Worm Breeder's Gazette 8(3): 56
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
In tracking down the reason for pronounced (10-fold) deficiency of lysosomal cathepsin D in daf-4 mutants, we have noticed that the endocytosis of exogenous proteins into intestinal cells occurs very slowly in these mutants. We feed young adult worms RITC-BSA for 6-8 hours in buffer, then wash and place the animals on lawns to flush the intestinal lumens for 4-6 hours. N2 shows a bright red fluorescence in the characteristic lysosomal granules in the gut cells, as well as the blue autofluorescence attributed to lipofuscins ('age pigment'). By contrast, mutants homozygous for daf-4 alleles e1364 or m72 show almost no incorporated red fluorescence, and greatly reduced blue fluorescence. Heterozygotes (daf-4/+) show normal incorporation, as do animals in which an amber allele (m72) is suppressed by sup-7. When daf-4 is suppressed by an epistatic dauer-defective daf-5 mutation, endocytosis rate and autofluorescence remain at the low levels characteristic of daf-4. Although we realize the dangers of inferring causal relationships amongst the pleiotropic phenotypes of these mutants, it seems plausible that the endocytosis defect is the primary lesion in daf-4 mutants. We propose that the dauer- constitutive phenotype results from failure to take adequate amounts of foodstuffs into the intestinal cells. In this view, the epistatic dauer-defective mutations (e.g., daf-5) could be understood as simply preventing dauer formation from being triggered by any nutrition- related signal, rather than as direct physiological suppressors of the primary defect. This is consistent with the observation that the 'small' phenotype of daf-4 mutants is not suppressed by daf-5, and with the fact that daf-5 is epistatic to most known dauer- constitutives. We have yet to test the effects of other dauer- constitutive or dauer-defective mutations on endocytosis. We believe the endocytosis defect could account for the extreme deficiency of cathepsin D. We know that cathepsin D autolyzes at acid pH both in crude extracts and in pure form. If there were little exogenous protein entering the lysosomes of a daf-4 mutant, cathepsin D would presumably autolyze for want of other proteins to act as competing substrates. This situation is mimicked in acutely starved wild-type worms, where we find a 4-5 fold decrease in cathepsin D level within about 4 hours. The cathepsin D deficiency correlates well with poor endocytosis, since cathepsin D levels remain depressed in endocytosis- defective daf-4 ; af-5 double homozygotes, but are restored to nearly normal when daf-4 (m72) is suppressed by sup-7.