Qualitative and quantitative characterization of autophagy in Caenorhabditis elegans by electron microscopy

Caenorhabditis elegans has been introduced relatively late into the field of autophagy with no previous results by classical methods. Therefore, it has to be studied in parallel with both traditional electron microscopy and modern molecular approaches. In general, correct identification of autophagic elements by electron microscopy is indispensable to establish a firm basis for our understanding of the process. The principles and the method for identification, applied also for C. elegans, are summarized first in this article, to facilitate their utilization both for further studies and the analysis of new cell types and to support researchers new to electron microscopy techniques. Studying autophagy in the worm by electron microscopy has required the development of special handling and sampling techniques in addition to overcoming the general technical difficulties due to the nature of C. elegans samples. These are described in detail, together with some initial qualitative and quantitative results obtained by them. The feasibility of the presented method is supported by data which show that in continuously fed worms the autophagic compartment is in the lower range of the 10(-2)% order of magnitude of the cytoplasmic volume, while immediately after molting or upon starvation in the second larval period, usually more than a 10-fold increase can be measured. In dauer larvae, individual variation of the autophagic compartment is very high. The predauer stage in daf-2 mutants does not seem to show significant constitutive autophagic activity. Some autophagy-related gene mutants show characteristic ultrastuctural features, such as autophagosomes with membrane abnormalities (unc-51/Atg1) or the hypertrophy of multivesicular bodies (let-512/Vps34, bec-1/Atg6).