The 270 kDa splice variant of erythrocyte β-spectrin(βΙΣ2) segregates in vivo and in vitro to specific domains of cerebellar neurons (1)
Nervous tissue displays many spectrin isoforms. Two developmentally regulated spectrins have been identified in chicken and mouse brain. The basic unit of both is an αβ het- erodimer. The most widely distributed form, also known as fodrin is abundant during fetal development, and is largely confined to axons and presynaptic terminals in mature brain. A second form of spectrin also exists in brain and muscle tissue, based on cross-reactivity with antibodies to erythroid αβ-spectrin.
To prepare sequence-specific anti- bodies against βΙΣ2 spectrin, the synthetic peptide ‘βΙΣ2-A’ (Pro- Gly-Gln-His-Lys-Asp-Gly-Gln-Lys-Ser-Thr-Gly-Asp-Glu-Arg- Pro-Thr) with Gly-Gly-Cys added at the carboxyl terminus, was prepared.
By western blotting, this antibody demonstrated specificity for a single 270 kDa protein in rat cerebellum and recognized no proteins in ghosts. This protein band was distinct from the position of MAP2, as marked by western blotting with mAb AP20.
The distribution of βΙΣ2 spectrin was highly concentrated in the soma of cells in the granular layer, and in scattered stellate cells within the molecular layer. Notably, the soma of Purkinje cells did not contain appreciable βΙΣ2 spectrin. The distribution of βΙΣ2 spec- trin was also compared to MAP2 in cultured granule cells. MAP2 stains the soma of these cells in a pattern coincident to that of βΙΣ2 spectrin, but unlike this spectrin it is also abundant along neurites.
Immunoprecipitation experiments were performed with antibodies to either βΙΣ2 spectrin or to βΙΙΣ1 spectrin. Blotting of these precipitates with antibodies to βΙΙΣ1, βΙΣ2 or αΙΙΣ∗ spectrin indicated that while αΙΙΣ∗ spectrin associated with both β-spectrins, presumably forming heterodimers and tetramers, there were no detectable complexes containing both βΙΣ2 and βΙΙΣ1 spectrin.
Cerebellar primary culture after ten days growth in vitro, βΙΣ2 spectrin was diffusely present in the cell body cytoplasm and along short cell processes in a pattern indistinguishable from granular cells in vivo. All anti-βΙΣ2 spectrin staining could be inhibited by pre-incubation of the antibody with 200 μg/ml peptide immunogen, confirming the specificity of the staining pattern.
A specific alternative transcript of the β-spectrin gene on (human) chromosome 2 that was first identified in skeletal muscle is: (1) expressed in most but not all cells of the rat cere- bellar cortex, (2) is highly polarized in a soma-dendritic pattern, (3) is concentrated on the plasma membrane only at the PSD, (4) may be associated in the cytoplasm with various organelles and cytoskeletal structures, including some (but not all) microtubules and (5) is expressed and polarized in cultured cerebellar granule cells. The erythrocyte β-spectrin related isoform described in muscle that is associated with the acetylcholine receptor also reacts with some erythrocyte spectrin antibodies. Spectrin may also directly link other soluble and membrane proteins to microtubules to facilitate their transport to synapses along soma and dendrites. In this model, the multifunctionality of spectrin allows it to participate as a key organizing center about which macromolecular complexes containing both cytoplasmic and integral membrane proteins can form, and links them to other cytoskeletal elements such as microfilaments or microtubules.