Organization of Postsynaptic Proteins via the Neuronal Cytoskeleton
Allison, Daniel Ward
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https://hdl.handle.net/2142/86757
Description
Title
Organization of Postsynaptic Proteins via the Neuronal Cytoskeleton
Author(s)
Allison, Daniel Ward
Issue Date
2000
Doctoral Committee Chair(s)
Ann Marie Craig
Vladimir I. Gelfand
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Cell
Language
eng
Abstract
"The mechanisms responsible for anchoring molecular components of postsynaptic specializations in the mammalian brain are not well understood but are presumed to involve associations with cytoskeletal elements. We used cytoskeletal perturbing agents and detergent extraction of primary hippocampal cultures to test directly the role of the cytoskeleton in localizing neurotransmitter receptors, and potential anchoring and signal transducing proteins at postsynaptic sites. Neurons were treated with latrunculin A to depolymerize actin, vincristine to depolymerize microtubules, or Triton X-100 to extract soluble proteins. Depolymerization of F-actin led to a decrease in both the number of synaptic NMDAR1 clusters and the number of GluR1-labeled spines. On GABAergic neurons, GluR1 clusters redistributed to non-synaptic sites, whereas on pyramidal neurons the clusters appeared to disperse. Furthermore, in control neurons AMPA receptors were extractable from pyramidal cell spines, whereas AMPA receptors on GABAergic neurons were unextractable. The synaptic clustering of PSD-95, a putative NMDA receptor anchoring protein and a core component of the postsynaptic density (PSD), was unaffected by actin depolymerization, microtubule depolymerization, or detergent extraction. The same was largely true for GKAP, a PSD-95 interacting protein. In contrast, the synaptic clustering of CaMKIIalpha, another core component of the PSD, was completely dependent upon an intact actin cytoskeleton and was partially disrupted by detergent. Drebrin and alpha-actinin-2, actin-binding proteins concentrated in spines, were dependent upon F-actin for synaptic localization, but were unaffected by detergent extraction. Surprisingly, the subcellular distributions of the inhibitory synaptic proteins GABAAR and gephyrin, which has a tubulin binding motif, were unaffected by depolymerization of the cytoskeleton, or detergent extraction. These studies reveal an unsuspected heterogeneity in the modes of attachment of postsynaptic proteins to the cytoskeleton, supporting the idea that PSD-95 and gephyrin may be core scaffolding components independent of the actin or tubulin cytoskeleton. This leads to a model in which there are two structural entities within the PSD, one actin-dependent ""regulatory"" complex as well as a core complex responsible for the organization of the PSD, independent of actin filaments. We have also demonstrated that microtubules do not play a major role in the localization of postsynaptic proteins to the PSD."
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