaling

Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin alphavbeta3 (K(D) about 1 microm). Antagonists to alphavbeta3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. alphavbeta3 was the predominant, if not the only, integrin that bound to FGF1, because FGF1 bound only weakly to several beta1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of beta3 plays a role in FGF1 binding. We found that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1 and FRS2alpha and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful tool to identify the role of integrins in FGF signaling.     

Targeting of the Sendai virus C protein to the plasma membrane via a peptide-only membrane anchor

Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPSFLKKILKLRGRR . . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic alpha-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.     

Synthesis of cetuximab-immunoliposomes via a cholesterol-based membrane anchor for targeting of EGFR

The objective of the present study was to construct epidermal growth factor receptor (EGFR) targeting cetuximab-immunoliposomes (ILs) for targeted delivery of boron compounds to EGFR(+) glioma cells for neutron capture therapy. The ILs were synthesized by using a novel cholesterol-based membrane anchor, maleimido-PEG-cholesterol (Mal-PEG-Chol), to incorporate cetuximab into liposomes by either surface conjugation or a post-insertion method. For post-insertion, the transfer efficiency of MAb conjugates from micelles to liposome was examined at varying temperatures, mPEG2000-DSPE ratios, and micelle-to-liposome lipid ratios. Following this, the cetuximab-ILs were evaluated for targeted delivery of the encapsulated boron anion, dodecahydro-closo-dodecaborate (2-) (B12H122-), to human EGFR gene transfected F98EGFR glioma cells as potential delivery agents for boron neutron capture therapy (BNCT). In addition, cellular uptake of cetuximab-ILs, encapsulating a fluorescence dye, was analyzed by confocal fluorescence microscopy and flow cytometry, and boron content was quantified by ICP-MS. Much greater ( approximately 8-fold) cellular uptake of boron was obtained using cetuximab-ILs in EGFR(+) F98EGFR compared with nontargeted human IgG-ILs. On the basis of these observations, we have concluded that cholesterol can serve as an effective anchor for MAb in liposomes, and cetuximab-ILs are potentially useful delivery vehicles for BNCT of gliomas.     

Sensitization of epidermal growth factor-induced signaling by bradykinin is mediated by c-Src. Implications for a role of lipid microdomains

Communication between receptor tyrosine kinase (RTK)- and G protein-coupled receptor (GPCR)-mediated signaling systems has received increasing attention in recent years. Here, we report that activation of G protein-coupled bradykinin B2 receptor induces an up-regulation of cellular responses mediated by epidermal growth factor receptor (EGFR) and provide essential mechanistic characteristics of this sensitization process. EGF, which failed to evoke detectable amount of calcium increase and neurotransmitter release when administrated alone in primary cultures of rat adrenal chromaffin cells and PC12 cells, became capable of inducing these responses specifically after bradykinin pretreatment. Both EGFR and non-receptor tyrosine kinase p60Src, whose kinase activities were required in the sensitization, were found to be enriched in cholesterol-rich lipid rafts. Bradykinin caused activation of p60Src and Src-dependent phosphorylation of the EGFR on Tyr-845 in lipid rafts, as well as recruitment of phospholipase C (PLC) gamma1 to the rafts. Depletion of cholesterol by methyl-beta-cyclodextrin disrupted the raft localization of EGFR and Src, as well as bradykinin-induced translocation of PLCgamma1. Furthermore, sensitization, which was impaired by cholesterol depletion, was restored by repletion of cholesterol. Therefore, we suggest that lipid rafts are essential participants in the regulation of receptor-mediated signal transduction and cross-talk via organizing signaling complexes in membrane microdomains.