Yutaka Furutani

Interaction between Telencephalin and ERM Family Proteins Mediates Dendritic Filopodia Formation

Dendritic filopodia are long, thin, actin-rich, and dynamic protrusions that are thought to play a critical role as a precursor of spines during neural development. We reported previously that a telencephalon-specific cell adhesion molecule, telencephalin (TLCN) [intercellular adhesion molecule-5 (ICAM-5)], is highly expressed in dendritic filopodia, facilitates the filopodia formation, and slows spine maturation. Here we demonstrate that TLCN cytoplasmic region binds ERM (ezrin/radixin/moesin) family proteins that link membrane proteins to actin cytoskeleton. In cultured hippocampal neurons, phosphorylated active forms of ERM proteins are colocalized with TLCN in dendritic filopodia, whereas α-actinin, another binding partner of TLCN, is colocalized with TLCN at surface membranes of soma and dendritic shafts. Expression of constitutively active ezrin induces dendritic filopodia formation, whereas small interference RNA-mediated knockdown of ERM proteins decreases filopodia density and accelerates spine maturation. These results indicate the important role of TLCN–ERM interaction in the formation of dendritic filopodia, which leads to subsequent synaptogenesis and establishment of functional neural circuitry in the developing brain.

Small GTPase Rab17 Regulates Dendritic Morphogenesis and Postsynaptic Development of Hippocampal Neurons

Background: Rab-type small GTPases are conserved membrane trafficking proteins in all eukaryotes. Results: Knockdown of Rab17 in mouse hippocampal neurons results in a marked reduction in the number of dendritic branches and total dendrite length. Conclusion: Rab17 regulates dendritic morphogenesis and postsynaptic development in hippocampal neurons. Significance: Our findings reveal the first molecular link between membrane trafficking and dendritogenesis. Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.

An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death.

Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2’s multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.

A Simple Assay and Histochemical Localization of Transglutaminase Activity Using a Derivative of Green Fluorescent Protein as Substrate

SUMMARY Histidine-tagged green fluorescent protein (His6-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His6-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His6-Xpress). His6-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247–258, 2001)

Protein Stretching II*1: Results for Carbonic Anhydrase

The force curve measurement mode of an atomic force microscope was used to record the force required to stretch a protein molecule that was covalently sandwiched through gold-thiol bonds between a mica substrate and a silicon nitride tip, both coated with gold. In one experiment, 8 ± 1 out of 20 lysyl residues of bovine carbonic anhydrase B were randomly derivatized to give free thiols, and grafted on an atomically flat gold (111) surface on mica. Force curves taken on the surface covered with protein molecules using a gold coated tip occasionally showed a large downward deflection indicating trapping and subsequent stretching of protein molecules between the tip and the substrate. In another experiment, the same protein was genetically engineered so that cysteine residues were introduced at both the amino and the carboxyl terminus. Force curves taken in a similar manner as in the first experiment indicated almost complete extension of a linear polypeptide chain. The result was explained in terms of extension of a pseudo-three-dimensional get in both cases, with additional stretching of a linear chain in the second case.

Androgen-Dependent Expression, Gene Structure, and Molecular Evolution of Guinea Pig Caltrin II, a WAP-Motif Protein

Abstract We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions.

Vitronectin Induces Phosphorylation of Ezrin/Radixin/Moesin Actin-binding Proteins through Binding to Its Novel Neuronal Receptor Telencephalin

Background: Telencephalin is an adhesion molecule specifically expressed on dendrites of telencephalic neurons. Results: Vitronectin binds telencephalin and induces phosphorylation of ezrin/radixin/moesin, accumulation of F-actin and PI(4,5)P2, and formation of phagocytic cup-like dendritic protrusions. Conclusion: Telencephalin is a novel neuronal receptor for vitronectin. Significance: This study demonstrates the functional interaction between vitronectin and telencephalin for cytoskeletal reorganization in dendritic protrusions. Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.

Identification and characterization of nCLP2, a novel C1q family protein expressed in the central nervous system

The C1q family is characterized by the C-terminally conserved globular C1q (gC1q) domain. Although more than 30 C1q family proteins have been identified in mammals, many of them remain ill-defined with respect to their molecular and biological properties. Here, we report on a novel C1q family protein specifically expressed in the central nervous system (CNS), which we designated neural C1q-like protein (nCLP) 2. nCLP2 was secreted as disulphide-bonded multimers comprising trimeric units. The multimers were stabilized by interchain disulphide bonds involving the cysteine residues in the N-terminal variable region and the C-terminal gC1q domain. The expression of nCLP2 was restricted to several brain regions and retina, including regions associated with memory formation (i.e. hippocampus, entorhinal cortex, anterodorsal thalamic nucleus). Immunoelectron microscopy revealed that nCLP2 was localized in the mossy fibre axons of hippocampal granule cells and their synaptic boutons and clefts, implying that nCLP2 was anterogradely transported in mossy fibres and secreted from the presynaptic termini. These results suggest that nCLP2 plays roles in synaptic function and maintenance in the CNS.

Evolution of trappin genes in mammals

BackgroundTrappin is a multifunctional host-defense peptide that has antiproteolytic, antiinflammatory, and antimicrobial activities. The numbers and compositions of trappin paralogs vary among mammalian species: human and sheep have a single trappin-2 gene; mouse and rat have no trappin gene; pig and cow have multiple trappin genes; and guinea pig has a trappin gene and two other derivativegenes. Independent duplications of trappin genes in pig and cow were observed recently after the species were separated. To determine whether these trappin gene duplications are restricted only to certain mammalian lineages, we analyzed recently-developed genome databases for the presence of duplicate trappin genes.ResultsThe database analyses revealed that: 1) duplicated trappin multigenes were found recently in the nine-banded armadillo; 2) duplicated two trappin genes had been found in the Afrotherian species (elephant, tenrec, and hyrax) since ancient days; 3) a single trappin-2 gene was found in various eutherians species; and 4) no typical trappin gene has been found in chicken, zebra finch, and opossum. Bayesian analysis estimated the date of the duplication of trappin genes in the Afrotheria, guinea pig, armadillo, cow, and pig to be 244, 35, 11, 13, and 3 million-years ago, respectively. The coding regions of trappin multigenes of almadillo, bovine, and pig evolved much faster than the noncoding exons, introns, and the flanking regions, showing that these genes have undergone accelerated evolution, and positive Darwinian selection was observed in pig-specific trappin paralogs.ConclusionThese results suggest that trappin is an eutherian-specific molecule and eutherian genomes have the potential to form trappin multigenes.