title: The 270 kDa splice variant of erythrocyte beta-spectrin (beta I sigma 2) segregates in vivo and in vitro to specific domains of cerebellar neurons.
abstract: Spectrin isoforms arise from four distinct genes, three of which generate multiple alternative transcripts. With no biochemical restrictions on the assembly of alpha beta heterodimers, more than 25 distinct heterodimeric spectrin species may exist. Whether (and why) this subtle but substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively spliced regions of alpha- and beta-spectrin have been prepared. Reported here is the localization in rat cerebellar neurons at light and electron microscopic levels of an antibody against a unique sequence (beta I sigma 2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell beta-spectrin gene (spectrin beta I sigma 2). In this version, the 3' sequence of erythroid beta-spectrin (beta I sigma 1) is replaced with an alternative sequence that shares substantial homology with the 3' sequence of non-erythroid beta-spectrin (beta II sigma 1). The antibody to beta I sigma 2-A stains a single protein band at 270 kDa, determined by western blotting, in both rat cerebellum and in cultured cerebellar granule cells, and does not react with beta II sigma 1 spectrin (beta-fodrin). This antibody stains the dendritic spines of Purkinje cells in the molecular layer, and is concentrated at postsynaptic densities (PSDs) adjacent to synapsin I (which is confined to the presynaptic membrane). The soma of Purkinje cells do not stain. In the granular layer, cytoplasmic organelles and the postsynaptic densities of granular cells stain strongly. Astrocytes are also stained. In all cells, plasma membrane staining is confined to postsynaptic densities (PSD). The beta I sigma 2 isoform co-immunoprecipitates with non-erythroid alpha-spectrin (alpha II sigma), even though the distribution of alpha II sigma within neurons only partially overlaps that of beta I sigma 2. No hybrid beta I sigma 2 and beta II sigma 1 (beta-fodrin) spectrin complexes appear to exist. Spectrin beta I sigma 2 is also polarized in cultured rat cerebellar granule cells, where it is abundant in cell bodies but not neurites. The overall distribution of beta I sigma 2 is as a subset of the distribution of spectrins 240/235E previously detected with a generally reactive erythrocyte alpha beta-spectrin antibody. These findings establish the highly precise segregation of a beta-spectrin isoform to distinct cytoplasmic and membrane surface domains, indicate that it is complexed (partially) with non-erythroid alpha-spectrin, and demonstrate that cytoskeletal targeting mechanisms are preserved in cultured granular cells.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Laboratory of Ultrastructure, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Malchiodi-Albedi
authors.forename: M
authors.initials: M
authors.lastname: Ceccarini
authors.forename: J C
authors.initials: JC
authors.lastname: Winkelmann
authors.forename: J S
authors.initials: JS
authors.lastname: Morrow
authors.forename: T C
authors.initials: TC
authors.lastname: Petrucci
mesh_terms: D000595:Amino Acid Sequence; D001253:Astrocytes / Q000737:chemistry / Q000648:ultrastructure*; D002525:Cerebellar Cortex / Q000166:cytology*; D004912:Erythrocytes / Q000737:chemistry*; D008869:Microtubule-Associated Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure*; D011689:Purkinje Cells / Q000737:chemistry; D012326:RNA Splicing; D013049:Spectrin / Q000235:genetics / Q000378:metabolism*; D013569:Synapses / Q000737:chemistry; D016704:Synapsins / Q000032:analysis
chemical_list: D008869:Microtubule-Associated Proteins; D016704:Synapsins; D013049:Spectrin