The molecular biology of lysosome fusion: mapping and cloning the beige gene

Chediak-Higashi syndrome is an autosomal recessive disorder of humans that has profound effects upon lysosome morphology and function. This fatal disorder results in abnormally large lysosomes that are thought to be caused by misregulated vesicle fusions. This dissertation investigates the molecular mechanism(s) responsible for this disorder using cellular and genetic methodologies in order to better understand how fusion events in the endocytic pathway are regulated. Chediak-Higashi syndrome, the beige mouse, and the Aleutian mink are phenotypically similar disorders that are thought to contain defects in homologous genes. In this dissertation, an in vitro assay for lysosome morphology was developed that allows the mutant and wild type cells to be distinguished. Somatic cell fusions between normal and mutant cells showed that the mutant phenotype was complemented in these heterokaryons. Fusion between any two mutants failed to complement, demonstrating that mice, minks, and humans have defects within homologous genes. It had previously been shown that lysosome function and morphology in macrophages was dependent upon microtubules and microtubule motor proteins. We tested the hypothesis that microtubule motor protein function in beige mouse macrophages was defective by inducing lysosome movements using cytoplasmic acidification or PMA treatment. Beige mouse macrophages were capable of normal lysosome movements, suggesting that microtubule motor protein function was normal. In order to localize the defective gene and determine the molecular mechanism defective in beige, an interspecific backcrossed panel of beige mice was used to map the beige gene to a 3cM interval on mouse chromosome 13. A Yeast Artificial Chromosome (YAC) contig covering the beige region was generated using nidogen as the initial probe. YACs were then introduced into beige mouse fibroblasts using spheroplast fusion and the resulting colonies examined for complementation of the mutant phenotype using our in vitro assay of lysosome morphology. Two partially overlapping YACs were isolated that complemented the beige phenotype. These YACs were further used to identify cDNAs in order to isolate the beige gene. The beige gene will then be used to isolate the human CHS gene by hybridization.