bead-aggregation-assays-for-characterization-putative-cell-adhesion

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Ohio State University

N-cadherin is a prominent component of developing and mature synapses, yet very little is known about its trafficking within neurons. To investigate N-cadherin dynamics in developing axons, we used in vivo two-photon time-lapse microscopy of N-cadherin--green fluorescent protein (Ncad-GFP), which was expressed in Rohon-Beard neurons of the embryonic zebrafish spinal cord. Ncad-GFP was present as either stable accumulations or highly mobile transport packets. The mobile transport packets were of two types: tubulovesicular structures that moved preferentially in the anterograde direction and discrete-punctate structures that exhibited bidirectional movement. Stable puncta of Ncad-GFP accumulated in the wake of the growth cone with a time course. Colocalization of Ncad-GFP puncta with synaptic markers suggests that N-cadherin is a very early component of nascent synapses. Expression of deletion mutants revealed a potential role of the extracellular domain in appropriate N-cadherin trafficking and targeting. These results are the first to characterize the trafficking of a synaptic cell-adhesion molecule in developing axons in vivo. In addition, we have begun to investigate the cell biology of N-cadherin trafficking and targeting in the context of an intact vertebrate embryo.

In vivo trafficking and targeting of N-cadherin to nascent presynaptic terminals.

The abundance of synaptic connectivity in the cultured hippocampal slice preparation allows measurements of the unitary excitatory connection between pairs of pyramidal neurons using simultaneous presynaptic and postsynaptic intracellular recordings. However, the useful yield of these recordings can be greatly reduced by the presence of polysynaptic inhibition that occludes the measurement of the monosynaptic excitatory postsynaptic current (EPSC). We have found that the traditional method of eliminating contaminating synaptic inhibition with GABA receptor antagonists is of limited usefulness because the recurrent excitatory connections in organotypic slices cause epileptiform bursting in the absence of inhibitory function. This bursting obscures EPSCs to an even greater extent than the normally occurring polysynaptic inhibitory transmission. Here, we report a new method for isolating monosynaptic EPSCs using the mu-opioid agonist peptide DAMGO to reduce polysynaptic inhibition during these recordings. Activation of mu-opioid receptors is known to hyperpolarize inhibitory neurons. We found that DAMGO application reduces the amplitude and frequency of polysynaptic inhibition, allowing isolation of the excitatory connection between the two neurons being recorded. Furthermore, because inhibitory function is not completely eliminated by DAMGO application, epileptiform bursting very rarely develops. Therefore, the use of DAMGO to prevent polysynaptic inhibition without causing epileptiform bursting provides a useful tool to substantially increase the yield of experiments measuring the unitary excitatory connection between pyramidal neurons in the cultured hippocampal slice preparation.

Blocking polysynaptic inhibition via opioid receptor activation isolates excitatory synaptic currents without triggering epileptiform activity in organotypic hippocampal slices.

The pcdhalpha/CNR gene comprises a diverse array of neuronal cell-surface proteins of the cadherin superfamily, although very little is known about their role in neural development. Here we provide the first in-depth characterization of pcdh1alpha in zebrafish. Whole-mount immunocytochemistry demonstrates that a large proportion of endogenous cytoplasmic domain immunoreactivity is present in the nucleus, suggesting that endoproteolytic cleavage and nuclear translocation of the intracellular domain are important aspects of pcdh1alpha activity in vivo. Using whole-mount immunocytochemistry and BAC-based expression of Pcdh1alpha-GFP fusion proteins, we find that Pcdh1alpha does not appear to form stable, synaptic puncta at early stages of synaptogenesis. We also demonstrate that the presence of the Pcdh1alpha cytoplasmic domain is essential for normal function. Truncation of Pcdh1alpha proteins, using splice-blocking antisense morpholinos to prevent the addition of the common intracellular domain to the entire pcdh1alpha cluster, results in neuronal apoptosis throughout the developing brain and spinal cord, demonstrating an essential role for pcdh1alpha in early neural development. This cell death phenotype can be attenuated by the expression of a soluble Pcdh1alpha cytoplasmic domain.

Inhibition of protocadherin-alpha function results in neuronal death in the developing zebrafish.

One of the earliest stages of brain morphogenesis is the establishment of the neural tube during neurulation. While some of the cellular mechanisms responsible for neurulation have been described in a number of vertebrate species, the underlying molecular processes are not fully understood. We have identified the zebrafish homolog of protocadherin-19, a member of the cadherin superfamily, which is expressed in the anterior neural plate and is required for brain morphogenesis. Interference with Protocadherin-19 function with antisense morpholino oligonucleotides leads to a severe disruption in early brain morphogenesis. Despite these pronounced effects on neurulation, axial patterning of the neural tube appears normal, as assessed by in situ hybridization for otx2, pax2.1 and krox20. Characterization of embryos early in development by in vivo 2-photon timelapse microscopy reveals that the observed disruption of morphogenesis results from an arrest of cell convergence in the anterior neural plate. These results provide the first functional data for protocadherin-19, demonstrating an essential role in early brain development.

Protocadherin-19 is essential for early steps in brain morphogenesis.

Many synapses undergo immediate and persistent activity-dependent changes in strength via processes that fall under the umbrella of synaptic plasticity. It is known that this type of synaptic plasticity exhibits an underlying state dependence; that is, as synapses change in strength they move into distinct 'states' that are defined by the mechanism and ability to undergo future plasticity. In this study, we have investigated the molecular mechanisms that underlie state-dependent synaptic plasticity. Using intracellular application of peptides that mimic the C-terminal tail sequences of GluR1 and GluR2 AMPA receptor subtypes, combined with paired recordings of minimal synaptic connections, we have shown that AMPA receptor subtypes present in the membrane at a given time confer some properties of plasticity states. These data show that during synaptic plasticity, AMPA receptor subtypes are differentially stabilized by postsynaptic density proteins in or out of the postsynaptic membrane, and this differential synaptic expression of different AMPA receptor subtypes defines distinct synaptic states.

AMPA receptor subunits define properties of state-dependent synaptic plasticity.

The protocadherins comprise the largest subgroup within the cadherin superfamily, yet their cellular and developmental functions are not well understood. In this study, we demonstrate that pcdh 19 (protocadherin 19) acts synergistically with n-cadherin (ncad) during anterior neurulation in zebrafish. In addition, Pcdh 19 and Ncad interact directly, forming a protein-protein complex both in vitro and in vivo. Although both molecules are required for calcium-dependent adhesion in a zebrafish cell line, the extracellular domain of Pcdh 19 does not exhibit adhesive activity, suggesting that the involvement of Pcdh 19 in cell adhesion is indirect. Quantitative analysis of in vivo two-photon time-lapse image sequences reveals that loss of either pcdh 19 or ncad impairs cell movements during neurulation, disrupting both the directedness of cell movements and the coherence of movements among neighboring cells. Our results suggest that Pcdh 19 and Ncad function together to regulate cell adhesion and to mediate morphogenetic movements during brain development.

Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation.

During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.

A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion.

The embryonic zebrafish is a nearly ideal model system in which to use time-lapse imaging to study the development of the vertebrate nervous system in vivo. The embryos are small and transparent, they develop externally and rapidly, and the embryonic central nervous system is relatively simple and highly stereotyped. With the refinement of green fluorescent protein (GFP) as a genetically encoded fluorescent tag of neuronal proteins, along with advances in imaging technology, it is possible to follow the cellular and molecular events underlying development as they occur in the living embryo. This article describes strategies for imaging synapse formation in the embryonic zebrafish.

In vivo imaging of synaptogenesis in zebrafish.

The embryonic zebrafish is a nearly ideal model system in which to use time-lapse imaging to study the development of the vertebrate nervous system in vivo. The embryos are small and transparent, they develop externally and rapidly, and the embryonic central nervous system is relatively simple and highly stereotyped. With the refinement of green fluorescent protein (GFP) as a genetically encoded fluorescent tag of neuronal proteins, along with advances in imaging technology, it is possible to follow the cellular and molecular events underlying development as they occur in the living embryo. This protocol describes methods for imaging synapse formation in embryonic zebrafish. Injection of DNA into early embryos is followed by mounting of the transgenic embryos in agarose and then time-lapse data collection.

Fluorescence imaging of transgenic zebrafish embryos.

The proper assembly of neural circuits during development requires the precise control of axon outgrowth, guidance, and arborization. Although the protocadherin family of cell surface receptors is widely hypothesized to participate in neural circuit assembly, their specific roles in neuronal development remain largely unknown. Here we demonstrate that zebrafish pcdh18b is involved in regulating axon arborization in primary motoneurons. Although axon outgrowth and elongation appear normal, antisense morpholino knockdown of pcdh18b results in dose-dependent axon branching defects in caudal primary motoneurons. Cell transplantation experiments show that this effect is cell autonomous. Pcdh18b interacts with Nap1, a core component of the WAVE complex, through its intracellular domain, suggesting a role in the control of actin assembly. Like that of Pcdh18b, depletion of Nap1 results in reduced branching of motor axons. Time-lapse imaging and quantitative analysis of axon dynamics indicate that both Pcdh18b and Nap1 regulate axon arborization by affecting the density of filopodia along the shaft of the extending axon.

Article	Year
In vivo trafficking and targeting of N-cadherin to nascent presynaptic terminals. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 15483121	2004
Blocking polysynaptic inhibition via opioid receptor activation isolates excitatory synaptic currents without triggering epileptiform activity in organotypic hippocampal slices. Journal of neuroscience methods\| PubMed ID: 16081163	2006
Inhibition of protocadherin-alpha function results in neuronal death in the developing zebrafish. Developmental biology\| PubMed ID: 18602383	2008
Protocadherin-19 is essential for early steps in brain morphogenesis. Developmental biology\| PubMed ID: 19615992	2009
AMPA receptor subunits define properties of state-dependent synaptic plasticity. The Journal of physiology\| PubMed ID: 20351044	2010
Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation. The Journal of cell biology\| PubMed ID: 21115806	2010
A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion. The Journal of cell biology\| PubMed ID: 22184198	2011
In vivo imaging of synaptogenesis in zebrafish. Cold Spring Harbor protocols\| PubMed ID: 22550294	2012
Fluorescence imaging of transgenic zebrafish embryos. Cold Spring Harbor protocols\| PubMed ID: 22550295	2012
Protocadherin-18b interacts with Nap1 to control motor axon growth and arborization in zebrafish. Molecular biology of the cell\| PubMed ID: 24371087	2014

Michelle R. Emond

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