Overexpression of a neural-specific rho family GTPase, cRac1B, selectively induces enhanced neuritogenesis and neurite branching in primary neurons.

Rho family GTPases have been implicated in cytoskeletal reorganization during neuritogenesis. We have recently identified a new gene of this family, cRac1B, specifically expressed in the chicken developing nervous system. This GTPase was overexpressed in primary neurons to study the role of cRac1B in the development of the neuronal phenotype. Overexpression of cRac1B induced an increment in the number of neurites per neuron, and dramatically increased neurite branching, whereas overexpression of the highly related and ubiquitous cRac1A GTPase did not evidently affect neuronal morphology. Furthermore, expression of an inactive form of cRac1B strikingly inhibited neurite formation. The specificity of cRac1B action observed in neurons was not observed in fibroblasts, where both GTPases produced similar effects on cell morphology and actin organization, indicating the existence of a cell type-dependent specificity of cRac1B function. Molecular dissection of cRac1B function by analysis of the effects of chimeric cRac1A/cRac1B proteins showed that the COOH-terminal portion of cRac1B is essential to induce increased neuritogenesis and neurite branching. Considering the distinctive regulation of cRac1B expression during neural development, our data strongly support an important role of cRac1B during neuritogenesis, and they uncover new mechanisms underlying the functional specificity of distinct Rho family GTPases.

Differential expression of distinct members of Rho family GTP-binding proteins during neuronal development: identification of Rac1B, a new neural-specific member of the family.

Previous studies on small GTP-binding proteins of the Rho family have revealed their involvement in the organization of cell actin cytoskeleton. The function of these GTPases during vertebrate development is not known. With the aim of understanding the possible role of these proteins during neuronal development, we have cloned and sequenced five members expressed in developing chick neural retinal cells. We have identified four chicken genes, cRhoA, cRhoB, cRhoC, and cRac1A, homologous to known human genes, and a novel Rac gene, cRac1B. Analysis of the distribution of four of the identified transcripts in chicken embryos shows for the first time high levels of expression of Rho family genes in the vertebrate developing nervous system, with distinct patterns of distribution for the different transcripts. In particular, cRhoA and cRac1A gene expression appeared ubiquitous in the whole embryo, and the cRhoB transcript was more prominent in populations of neurons actively extending neurites, whereas the newly identified cRac1B gene was homogeneously expressed only in the developing nervous system. Temporal analysis of the expression of the five genes suggests a correlation with the morphogenetic events occurring within the developing retina and the retinotectal pathway. Expression of an epitope-tagged cRac1B in retinal neurons showed a diffuse distribution of the protein in the cell body and along neurites. Taken as a whole, our results suggest important roles for ubiquitous and neural-specific members of the Rho family in the acquisition of the mature neuronal phenotype.

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.