ABSTRACT
Cdc42, a member of the Rho family of GTP binding proteins, functions in the formation of polarized actin structures, in elongation of cell shape, and in cell signaling. Although genetic mutations previously have not been available in multicellular organisms, studies have attempted to discern Cdc42 functions in organisms, including Drosophila, using dominant active or interfering alleles. Here, for the first time, we examine the functions of Cdc42 in developing tissues using loss-of-function mutations in the Drosophila Cdc42 gene. We find that Cdc42(-) epithelial cells fail to elongate into a columnar cell shape and cannot maintain a monolayered epithelial structure. In contrast to previous studies, we find no requirement for Cdc42 in cell division or in activation of the Jun N-terminal kinase pathway. In addition, Cdc42 function is not required for cytoplasmic actin filament assembly in the nurse cells during oogenesis, although it may facilitate this process. Furthermore, our results indicate that Cdc42 plays a role in intercellular interactions between the germ line and the somatic follicle cells. These results confirm the role of Cdc42 in actin filament assembly and provide new insights into its functions in epithelial morphogenesis and regulating intercellular signaling events.
Subject(s)
Actins/metabolism , Drosophila/embryology , cdc42 GTP-Binding Protein/metabolism , Animals , Animals, Genetically Modified , Body Patterning , Cell Size , Drosophila/genetics , Epithelium/embryology , Eye/embryology , Fluorescent Antibody Technique , Germ Cells/metabolism , In Situ Hybridization , JNK Mitogen-Activated Protein Kinases , Microscopy, Electron, Scanning , Mitogen-Activated Protein Kinases/metabolism , Mutation , Nervous System/embryology , Phenotype , Signal Transduction , Wings, Animal/embryology , cdc42 GTP-Binding Protein/geneticsABSTRACT
The differences in rates of frameshift mutations between a dinucleotide repeat sequence [(CA)(17)] and a tetranucleotide repeat sequence [(GAAA)(17)] have been determined in immortalized, non-tumorigenic, mismatch repair-proficient mouse cells and in mismatch repair-defective human colorectal cancer cells. Clones with mutations were selected on the basis of restoration of activity of a bacterial neomycin resistance gene whose reading frame was disrupted by insertion of the microsatellite upstream of the translation initiation codon. This gene was introduced into the cells on a plasmid, which integrated into the genome of the host cells. Mutation rates of the tetra-nucleotide repeat were much lower than those of the dinucleotide repeat in both cell types. In addition, independent subclones of the colorectal cancer cell line were assayed by PCR for instability of endo-gen-ous tetranucleotide and dinucleotide repeat sequen-ces. In all cases, the mutation frequencies of the dinucleotide repeats were higher than those of the tetranucleotide repeats.
