Research reportRole of actin in the organisation of brain postsynaptic densities
Introduction
The postsynaptic density (PSD) is a disc-shaped proteinaceous structure that is embedded in the postsynaptic membrane at synaptic junctions in the brain 13, 14, 27. In the developing brain the appearance of morphologically distinct junctions between the pre- and post-synaptic membranes coincides with the deposition of PSD material [17], suggesting that one of the functions of the PSD is to define the site and extent of junctional contacts. Morphologically intact PSDs can be isolated from adult brain by dissolving synaptic plasma membranes in detergents, indicating that their structural integrity is intrinsically determined 7, 8, 34. Such preparations contain high concentrations of neurotransmitter receptors, suggesting that a further function of the PSD is to maintain receptors at the postsynaptic site 9, 25, 35. The close association of PSDs with neurotransmitter receptors provides a useful reference point for the analysis of their molecular structure. The investigation of the postsynaptic structure of central synapses has proved problematic because isolated PSDs contain a complex mixture of proteins 1, 7, 26, 33making it difficult to identify the major structural components. One that has been widely considered for this role is a 51 kDa protein originally described as the major PSD protein 8, 19, and subsequently identified as the α subunit of Ca2+/calmodulin-dependent protein kinase II (CaMK IIα) 12, 20, 21. A second prominent component that might also be involved is actin 4, 19, 24. The exact relationship of these proteins to the PSD structure has yet to be determined.
The relationship between neurotransmitter receptors and the postsynaptic site is far better understood at the neuromuscular junction, where the clustering of nicotinic acetylcholine receptors (AChR) involves several postsynaptic proteins, including a 43 kDa component 11, 29, cytoplasmic actin 2, 15, and β-spectrin 2, 3. Removal of actin and β-spectrin from myotube membranes results in the dispersal of AChR into microclusters, implying a role for actin cytoskeletal proteins in the overall organisation of neurotransmitter receptor proteins at the postsynaptic site 2, 10. Since, in addition to actin, spectrin is also present in isolated brain PSDs 5, 36, it is possible that these molecules also play a significant role in maintaining neurotransmitter receptors at postsynaptic junctional sites in the brain. An important step in the characterisation of receptor-related proteins at the neuromuscular junction was their selective extraction from synapse-rich membrane preparations (see review by Froehner [10]). We have applied this approach to brain PSDs treating them with LIS. After removal of the entire actin content of brain PSDs by LIS extraction [30]the excitatory and inhibitory receptors and CaMK IIα remained associated with PSD. Although PSDs were fragmented by this treatment their fundamental morphological structure, consisting of a lattice-like network — the postsynaptic junctional lattice remained intact. These findings suggest that, as with the organisation of receptor microclusters at the neuromuscular junction, actin may operate to organise postsynaptic junctional lattice components into the PSD structure at central synapses.
Section snippets
Subcellular fractionation
Synaptosomal plasma membranes (SPM) were prepared from pig forebrain by combined flotation–sedimentation density gradient centrifugation [18]. PSD fractions were isolated from them as described by Matus and Taff-Jones [26]except that after treatment of the SPM fraction with 2% deoxycholate the detergent insoluble material was collected by centrifugation through a 35% sucrose cushion at 60 000×g for 2 h as suggested by Cohen and colleagues [7]. Protein concentrations were determined by using a
Differential extraction of cytoskeletal proteins from isolated PSDs
Treatment of PSDs with 20 mM LIS selectively extracted a small number of proteins, the most prominent of which was identified as actin on the basis of its apparent molecular mass (ca. 45 kDa; Fig. 1A) and its reaction with an anti-actin antibody on immunoblots (Fig. 1B). This is in agreement with previous evidence identifying this component of PSDs as actin 4, 24. All but trace amounts of PSD actin were solubilized by LIS treatment (Fig. 1B). Another prominent protein in the LIS-extractable
Discussion
The PSD is a prominent feature of the majority of forebrain synaptic junctions. Its coincidence with the junctional area of the postsynaptic membrane, its close correlation with nascent junctions in the developing brain and its survival as an independent structure containing high concentrations of neurotransmitter receptors after the solubilisation of synaptic plasma membranes all suggest that it is the key organising structure in determining the site and extent of synaptic contacts. Despite
Acknowledgements
We thank the following people for generously providing antibodies: Anne Stephenson (NMDA-R1 glutamate receptor), Peter Streit (Glu-RB glutamate receptor), Grayson Richards (GABAA receptor), Ute Groeschel-Stewart (actin), Beat Riederer (spectrin). We also thank Andreas Hefti and Markus Dürrenberger for assistance with the electron microsopy.
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