How autophagy, an evolutionarily conserved intracellular catabolic system for bulk degradation, selectively degrades protein aggregates is poorly understood. Here, we show that several maternally derived germ P granule components are selectively eliminated by autophagy in somatic cells during C. elegans embryogenesis. The activity of sepa-1 is required for the degradation of these P granule components and for their accumulation into aggregates, termed PGL granules, in autophagy mutants. SEPA-1 forms protein aggregates and is also a preferential target of autophagy. SEPA-1 directly binds to the P granule component PGL-3 and also to the autophagy protein LGG-1/Atg8. SEPA-1 aggregates consistently colocalize with PGL granules and with LGG-1 puncta. Thus, SEPA-1 functions as a bridging molecule in mediating the specific recognition and degradation of P granule components by autophagy. Our study reveals a mechanism for preferential degradation of protein aggregates by autophagy and emphasizes the physiological significance of selective autophagy during animal development.

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Autophagy is an evolutionarily conserved intracellular catabolic system for degradation of long-lived proteins or damaged organelles. In this study, we have identified and characterized a new gene, epg-1, that plays a role in the autophagy pathway in C. elegans. Loss of function of epg-1 causes defects in various autophagy-regulated processes, including degradation of aggregate-prone proteins and optimal survival of animals during starvation. epg-1 encodes a novel protein that shows limited sequence similarity to the yeast autophagy protein Atg13. epg-1 displays a similar expression pattern to, and directly interacts with, the C. elegans Atg1 homolog UNC-51, suggesting that epg-1 encodes a divergent functional homolog of Atg13 in C. elegans.

Cofilin has been reported to depolymerize F-actin alternately by either severing filaments to increase the number of depolymerizing ends or by increasing the off-rate of monomers from F-actin without increasing the number of filament ends. We have compared directly the ability of native and recombinant cofilins from Dictyostelium to sever F-actin. Our results demonstrate that native cofilin has a higher level of severing activity than recombinant cofilin. Significantly, the measurement of cofilin's severing activity by two independent methods, direct visualization with an improved light microscope assay and by scoring of the number of pointed ends by DNase I binding, clearly shows that both native and recombinant cofilins sever F-actin but to different extents. The severing activity in preparations of recombinant cofilin is variable depending on the method of preparation and, in some cases, is difficult to detect by microscopy assays. This latter point is particularly significant because it may lead to the conclusion that cofilin severs weakly or not at all depending on its method of isolation.

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

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