Biochemical and morphological analysis on the localization of Rac1 in neurons

The acquisition of cell type-specific morphologies is a central feature of neuronal differentiation. Many extra- and intracellular signals are known to cause the morphological changes of neuronal cells through the reconstruction of the microfilaments underneath the cell membrane. The membrane microdomain called "raft" has been paid much attention, for this domain contains many signal-transducing molecules including trimeric G proteins and cytoskeletal proteins. The raft domain is recovered in a low-density fraction after the treatment of the membrane with the non-ionic detergent such as Triton X-100 and the enrichment of cholesterol and sphingolipids is ascribed to be responsible for the detergent insolubility. In contrast to the well-known localization of trimeric G proteins in raft, the localization of small G proteins in the raft is poorly characterized. Since Rho family small G proteins (Rho, Rac, and Cdc42) regulate the microfilament system, we studied the localization of Rho family small G proteins in the raft of rat brain with western blotting. Specific localization of Rac1 was detected in the raft from 10-day-old and 8-week-old rat whole brain, and also in the raft prepared from the growth cone and synaptic plasma membrane fractions. Rho and Cdc42 were, in contrast, recovered in the Triton soluble fraction. Double immunostaining of cultured hippocampal neurons with antibodies to Rac1 and MAP-2, or Rac1 and tau, showed punctate distribution of Rac1 in axons as well as in dendrites.