| Popis: |
Autophagy (cellular self-eating) is a highly regulated, lysosome-mediated catabolic process of eukaryotic cells to segregate by a special membrane and subsequently degrade their own constituents during development or starvation. Electron microscopy analysis reveals autophagic elements in various cell types of the nematode Caenorhabditis elegans, whose genome contains counterparts of several yeast genes involved in autophagy. Genetic manipulation inactivating autophagy-related genes in C. elegans causes defects in development, affects dauer larval morphogenesis, accelerates aging thereby shortening life span, reduces cell size, decreases survival during starvation, promotes apoptotic cell death, and protects neurons from undergoing hyperactive ion channel- or neurotoxin-induced degeneration. These results implicate autophagy in various developmental and cellular functions such as reproductive growth, aging, and cell growth, as well as cell survival and loss. This chapter discusses methods of inactivating C. elegans autophagy genes by RNA interference, testing the resistance of autophagy-deficient nematodes to starvation-induced stress, handling mutants carrying a deletion in the autophagy pathway, and monitoring autophagic activity by using LysoTracker Red dye or reporters labeled with green fluorescent protein. Such methods may be adaptable to identify additional roles of autophagy in development and cellular function, and may also help to detect the intracellular accumulation of autophagy proteins and monitor autophagosome formation. |
