Precise tracking of the dynamics of multiple proteins in endocytic events.

Endocytosis is a complex and dynamic process that involves dozens of different proteins to define the site of endocytosis, form a membrane invagination, and pinch off a membrane vesicle into the cytoplasm. Fluorescent light microscopy is a powerful tool to visualize the dynamic behaviors of the proteins taking part in the endocytic process. The resolution of light microscopy is, however, a serious limitation. Here, we detail a fluorescence microscope method that we have developed to visualize the dynamics of the clathrin-mediated endocytic protein machinery in yeast cells. This method is based on subpixel centroid tracking of endocytic proteins. For each endocytic protein, the centroid trajectories obtained from multiple endocytic events are used to compute an average trajectory that describes, at nanometer scale, the assembly and movement of the protein during endocytosis. The average trajectories of the different endocytic proteins are then aligned together in space and time to reconstruct how the different proteins behave relative to each other during the endocytic process.

Transgenic expression of syndecan-1 uncovers a physiological control of feeding behavior by syndecan-3.

Transgenic expression in the hypothalamus of syndecan-1, a cell surface heparan sulfate proteoglycan (HSPG) and modulator of ligand-receptor encounters, produces mice with hyperphagia and maturity-onset obesity resembling mice with reduced action of alpha melanocyte stimulating hormone (alphaMSH). Via their HS chains, syndecans potentiate the action of agouti-related protein and agouti signaling protein, endogenous inhibitors of alphaMSH. In wild-type mice, syndecan-3, the predominantly neural syndecan, is expressed in hypothalamic regions that control energy balance. Food deprivation increases hypothalamic syndecan-3 levels several-fold. Syndecan-3 null mice, otherwise apparently normal, respond to food deprivation with markedly reduced reflex hyperphagia. We propose that oscillation of hypothalamic syndecan-3 levels physiologically modulates feeding behavior.

Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles.

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

Heparin-binding proteins HB-GAM (pleiotrophin) and amphoterin in the regulation of cell motility.

Fractionation of proteins from perinatal rat brain was monitored using a neurite outgrowth assay. Two neurite-promoting proteins, HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin) and amphoterin, were isolated, cloned and produced by baculovirus expression for structural and functional studies. HB-GAM is highly expressed in embryonic and early post-natal fiber pathways of the nervous system, and it enhances axonal growth/guidance by binding to N-syndecan (syndecan-3) at the neuron surface. N-syndecan in turn communicates with the cytoskeleton through the cortactin/src-kinase pathway to enhance neurite extension. In addition to N-syndecan, the chondroitin sulfate proteoglycan RPTP beta/zeta (receptor-type tyrosine phosphatase beta/zeta) is implicated in the receptor mechanism of HB-GAM. HB-GAM is also prominently expressed in developing and regenerating bone as a matrix-bound cue for migration of osteoblasts/osteoblast precursors to the site of bone deposition. HB-GAM is suggested to regulate motility in osteoblasts through a similar mechanism as in neurons. Structural studies using heteronuclear NMR reveal two similar protein domains in HB-GAM, both consisting of three anti-parallel beta-strands. Search of sequence databases shows that the beta structures of HB-GAM and of the similar domains of MK (midkine) correspond to the thrombospondin type I (TSR) sequence motif. We suggest that the TSR sequence motif, found in several neurite outgrowth-promoting and other cell surface and matrix-binding proteins, defines a beta structure similar to those found in HB-GAM and MK. In general, amphoterin is highly expressed in immature and transformed cells. We suggest a model, according to which amphoterin is an autocrine/paracrine regulator of invasive migration. Amphoterin binds to RAGE (receptor of advanced glycation end products), an immunoglubulin superfamily member related to N-CAM (neural cell adhesion molecule), that communicates with the GTPases Cdc42 and Rac to regulate cell motility. In addition, ligation of RAGE by amphoterin activates NF-kappaB to regulate transcription.

Heparin-binding growth-associated molecule contains two heparin-binding beta -sheet domains that are homologous to the thrombospondin type I repeat.

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.

Heparan sulphate and HB-GAM (heparin-binding growth-associated molecule) in the development of the thalamocortical pathway of rat brain.

Extracellular matrix (ECM) molecules, such as laminin, tenascin, chondroitin sulphate proteoglycans and heparan sulphate proteoglycans have been suggested to have 'signpost' and directing roles in the formation of axonal projections in cortical development. We show here that the expression of the neurite outgrowth-promoting protein heparin-binding growth-associated molecule (HB-GAM) and N-syndecan, a transmembrane heparan sulphate proteoglycan previously isolated as a receptor for HB-GAM, is spatiotemporally associated with the developing thalamocortical pathway in the rat brain. Using in situ hybridization, thalamic neurons were shown to express mRNA for N-syndecan, and in vitro, thalamic neurons grew more neurites on HB-GAM than on laminin. The HB-GAM-induced neurite outgrowth in thalamic neurons was inhibited by heparitinase, heparin, soluble N-syndecan and by an excess of soluble HB-GAM in the culture medium. In a pathway assay, thalamic neurons selectively preferred attaching and growing neurites on matrices containing HB-GAM than on those containing fibronectin or laminin alone, suggesting that HB-GAM may modulate the effect of other ECM proteins. On an unfixed brain slice preparation, thalamic neurons repeatedly showed a typical neurite outgrowth and attachment pattern resembling the expression pattern of HB-GAM. On the brain slices, the neurite outgrowth was significantly inhibited by heparitinase, heparin and soluble HB-GAM, thus displaying features of neurite outgrowth on matrix-bound HB-GAM. Our results suggest that HB-GAM is important for the neurite outgrowth of thalamic neurons and it may function as an ECM-bound guidance cue for thalamic neurons that possess HB-GAM-binding heparan sulphates on their cell membrane.

Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM).

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell- matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM-induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

N-syndecan and HB-GAM (heparin-binding growth-associated molecule) associate with early axonal tracts in the rat brain.

Heparin-Binding Growth-Associated Molecule (HB-GAM)/pleiotrophin is an 18 kDa extracellular matrix- and cell-surface-associated protein shown to enhance neurite outgrowth of perinatal forebrain neurones in vitro. The heparan sulphate proteoglycan N-syndecan (Raulo et al., 1994) has been isolated as a receptor/coreceptor for the HB-GAM. We have investigated, whether HB-GAM and N-syndecan could have a similar role in neurite outgrowth and axon guidance in early axonal tracts of brain. In the present study N-syndecan was found to be spatiotemporally associated with the developing axonal tracts already on embryonic day 9 in rat, as revealed by coexpression with class III beta-tubulin, which is one of the earliest neuronal markers (Easter et al., 1993; Brittis et al., 1995). Later, N-syndecan and HB-GAM were detected in the first afferent serotonergic projections arising from the pontine raphe nuclei. The expression pattern of HB-GAM peaked in the developing rhombencephalon at embryonic stage (E) 13-14. At the same time, N-syndecan was expressed in the developing raphe neurones growing neurites towards the diencephalon along HB-GAM immunoreactive pathways. When rhombencephalic neurones were cultured on decreasing concentrations of substrate-bound HB-GAM, E13 neurones showed a significantly better neurite outgrowth response than E11, E16 or E18 neurones. The neurite outgrowth of raphe neurones in vitro was inhibited by adding soluble heparin or N-syndecan into the culture medium, whereas addition of chondroitin sulphate had no effect. In a simple pathway assay, E13 raphe neurones selectively preferred attaching and growing neurites on pathways containing HB-GAM as compared with regions containing either laminin or fibronectin alone. Our results suggest that HB-GAM may function as a developmentally regulated cue for rhombencephalic neurones that possess N-syndecan on their cell membrane.

Regulation of mRNA localization by transmembrane signalling: local interaction of HB-GAM (heparin-binding growth-associated molecule) with the cell surface localizes beta-actin mRNA.

Localization of mRNAs is currently thought to be partially responsible for molecular sorting to specific compartments within the cell. In mammalian cells the best-studied example is the beta-actin mRNA that is localized to the cell processes, and its localization is necessary in migratory responses of cells. It is reasonable to assume that mRNA localization within cells is coupled to transmembrane signalling due to extracellular factors, but little is known about such putative mechanisms. We show here that HB-GAM, an extracellular matrix-associated factor that enhances migratory responses in cells, is able to localize beta-actin mRNA when locally applied to cells via microbeads. The HB-GAM-induced mRNA localization is specifically inhibited by low concentrations of heparin and by heparitinase treatment of cells, showing that cell-surface heparin-type glycans are required for the effect. The finding that soluble N-syndecan is also inhibitory suggests that the transmembrane proteoglycan N-syndecan, previously identified as an HB-GAM receptor, is involved in the mRNA-localizing effect of HB-GAM. Inhibition of the mRNA localization by the src-kinase inhibitor PP1 is compatible with an N-syndecan-mediated effect since the receptor function of N-syndecan has been recently found to depend on the src-kinase signalling pathway. The mRNA-localizing activity of N-syndecan is also suggested by the finding that affinity-purified anti-N-syndecan antibodies coated on microbeads are able to localize beta-actin mRNA.

Cortactin-Src kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth.

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway.

Developmentally regulated neurite outgrowth response from dorsal root ganglion neurons to heparin-binding growth-associated molecule (HB-GAM) and the expression of HB-GAM in the targets of the developing dorsal root ganglion neurites.

Heparin-binding growth-associated molecule (HB-GAM) is a highly conserved cell surface- and extracellular matrix-associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB-GAM. We discovered that the neurite outgrowth response to HB-GAM is maximal at E5.5-6.5 (St 28-30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB-GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB-GAM protein expression there is at its peak. Though HB-GAM is a secreted protein, at the RNA level the timing of HB-GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate-bound HB-GAM in a developmentally regulated manner, and that the expression of both HB-GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB-GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.

Co-expression of heparin-binding growth-associated molecule (HB-GAM) and N-syndecan (syndecan-3) in developing rat brain.

Biochemical and cell biological studies have previously identified N-syndecan as a neuronal cell surface receptor in neurite outgrowth induced by heparin-binding growth-associated molecule (HB-GAM). In the present study we have compared temporal and spatial expression patterns of N-syndecan and HB-GAM using Northern and Western blotting and immunohistochemistry. Expression of N-syndecan mRNA and protein peaks during the perinatal developmental stage of the brain in the same manner as the expression of HB-GAM mRNA and protein. In addition, both proteins are preferentially localized to fiber tracts of developing brain. We suggest that HB-GAM and N-syndecan form ligand-receptor complexes in developing axon tracts of brain.