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.

A Defect in Endocytosis into Intestinal Cells of daf-4 Mutants

G. Clokey, L. Jacobson

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.