Quantification of HER2 in COS7 cells using quantum weak measurement.

A Mach-Zehnder interferometer system based on weak measurement was set up to determinate the concentration variation of molecule by measuring the phase difference change between the two optical paths. The spectrum of the light was recorded to monitor the concentration of trastuzumab (Herceptin), which is a humanised monoclonal antibody, targeted to human epidermal growth factor receptor 2 (HER2). The trastuzumab targeting to HER2 was real-time detected and continuously monitored, the HER2 numbers of COS7 cells on a coverslip was determined at pico-molar level. Our weak measurement enabled method proposes an alternative approach for the concentration detection of molecules, providing a promising functional tool for the quantification of HER2 in cancer cells, possibly promoting fields such as the diagnosis and treatment of cancer.

A human dynamin-related protein controls the distribution of mitochondria.

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.

Lack of association between receptor protein tyrosine phosphatase RPTP mu and cadherins.

RPTP mu is a receptor-like protein tyrosine phosphatase that mediates homophilic cell-cell interactions. Surface expression of RPTP mu is restricted to cell-cell contacts and is upregulated with increasing cell density, suggesting a role for RPTP mu in contact-mediated signaling. It was recently reported (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977-986) that RPTP mu binds directly to cadherin/catenin complexes, and thus may regulate the tyrosine phosphorylation of such complexes. Here we report that this concept needs revision. Through reciprocal precipitations using a variety of antibodies against RPTP mu, cadherins, and catenins, we show that RPTP mu does not interact with cadherin/catenin complexes, even when assayed under very mild lysis conditions. We find that the anti-RPTP mu antiserum used by others precipitates cadherins in a nonspecific manner independent of RPTP mu. We conclude that, contrary to previous claims, RPTP mu does not interact with cadherin complexes and thus is unlikely to directly regulate cadherin/catenin function.

Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex.

Cadherins are calcium-dependent cell adhesion molecules that play fundamental roles in embryonic development, tissue morphogenesis, and cancer. A prerequisite for their function is association with the actin cytoskeleton via the catenins. Tyrosine phosphorylation of beta-catenin, which correlates with a reduction in cadherin-dependent cell adhesion, may provide cells with a mechanism to regulate cadherin activity. Here we report that beta-catenin immune precipitates from PC12 cells contain tyrosine phosphatase activity which dephosphorylates beta-catenin in vitro. In addition, we show that a member of the leukocyte antigen-related protein (LAR)-related transmembrane tyrosine phosphatase family (LAR-PTP) associates with the cadherin-catenin complex. This association required the amino-terminal domain of beta-catenin but does not require the armadillo repeats, which mediate association with cadherins. The interaction also is detected in PC9 cells, which lack alpha-catenin. Thus, the association is not mediated by alpha-catenin or by cadherins. Interestingly, LAR-PTPs are phosphorylated on tyrosine in a TrkA-dependent manner, and their association with the cadherin-catenin complex is reduced in cells treated with NGF. We propose that changes in tyrosine phosphorylation of beta-catenin mediated by TrkA and LAR-PTPs control cadherin adhesive function during processes such as neurite outgrowth.

Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex.

We have identified a protein named pinin that is associated with the mature desmosomes of the epithelia (Ouyang, P., and S.P. Sugrue. 1992. J. Cell Biol. 118:1477-1488). We suggest that the function of pinin is to pin intermediate filaments to the desmosome. Therefore, pinin may play a significant role in reinforcing the intermediate filament-desmosome complex. cDNA clones coding for pinin were identified, using degenerative oligonucleotide probes that were based on the internal amino acid sequence of pinin for the screening of a cDNA library. Immunoblotting of expressed recombinant proteins with the monoclonal 08L antibody localized the 08L epitope to the carboxyl end of the protein. Polyclonal antibodies directed against fusion proteins immunoidentified the 140-kD protein in tissue extracts. Immunofluorescence analysis, using the antifusion protein antibody, demonstrated pinin at lateral epithelial boundaries, which is consistent with desmosomal localization. The conceptual translation product of the cDNA clones contained three unique domains: (a) a serine-rich domain; (b) a glutamine-proline, glutamine-leucine repeat domain; and (c) an acidic domain rich in glutamic acid. Although the 3' end of the open reading frame of the clone for pinin showed near identity to a partial cDNA isolated for a pig neutrophil phosphoprotein (Bellavite, P., F. Bazzoni, et al. 1990. Biochem. Biophys. Res. Commun. 170:915-922), the remaining sequence demonstrated little homology to known protein sequences. Northern blots of mRNA from chicken corneal epithelium, MDCK cells, and various human tissues indicated that pinin messages exhibit tissue-specific variation in size, ranging from 3.2 to 4.1 kb. Genomic Southern blots revealed the existence of one gene for pinin, suggesting alternative splicing of the mRNA. Expression of the full-length cDNA clones in human 293 cells and monkey COS-7 cells demonstrated that a 140-kD immunoreactive species on Western blots corresponded to pinin. Pinin cDNA transfected into the transformed 293 cells resulted in enhanced cell-cell adhesion. Immunofluorescence staining revealed that the expressed pinin protein was assembled to the lateral boundaries of the cells in contact, which is consistent with the staining pattern of pinin in epithelial cells.

Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.

We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").

Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools.

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)delta PH domain-green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCdelta PH domain known to form critical contacts with PtdIns(4, 5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol- labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

Disulfide-linked head-to-head multimerization in the mechanism of ion channel clustering by PSD-95.

The PSD-95/SAP90 family of PDZ-containing proteins is directly involved in the clustering of specific ion channels at synapses. We report that channel clustering depends on a conserved N-terminal domain of PSD-95 that mediates multimerization and disulfide linkage of PSD-95 protomers. This N-terminal multimerization domain confers channel clustering activity on a single PDZ domain. Thus, channel clustering depends on aggregation of PDZ domains achieved by head-to-head multimerization of PSD-95, rather than by concatenation of PDZ domains in PSD-95 monomers. This mechanism predicts that PSD-95 can organize heterogeneous membrane protein clusters via differential binding specificities of its three PDZ domains. PSD-95 and its relative chapsyn-110 exist as disulfide-linked complexes in rat brain, consistent with head-to-head multimerization of these proteins in vivo.

Protein topology of presenilin 1.

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.

Assignment of early caudal identity to neural plate cells by a signal from caudal paraxial mesoderm.

The early patterning of the vertebrate central nervous system involves the generation of progenitor cells with distinct fates at rostral and caudal levels of the neuraxis. We provide evidence that the assignment of early rostrocaudal differences in progenitor cell properties is established by spatial restrictions in the signaling properties of the paraxial mesoderm and epidermal ectoderm. Caudal level paraxial mesoderm secretes a factor, distinct from retinoic acid or fibroblast growth factors (FGFs), that can impose caudal fates on prospective anterior proencephalic progenitors. The caudalizing activity of the paraxial mesoderm can, however, be induced by FGF signaling. The distinct properties of cells at rostral and caudal levels of the neural plate appear to depend, in addition, on the early exclusion of bone morphogenetic proteins (BMPs) from rostral level epidermal ectoderm. Thus, differences in the signaling properties of cell groups that flank the neural plate appear to contribute to the early rostrocaudal identity of neural cells, distinguishing progenitor cells at prospective anterior proencephalic regions from those at more caudal levels of the neuraxis.

Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis.

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1-Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin's GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

Low-density lipoprotein from apolipoprotein E-deficient mice induces macrophage lipid accumulation in a CD36 and scavenger receptor class A-dependent manner.

OBJECTIVE: To investigate the potential of circulating low-density lipoprotein (LDL), isolated from apolipoprotein E (apoE)-deficient mice (E-/-LDL) and from LDL receptor-deficient mice (Lr-/-LDL), to induce foam cell formation. METHODS AND RESULTS: Binding studies using COS-7 cells overexpressing CD36, J774 cells, and mouse peritoneal macrophages (MPMs) unexpectedly showed for the first time that E-/-LDL, which is enriched in cholesterol, is a high-affinity ligand for CD36 and exhibited greater macrophage uptake than Lr-/-LDL or normal LDL. Minimal copper-mediated oxidization of Lr-/-LDL or C57LDL in vitro resulted in increased ligand internalization, although cell uptake of these oxidized LDLs was lower than that of E-/-LDL, even at oxidation levels similar to that found in E-/-LDL. Treatment of MPMs with E-/-LDL and Lr-/-LDL (to a 2- to 3-fold lesser extent), but not normal LDL, resulted in significant cellular cholesteryl ester accumulation and foam cell formation. Experiments using MPMs lacking CD36, scavenger receptor class A (SR-A), or both, indicated a major contribution of CD36 ( approximately 50%), and to a lesser extent, SR-A (24% to 30%), to E-/-LDL uptake. CONCLUSIONS: Because of its increased state of oxidation and high cholesterol content, LDL in apoE-deficient mice acts in a proatherogenic manner, without requiring further modification in the vascular wall, to induce foam cell formation through its uptake by scavenger receptors.

Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein.

OBJECTIVE: Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein-coupled receptor transport from the endoplasmic reticulum to the cell surface. METHODS AND RESULTS: Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum-export signal. Using a yeast two-hybrid system, we identified a Golgi membrane-associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor. CONCLUSIONS: Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.

Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.

AGPAT (1-acyl-sn-glycerol 3-phosphate acyltransferase) exists in at least five isoforms in humans, termed as AGPAT1, AGPAT2, AGPAT3, AGPAT4 and AGPAT5. Although they catalyse the same biochemical reaction, their relative function, tissue expression and regulation are poorly understood. Linkage studies in humans have revealed that AGPAT2 contributes to glycerolipid synthesis and plays an important role in regulating lipid metabolism. We report the molecular cloning, tissue distribution, and enzyme characterization of mAGPATs (murine AGPATs) and regulation of cardiac mAGPATs by PPARalpha (peroxisome-proliferator-activated receptor alpha). mAGPATs demonstrated differential tissue expression profiles: mAGPAT1 and mAGPAT3 were ubiquitously expressed in most tissues, whereas mAGPAT2, mAGPAT4 and mAGPAT5 were expressed in a tissue-specific manner. mAGPAT2 expressed in in vitro transcription and translation reactions and in transfected COS-1 cells exhibited specificity for 1-acyl-sn-glycerol 3-phosphate. When amino acid sequences of five mAGPATs were compared, three highly conserved motifs were identified, including one novel motif/pattern KX2LX6GX12R. Cardiac mAGPAT activities were 25% lower (P<0.05) in PPARalpha null mice compared with wild-type. In addition, cardiac mAGPAT activities were 50% lower (P<0.05) in PPARalpha null mice fed clofibrate compared with clofibrate fed wild-type animals. This modulation of AGPAT activity was accompanied by significant enhancement/reduction of the mRNA levels of mAGPAT3/mAGPAT2 respectively. Finally, mRNA expression of cardiac mAGPAT3 appeared to be regulated by PPARalpha activation. We conclude that cardiac mAGPAT activity may be regulated by both the composition of mAGPAT isoforms and the levels of each isoform.

Regulation of adenylate cyclase type VIII splice variants by acute and chronic Gi/o-coupled receptor activation.

We previously reported that acute agonist activation of G(i/o)-coupled receptors inhibits adenylate cyclase (AC) type VIII activity, whereas agonist withdrawal following chronic activation of these receptors induces AC-VIII superactivation. Three splice variants of AC-VIII have been identified, which are called AC-VIII-A, -B and -C (with AC-VIII-B missing the glycosylation domain and AC-VIII-C lacking most of the C1b area). We report here that AC-VIII-A and -B, but not -C, are inhibited by acute mu-opioid and dopaminergic type D2 receptor activation, indicating that the C1b area of AC-VIII has an important role in AC inhibition by G(i/o)-coupled receptor activation. On the other hand the glycosylation sites in AC-VIII did not play a role in AC-VIII regulation. Although AC-VIII-A and -C differed in their capacity to be inhibited by acute agonist exposure, agonist withdrawal after prolonged treatment led to a similar superactivation of all three splice variants, with no significant change in AC-VIII expression. AC-VIII superactivation was not affected by pre-incubation with a cell permeable cAMP analogue, indicating that the superactivation does not depend on the agonist-induced reduction in cAMP levels. The superactivated AC-VIII-A, -B and -C were similarly re-inhibited by re-application of agonist (morphine or quinpirole), returning the activity to control levels. These results demonstrate marked differences in the agonist inhibition of the AC-VIII splice variants before, but not after, superactivation.

Analysis of the ATM protein in wild-type and ataxia telangiectasia cells.

Ataxia telangiectasia (A-T) is a human disorder that results in a number of clinical symptoms, including cerebellar degeneration and increased cancer predisposition. Recently the gene that is defective in A-T has been cloned and designated ATM. Here, we describe the production of antisera raised against the approximately 350 kDa ATM protein. Antisera specificity is confirmed by them recognising a approximately 350 kDa polypeptide in wild-type cells but not in A-T cells containing mutations that truncate ATM upstream of the antibody binding sites. We show that ATM is almost exclusively nuclear and is expressed in all cell lines and tissues analysed. However, ATM levels are not regulated in response to u.v. or ionising radiation. These data are consistent with ATM being a component of the DNA damage detection apparatus rather than being an inducible downstream effector of the DNA damage response. In addition, we analyse ATM protein expression in a variety of A-T patients. Strikingly, ATM expression is reduced drastically or absent in all patients analysed, including those predicted to express proteins that should be detected by our antisera. Thus, the A-T phenotype may result not only from mutations that disrupt functional domains of ATM, but also from mutations that destabilise the ATM mRNA or protein. Finally, we report that a group of patients displaying an intermediate A-T phenotype express low levels of apparently full-length ATM. This suggests that the ATM pathway is partially active in these individuals and that there is a correlation between levels of residual ATM expression and disease severity.

Octameric stoichiometry of the KATP channel complex.

ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits-a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.

Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor.

KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945-951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4-. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.

Nuclear import of cellular retinoic acid-binding protein type I in mouse embryonic cells.

Using confocal microscopy we show that cellular retinoic acid-binding protein type I (CRABP I), expressed in several embryonic cell types, displays a compartmentalized subcellular distribution. The protein was excluded from the nucleus in some cells, while in others it accumulated in the nucleus. In the rat cerebellar cell line ST15A, which expresses CRABP I, the protein was found in the cytoplasm with a prominent nuclear exclusion. Addition of retinoic acid to embryos in vivo and to ST15 A cells in vitro did not affect the localization of the protein. Localization of CRABP I and CRABP I fused to a nuclear localization signal expressed in transfected cells, suggested that cell-specific factors may regulate nuclear import of CRABP I. The potential role of a CRABP I-controlled nuclear import of retinoic acid is discussed.

Ultrasensitive confocal fluorescence microscopy of C-reactive protein interacting with FcgammaRIIa.

BACKGROUND: C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcgammaRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcgammaRIIa may have caused false-positive results. METHODS AND RESULTS: To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcgammaRIIa. CRP indeed binds to FcgammaRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude. CONCLUSIONS: Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcgammaRIIa and explains and reconciles the diverse and conflicting data presented in the literature.