Sodium channel Nav1.6 is localized at nodes of Ranvier, dendrites, and synapses

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Nav1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Nav1.6-deficient mouse mutant strain med, whose nodes were negative for Nav1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Nav1.6 is the predominant channel type in this structure. In the central nervous system, Nav1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Nav1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

Real-time quantitative measurement of autocrine ligand binding indicates that autocrine loops are spatially localized

Autocrine ligands are important regulators of many normal tissues and have been implicated in a number of disease states, including cancer. However, because by definition autocrine ligands are synthesized, secreted, and bound to cell receptors within an intrinsically self-contained “loop,” standard pharmacological approaches cannot be used to investigate relationships between ligand/receptor binding and consequent cellular responses. We demonstrate here a new approach for measurement of autocrine ligand binding to cells, using a microphysiometer assay originally developed for investigating cell responses to exogenous ligands. This technique permits quantitative measurements of autocrine responses on the time scale of receptor binding and internalization, thus allowing investigation of the role of receptor trafficking and dynamics in cellular responses. We used this technique to investigate autocrine signaling through the epidermal growth factor receptor by transforming growth factor alpha (TGFα) and found that anti-receptor antibodies are far more effective than anti-ligand antibodies in inhibiting autocrine signaling. This result indicates that autocrine-based signals can operate in a spatially restricted, local manner and thus provide cells with information on their local microenvironment.

Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets

5′-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) of wheat (Triticum aestivum) were cloned and sequenced. The sequences of the two genes, Acc-1,1 and Acc-1,2, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by Acc-1,1 and Acc-1,2 is 93% identical with the maize plastid ACCase but only 80–84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5′-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the Acc-1 genes is at least 130–170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes Acc-1,1 and Acc-1,2, indicates that all three genes are transcriptionally active.

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

Na+/Cl−-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [35S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na+/Cl−-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.

Internal initiation of translation of five dendritically localized neuronal mRNAs

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5′ leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the α subunit of calcium–calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5′ untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and α-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5′ untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5′ leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

Multicopy plasmids are clustered and localized in Escherichia coli

We localized the multicopy plasmid RK2 in Escherichia coli and found that the number of fluorescent foci observed in each cell was substantially less than the copy number of the plasmid, suggesting that many copies of RK2 are grouped into a few multiplasmid clusters. In minimal glucose media, the majority of cells had one or two foci, with a single focus localized near midcell, and two foci near the 1/4 and 3/4 cell positions. The number of foci per cell increased with cell length and with growth rate, and decreased upon entering stationary phase, suggesting a coordination of RK2 replication or segregation with the bacterial cell cycle. Time-lapse microscopy demonstrated that partitioning of RK2 foci is achieved by the splitting of a single focus into two or three smaller foci, which are capable of separating with rapid kinetics. A derivative of the high-copy-number plasmid pUC19 containing the lacO array was also localized by tagging with GFP-LacI. Whereas many of the cells contained numerous, randomly diffusing foci, most cells exhibited one or two plasmid clusters located at midcell or the cell quarter positions. Our results suggest a model in which multicopy plasmids are not always randomly diffusing throughout the cell as previously thought, but can be replicated and partitioned in clusters targeted to specific locations.

Complementary DNA Cloning and Characterization of Ferredoxin Localized in Bundle-Sheath Cells of Maize Leaves1

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of −423 and −406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

Nucellain, a Barley Homolog of the Dicot Vacuolar-Processing Protease, Is Localized in Nucellar Cell Walls1

The nucellus is a complex maternal grain tissue that embeds and feeds the developing cereal endosperm and embryo. Differential screening of a barley (Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolation of two cDNA clones encoding nucellus-specific homologs of the vacuolar-processing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developing corn (Zea mays) grains was also identified. In dicots the vacuolar-processing enzyme is believed to be involved in the processing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projection, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of developing grains such as the testa or the pericarp. Using an antibody raised against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains. Immunogold-labeling experiments with this antibody localized the nucellain epitope not in the vacuoles, but in the cell walls of all nucellar cell types. We propose that nucellain plays a role in processing and/or turnover of cell wall proteins in developing cereal grains.

Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce1

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to an hrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s. pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn2+/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H2O2 in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit1

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

The ocular albinism type 1 gene product is a membrane glycoprotein localized to melanosomes.

Ocular albinism type 1 (OA1) is an inherited disorder characterized by severe reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium reveals the presence of macromelanosomes, suggesting a defect in melanosome biogenesis. The gene responsible for OA1 is exclusively expressed in pigment cells and encodes a predicted protein of 404 aa displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Using polyclonal antibodies we have identified the endogenous OA1 protein in retinal pigment epithelial cells, in normal human melanocytes and in various melanoma cell lines. Two forms of the OA1 protein were identified by Western analysis, a 60-kDa glycoprotein and a doublet of 48 and 45 kDa probably corresponding to unglycosylated precursor polypeptides. Upon subcellular fractionation and phase separation with the nonionic detergent Triton X-114, the OA1 protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OA1 protein, consistent with its possible involvement in melanosome biogenesis. The identification of a novel melanosomal membrane protein involved in a human disease will provide insights into the mechanisms that control the cell-specific pathways of subcellular morphogenesis.

Targeting of proteins to granule subsets is determined by timing and not by sorting: The specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells.

The mechanism of protein targeting to individual granules in cells that contain different subsets of storage granules is poorly understood. The neutrophil contains two highly distinct major types of granules, the peroxidase positive (azurophil) granules and the peroxidase negative (specific and gelatinase) granules. We hypothesized that targeting of proteins to individual granule subsets may be determined by the stage of maturation of the cell, at which the granule proteins are synthesized, rather than by individual sorting information present in the proteins. This was tested by transfecting the cDNA of the specific granule protein, NGAL, which is normally synthesized in metamyelocytes, into the promyelocytic cell line HL-60, which is developmentally arrested at the stage of formation of azurophil granules, and thus does not contain specific and gelatinase granules. Controlled by a cytomegalovirus promoter, NGAL was constitutively expressed in transfected HL-60 cells. This resulted in the targeting of NGAL to azurophil granules as demonstrated by colocalization of NGAL with myeloperoxidase, visualized by immunoelectron microscopy. This shows that targeting of proteins into distinct granule subsets may be determined solely by the time of their biosynthesis and does not depend on individual sorting information present in the proteins.

Ancestral major histocompatibility complex DRB genes beget conserved patterns of localized polymorphisms.

Genes within the major histocompatibility complex (MHC) are characterized by extensive polymorphism within species and also by a remarkable conservation of contemporary human allelic sequences in evolutionarily distant primates. Mechanisms proposed to account for strict nucleotide conservation in the context of highly variable genes include the suggestion that intergenic exchange generates repeated sets of MHC DRB polymorphisms [Gyllensten, U. B., Sundvall, M. & Erlich, H. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3686-3690; Lundberg, A. S. & McDevitt, H. 0. (1992) Proc. Natl. Acad. Sci. USA 89, 6545-6549]. We analyzed over 50 primate MHC DRB sequences, and identified nucleotide elements within macaque and baboon DRB6-like sequences with deletions corresponding to specific exon 2 hypervariable regions, which encode a discrete alpha helical segment of the MHC antigen combining site. This precisely localized deletion provides direct evidence implicating segmental exchange of MHC-encoded DRB gene fragments as one of the evolutionary mechanisms both generating and maintaining MHC diversity. Intergenic exchange at this site may be fundamental to the diversification of immune protection in populations by permitting alteration in the specificity of the MHC that determines the repertoire of antigens bound.

Decreased food intake does not completely account for adiposity reduction after ob protein infusion.

The effects of recombinantly produced ob protein were compared to those of food restriction in normal lean and genetically obese mice. Ob protein infusion into ob/ob mice resulted in large decreases in body and fat-depot weight and food intake that persisted throughout the study. Smaller decreases in body and fat-depot weights were observed in vehicle-treated ob/ob mice that were fed the same amount of food as that consumed by ob protein-treated ob/ob mice (pair feeding). In lean mice, ob protein infusion significantly decreased body and fat-depot weights, while decreasing food intake to a much lesser extent than in ob/ob mice. Pair feeding of lean vehicle-treated mice to the intake of ob protein-treated mice did not reduce body fat-depot weights. The potent weight-, adipose-, and appetite-reducing effects exerted by the ob protein in ob protein-deficient mice (ob/ob) confirm hypotheses generated from early parabiotic studies that suggested the existence of a circulating satiety factor of adipose origin. Pair-feeding studies provide compelling evidence that the ob protein exerts adipose-reducing effects in excess of those induced by reductions in food intake.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.