TROPOMYOSIN LOCALIZATION REVEALS DISTINCT POPULATIONS OF MICROFILAMENTS IN NEURITES AND GROWTH CONES

The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majori ty of tropomyosin was found in a microfilament-enriched fraction of cu ltured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfi laments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tm-s gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, react s only with the neurite. A third antibody which reacts with the Tm5a/5 b isoforms encoded by a separate gene from alpha Tm-s was strongly rea ctive with both neurites and growth cones in 16-h cultures but only wi th the neurite shaft in 40-h cultures. Treatment of neurons with cytoc halasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Remov al of the drug resulted in the disappearance of Tm5NM1-2 from the grow th cone, indicating that isoform segregation is an active process depe ndent on intact microfilaments. Treatment of 40-h cultures with nocoda zole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a /5b now spread back into the growth cone. We conclude that the organiz ation of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dep endent on microtubule integrity. These results indicate that tropomyos in isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct popula tions of actin filaments in neurites and growth cones. Further, the co mposition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.