Introduction

Brain function hinges on the clustering of appropriate neurotransmitter receptors and associated signaling molecules at the postsynaptic membrane in perfect opposition to the presynaptic active zone where the release of a specific neurotransmitter occurs. Amazingly, this organization occurs along the dendrites, long distances away from the cell body where proteins are synthesized. Understanding how neurons organize this intricate network of synapses formed in early development is integral to understanding central nervous system function. In general, the formation of a synapse is thought to occur in three successive steps beginning with the synthesis of new proteins in the soma, followed by protein sorting and trafficking to axons and dendrites, and then by their assembly at nascent neuronal contacts (Figure 4.1 A). At postsynaptic sites, the intense clustering of neurotransmitter

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Figure 4.1. Protein Complexes at Excitatory Synapses. (A) An image of a neuron stained with the presynaptic marker synaptophysin (green), to identify synaptic contacts. This panel illustrates steps involved in the assembly of proteins at contact sites. Synapse formation is generally thought to involve three basic steps which include production of proteins in the cell soma (A-1), transport of these proteins to early sites of contact between axons and dendrites (A-2), and assembly of protein complexes at synapses (A-3). (B) The intense clustering of proteins seen at the PSD of excitatory synapses is highlighted in the electron micrograph shown in (B). A schematic diagram of this region is blown up in, illustrating the role of scaffolding molecules such as PSD-95 in assembly of large protein complexes. PSD-95 forms the core of the protein network, which is associated with the membrane through palmitoylation, and anchored within the postsynaptic compartment by several proteins that associate with actin. Coupling of PSD-95 to adhesion molecules such as neuroligins allows for trans-synaptic signaling. See Colorplate 4.

Figure 4.1. Protein Complexes at Excitatory Synapses. (A) An image of a neuron stained with the presynaptic marker synaptophysin (green), to identify synaptic contacts. This panel illustrates steps involved in the assembly of proteins at contact sites. Synapse formation is generally thought to involve three basic steps which include production of proteins in the cell soma (A-1), transport of these proteins to early sites of contact between axons and dendrites (A-2), and assembly of protein complexes at synapses (A-3). (B) The intense clustering of proteins seen at the PSD of excitatory synapses is highlighted in the electron micrograph shown in (B). A schematic diagram of this region is blown up in, illustrating the role of scaffolding molecules such as PSD-95 in assembly of large protein complexes. PSD-95 forms the core of the protein network, which is associated with the membrane through palmitoylation, and anchored within the postsynaptic compartment by several proteins that associate with actin. Coupling of PSD-95 to adhesion molecules such as neuroligins allows for trans-synaptic signaling. See Colorplate 4.

receptors, adhesion molecules, and signaling proteins at the postsynaptic density (PSD) can be clearly seen in electron micrographs of excitatory synapses (Figure 4.1B). Remarkably, the content and morphology of inhibitory postsynaptic sites is fundamentally different from that of excitatory contacts (see Chapter 19). A schematic diagram depicting some of the proteins identified at postsynaptic sites of excitatory synapses emphasizes the complexity and specificity of these structures (Figure 4.1). What drives clustering and assembly of diverse proteins at these specific locations? In this chapter we discuss some of the mechanisms implicated in protein trafficking, especially those driven by scaffolding molecules that induce protein clustering through multiple protein-protein interaction domains and protein multimerization.

Much of our understanding of protein targeting and clustering at synaptic sites has been gleaned from studies in cell culture. Cell culture systems have been used in scientific research for centuries, and have been the basis for the development of many biomedical advances such as vaccines1. Provided with the appropriate support, cells in culture display many of the properties of the differentiated cells from which they originated2. In general, cell culture systems can be divided into two categories, primary cell cultures that are prepared from fresh tissues and cell lines which can be split and repeatedly divide over long periods of time. Cell culture systems offer a well-defined, simplified homogenous system that avoids much of the inherent complexity in vivo and gains direct control over the environment of the cells. Another advantage of cultured cell systems is their receptiveness to gene transfer techniques. Gene transfer techniques (transfection) allow investigators to monitor and manipulate gene expression levels in cells, revealing information on the function of specific genes. The strength of this technique is amplified by the fusion of fluorescent probes or tags to the DNA before transfection, which allows for live imaging or immunohistochemical detection.

Although cultured cells provide a clean and powerful system for dissecting out mechanisms underlying protein assembly and contact formation, they may be unable to provide definitive answers about how synapses are formed in vivo. Because the natural surroundings of the cells have been removed, processes that depend on interaction with neighboring cells and the natural extracellular matrix may be altered in vitro. Unfortunately, along with the natural cellular environment in vivo comes inherent complexity. In vivo studies present more challenges for labeling, imaging, and manipulating individual cells amidst millions of others. However, as we discuss below, many of the techniques pioneered in vitro generated much of the current knowledge on how synapses assemble and cluster ion channels, cell adhesion molecules, and associated signaling and scaffolding proteins.

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