Proteome analysis of actin filament-associated proteins in the postnatal rat cerebellum

Abstract

The cerebellum contains more neurons than all other brain regions combined and these cells exhibit complex circuit development and dendritic elaboration during the postnatal period. Neural development, cellular morphogenesis, and synaptic plasticity are dependent on the dynamic regulation of the actin cytoskeleton by actin-binding proteins. The identification of the actin filament interactome, including proteins developmentally regulated in the postnatal cerebellum, could help define important regulators of actin cytoskeletal dynamics in developing cerebellar neurons. Affinity purification of cerebellar proteins on F-actin columns, combined with mass spectrometry, in total, 434 actin filament-associated proteins in postnatal rat cerebellum (P7) were identified. Furthermore, semi-quantitative RT-PCR was performed to screening postnatal developmentally regulated genes involved in actin dynamics and membrane trafficking in rat cerebellum (P0–P56). As the result, nine genes encoding members of the cerebellar F-actin interactome were developmentally regulated in the transcriptional level and at least five of them exhibited a similar pattern at the protein expression level by Western blot analysis. Further fluorescent immunohistochemical observations demonstrated that the actin-associated proteins Lethal(2) giant larvae protein homolog 1 (LLGL1) and metastasis suppressor 1 (MTSS1) were specifically upregulated in granule neurons and Purkinje cells during morphogenesis of axons and dendrites. This work defines a provisional actin filament interactome in rat postnatal cerebellum and identifies several candidate proteins that may be involved in the postnatal development of the cerebellum.