use-phluorin-to-assess-dynamics-axon-guidance-receptors-cell-culture

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

University of Lyon

Cell adhesion molecules of the immunoglobulin superfamily promote cell aggregation and neurite outgrowth via homophilic and heterophilic interactions. The transient axonal glycoprotein TAG-1 induces cell aggregation through homophilic interaction of its fibronectin repeats. We investigated the domains responsible for the neurite outgrowth promoting activity of TAG-1 as well as its interactions with other cell adhesion molecules. Binding experiments with Fc-chimeric proteins revealed that TAG-1 interacts with L1, NrCAM, and F3/contactin. The membrane-associated as opposed to the soluble form of TAG-1 behaves differently in these assays. We demonstrate that both the immunoglobulin as well as the fibronectin domains promote neurite outgrowth when used as substrates. Furthermore we investigated the putative role of L1 and NrCAM as the neuronal TAG-1 receptors mediating neurite extension. DRG neurons from L1-deficient mice were found to extend neurites on TAG-1 substrates and blocking NrCAM function did not diminish the TAG-1-dependent neurite outgrowth. These results indicate that neither L1 nor NrCAM are required for TAG-1-elicited neurite outgrowth.

Analysis of interactions of the adhesion molecule TAG-1 and its domains with other immunoglobulin superfamily members.

A general feature of the cell adhesion molecules belonging to the immunoglobulin family (Ig-CAMs) is to display a modular structure that provides a framework for multiple binding sites for other recognition molecules. Among this family, F3/contactin is a glycan phosphatidyl-inositol (GPI)-anchored molecule expressed by neurons that displays the distinctiveness to exert heterophilic but no homophilic binding activities. The Ig domains of F3/contactin were shown to interact with the L1 family of Ig-CAMs, including L1, NrCAM, and neurofascin. Binding between F3/contactin and NrCAM is known to modulate axonal elongation of the cerebellar granule cells and to control sensory axon guidance. F3/contactin mediates neuron-glial contacts through its association with extracellular matrix components (tenascin-R, tenascin-C) and RPTPbeta/phosphacan, influencing axonal growth and fasciculation. Another major role of F3/contactin is to organize axonal subdomains at the node of Ranvier of myelinated fibers in interplay with other Ig-CAMs, through its binding with caspr/paranodin at paranodes and the voltage-gated sodium channels in the nodal region. The F3/contactin deficient mice display a severe ataxia correlated with defects in axonal and dendritic projections in the cerebellum. These mice also display defects in nerve influx conduction due to the disruption of the axo-glial contacts at paranodes. Finally, the recent identification of a Drosophila homologue of F3/contactin indicated that this family of GPI-anchored CAMs plays a conserved function in axonal insulation.

F3/contactin, a neuronal cell adhesion molecule implicated in axogenesis and myelination.

During axon navigation, Semaphorin3A-induced growth cone retraction is correlated with endocytosis. Although its function remains elusive, we showed previously that the cell adhesion molecule of the immunoglobulin super family L1 associates with Neuropilin-1 (NP-1) the Sema3A-binding subunit of the receptor complex and is required for Sema3A to elicit axonal repulsive responses. We report here that upon Sema3A binding to NP-1, L1 and NP-1 are co-internalized through a clathrin-dependent mechanism mediated by L1. We show that in COS7 cells, L1/NP-1 endocytosis is correlated with a cell contraction similar to that observed with the Plexin (Plex)/NP-1 or Plex/NP1/L1 complexes. In neuronal cultures, a L1-mimetic peptide able to switch Sema3A repulsive responses to attraction blocks both endocytosis and growth cone collapse. Similarly, in the COS7 cell model, peptide application prevents both the Sema3-induced L1/NP-1 internalization and cell collapse. These studies demonstrate that the L1/NP-1 complex is able to confer a biological response to Sema3A with L1 mediating receptor internalization following ligand activation. They also reveal that endocytosis controlled by L1/NP-1 cis and trans interactions is pivotal in Sema3A-mediated axon guidance.

Semaphorin3A-induced receptor endocytosis during axon guidance responses is mediated by L1 CAM.

NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-beta-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.

NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts.

Neurone glial-related cell adhesion molecule (NrCAM) is a member of the L1 family of transmembrane cell adhesion receptors which are involved in the development and function of the mammalian nervous system. How these receptors interact with intracellular signalling pathways is not understood. To date the only identified binding partner to the cytoplasmic terminus of NrCAM is ankyrin G. We screened a developing rat brain cDNA yeast two-hybrid library with the cytoplasmic domain of NrCAM to identify further intracellular binding partners. We identified synapse associated protein 102 (SAP102) as a new binding partner for NrCAM. The interaction was confirmed biochemically using glutathione S-transferase (GST)-pull-down and tandem affinity purification, and also immunocytochemically as NrCAM and SAP102 co-localized in COS-7 and cerebellar granule cells. Binding was specific to NrCAM as neither neurofascin nor L1 bound SAP102, and this interaction was reliant on the last three amino acids of NrCAM. Additionally, NrCAM constructs whose last three amino acids had been deleted appeared to have a dominant negative effect on neurite extension of cerebellar granule cells. This is the first interaction reported for NrCAM, and its association with SAP102 suggests that it is part of a larger complex which can interact with many different signalling pathways.

Synapse associated protein 102 is a novel binding partner to the cytoplasmic terminus of neurone-glial related cell adhesion molecule.

To assess if membrane diffusion could affect the kinetics of receptor recruitment at adhesive contacts, we transfected neurons with green fluorescent protein-tagged immunoglobin cell adhesion molecules of varying length (25-180 kD), and measured the lateral mobility of single quantum dots bound to those receptors at the cell surface. The diffusion coefficient varied within a physiological range (0.1-0.5 microm(2)/s), and was inversely proportional to the size of the receptor. We then triggered adhesive contact formation by placing anti-green fluorescent protein-coated microspheres on growth cones using optical tweezers, and measured surface receptor recruitment around microspheres by time-lapse fluorescence imaging. The accumulation rate was rather insensitive to the type of receptor, suggesting that the long-range membrane diffusion of immunoglobin cell adhesion molecules is not a limiting step in the initiation of neuronal contacts.

Weak effect of membrane diffusion on the rate of receptor accumulation at adhesive contacts.

Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this activity to axon pathfinding is still unclear. Using genetic and culture models, we provide evidence that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, we show that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue.

Dual functional activity of semaphorin 3B is required for positioning the anterior commissure.

Semaphorins are major chemorepellents for developing neuronal projections. Their persistent expression at adult stages suggests that they may contribute to the functioning of neuronal circuits. We investigated the functional properties of semaphorin3A (Sema3A) in adult hippocampal neurons, and report that exogenous application of this cue decreases the efficacy of synaptic transmission evoked in the CA1 region of hippocampal slices. In situ hybridization, imaging and biochemical techniques showed that the Sema3A receptor component neuropilin-1 is present at hippocampal synapses and localizes in the presynaptic membrane. In differentiated cultured hippocampal neurons, Sema3A elicited Erk1/2 phosphorylation in somata and neuritic compartments. Furthermore, Sema3A application resulted in a striking reduction of synaptophysin and postsynaptic density 95 puncta without affecting the axon diameter. These observations reveal novel functional potentialities for secreted semaphorins, which suggest that these cues could modulate the morphology and function of synapses in the adult brain.

Semaphorin3A regulates synaptic function of differentiated hippocampal neurons.

During axon navigation, growth cones continuously interact with molecular cues in their environment, some of which control adherence and bundle assembly, others axon elongation and direction. Growth cone responses to these different environmental cues are tightly coordinated during the development of neuronal projections. Several recent studies show that axon sensitivity to guidance cues is modulated by extracellular and intracellular signals. This regulation may enable different classes of cues to combine their effects and may also represent important means for diversifying pathway choices and for compensating for the limited number of guidance cues. This chapter focuses on the modulation exerted by Ig Super-family cell adhesion molecules (IgSFCAMs) on guidance cues of the class III secreted semaphorins.

Modulation of semaphorin signaling by Ig superfamily cell adhesion molecules.

Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes.

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.

Sphingosine 1-phosphate (S1P), a lysophospholipid, plays an important chemotactic role in the migration of lymphocytes and germ cells, and is known to regulate aspects of central nervous system development such as neurogenesis and neuronal migration. Its role in axon guidance, however, has not been examined. We show that sphingosine kinase 1, an enzyme that generates S1P, is expressed in areas surrounding the Xenopus retinal axon pathway, and that gain or loss of S1P function in vivo causes errors in axon navigation. Chemotropic assays reveal that S1P elicits fast repulsive responses in retinal growth cones. These responses require heparan sulfate, are sensitive to inhibitors of proteasomal degradation, and involve RhoA and LIM kinase activation. Together, the data identify downstream components that mediate S1P-induced growth cone responses and implicate S1P signalling in axon guidance.

A role for S1P signalling in axon guidance in the Xenopus visual system.

Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4⁻/⁻ NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.

Wnt4 participates in the formation of vertebrate neuromuscular junction.

The Neurotrophic factor gdnf plays diverse developmental roles, supporting survival and also acting as a chemoattractant for axon and cell migration. We report that in the developing spinal cord, a focal source of gdnf is present in the floor plate (FP) where commissural axons cross the midline. Gdnf has no direct guidance properties but switches on the responsiveness of crossing commissural growth cones to the midline repellent Semaphorin3B by suppressing calpain-mediated processing of the Sema3B signaling coreceptor Plexin-A1. Analysis of single and double mutant mouse models indicates that although gdnf is the principal trigger of Sema3B midline repulsion, it acts with another FP cue, NrCAM. Finally, genetic and in vitro experiments provide evidence that this gdnf effect is RET independent and mediated by NCAM/GFRα1 signaling. This study identifies a regulator of midline crossing and reveals interplays between Semaphorin and gdnf signaling during axon guidance.

Julien Falk

University Claude Bernard Lyon1,
CGphiMC UMR CNRS 5534,
University Claude Bernard Lyon1, CGphiMC UMR CNRS 5534

University of Lyon

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Julien Falk

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}University Claude Bernard Lyon1,CGphiMC UMR CNRS 5534,University Claude Bernard Lyon1, CGphiMC UMR CNRS 5534

University of Lyon

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

University Claude Bernard Lyon1,
CGphiMC UMR CNRS 5534,
University Claude Bernard Lyon1, CGphiMC UMR CNRS 5534