Alpha-spectrins are major ubiquitinated proteins in rat hippocampal neurons and components of ubiquitinated inclusions in neurodegenerative disorders.

We have demonstrated that alpha-spectrins (alphaSpISigma* and alphaSpIISigma1) are major ubiquitinated proteins in terminally differentiated hippocampal neurons in culture. Western blotting experiments, using alphaSpISigma1, alphaSpIISigma1, and ubiquitin antibodies and lysates of 11-day-old cultured rat hippocampal neurons, have demonstrated that a single band comigrating with alphaSpISigma* and alphaSpIISigma1 in a 5% polyacrylamide sodium dodecyl sulfate gel is recognized by ubiquitin antibodies when (125)I-protein A is used for detection. Immunofluorescence staining of the 7- and 12 -day-old rat hippocampal neuron cultures using ubiquitin, alphaSpISigma1, and alphaSpIISigma1 antibodies demonstrated that all of these antibodies label neurons but not the astrocytes in the cultures. Immunoprecipitation of spectrin subunits in lysates of 12-day-old rat hippocampal neurons under stringent conditions (9.5 M urea) using alphaSpISigma1 and alphaSpIISigma1 antibodies followed by Western blot experiments of the immunoprecipitated spectrin subunits using alphaSpISigma1, alphaSpIISigma1 and ubiquitin antibodies confirmed that both alphaSpISigma* and alphaSpIISigma1 are ubiquitinated in rat hippocampal neurons. Furthermore, we demonstrated by immunohistochemistry that alpha-spectrins are components of the cytoplasmic ubiquitinated inclusions in hippocampal neurons in Alzheimer's and Parkinson's disease patients.

Kendrin/pericentrin-B, a centrosome protein with homology to pericentrin that complexes with PCM-1.

The centrosome is responsible for nucleating microtubules and performing other cellular roles. To define the organization of the centrosome more completely, a human anti-centrosome serum was used to screen a human cDNA library, and a cDNA encoding a >350 kDa centrosome protein was identified. Sequence analyses revealed that this novel centrosome protein contains two coiled-coil domains bounded by non-coiled regions. The N-terminal region of the protein, named pericentrin-B, shares 61% identity (75% similarity) with pericentrin, suggesting an evolutionary relationship between these proteins. Antibodies against pericentrin-B stain centrosomes at all stages of the cell cycle, and pericentrin-B remains associated with centrosomes following microtubule depolymerization. Immunodepletion of neither pericentrin-B nor PCM-1 from cellular extracts inhibited the ability of salt-stripped centrosomes to recover microtubule nucleation potential, demonstrating that neither protein plays a key role in microtubule nucleation processes. Moreover, the binding of both PCM-1 and pericentrin-B with salt-stripped centrosomes required intact microtubules, demonstrating that the association of PCM-1 and pericentrin-B with centrosomes is a late event in the centrosome maturation process. Finally, pericentrin-B and PCM-1 coimmunoprecipitate, suggesting that PCM-1 and pericentrin-B form a functional complex in cells. This observation may help to explain the generation of anti-centrosome autoantibodies in certain autoimmune patients and may be important for centrosome function.