The protein product of the neurofibromatosis 1 gene, neurofibromin, is abundantly expressed in the cerebral cortex during development, but its physiological role remains unknown. To gain insights into the functions of neurofibromin in neurons, we examined patterns of expression and subcellular localization of neurofibromin during neuronal differentiation. Western blot analysis of telencephali homogenates throughout chick embryogenesis revealed that neurofibromin expression increased during embryonic development. Further analysis showed that telencephalic neurons were also enriched in neurofibromin in culture and that a biphasic gain in expression correlated well with both phases of differentiation in culture, first with a massive outgrowth of processes and gains in neurotransmitter phenotype differentiation, and then with synapse formation. Compared to proteins associated with distinct cytoskeleton systems, the pattern of neurofibromin expression correlated closely with that of the cortical cytoskeleton protein paxillin. Moreover, analysis of immunofluorescence staining of neurofibromin showed that in the presence of a protein crosslinker which preserves both soluble and filamentous cytoskeleton proteins after extraction with Triton X-100, neurofibromin colocalized with F-actin only during the first differentiation phase. This colocalization persisted when the actin cytoskeleton was collapsed with cytochalasin D treatment. In contrast, during the second phase of differentiation neurofibromin colocalized with microtubules, but not F-actin, and the staining pattern was disrupted with nocodazole, but not cytochalasin. A constant finding under all conditions was the presence of neurofibromin in the nucleus, which supports the idea that the bipartite nuclear targeting sequence between residues 2555 and 2572 of neurofibromin may be functional. In summary, we have shown that telencephalic neurons and astroblasts are enriched in neurofibromin and that the subcellular targeting of neurofibromin toward the actin or the microtubule cytoskeleton is developmentally regulated.
- Confocal microscopy
- Microtubule cytoskeleton
- Neurofibromatosis 1 (NF1) gene product
- Subcellular targeting
- Telencephalic neuron