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Journal of Cell Science, Vol 101, 139-144, Copyright © 1992 by Company of Biologists
Submitted on September 2, 1991
Accepted on October 9, 1991
1 Laboratoire de Biologie Cellulaire (URA 1130 CNR)
2 Laboratoire de Biologie Cellulaire (URA 1130 CNR); and Laboratoire de Résonance Magnétique en Biologic et Médecine, Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires, 38041 Grenoble cedex, France
Author for correspondence
Methylene diphosphonate is taken up in Dictyostelium discoideum amoebae by fluid-phase pinocytosis, and it inhibits growth through the production of methylene analogs of adenosine triphosphate and diadenosine tetraphosphate. Methylene diphosphonate resistance was thus used as the basis of a screening strategy for the isolation of endocytosis mutants. Fifteen Dictyostelium mutants, whose growth was resistant to 7.5 mM methylene diphosphonate, were obtained and three of them were characterized in more detail. They were partially defective in fluid-phase pinocytosis (both the rate and extent of FITC-dextran entry were reduced to 40-50% of the parent type activity) and they had smaller amounts of several lysosomal enzymes, such as acid phosphatase, N-acetylhexosaminidase, 
-mannosidase (20-60% of the parent type activities). In contrast to the lysosomal hydrolases, the mutants had unchanged activities for enzyme markers selective for other compartments. They appeared phenotypically similar to the Dictyostelium mutant HMW570, which is defective in fluid-phase pinocytosis and oversecretes lysosomal enzymes. The methylene diphosphonate-resistant mutants were found to be unable to acidify fully their endosomal compartments and they have an increased endosomal pH, as shown by the use of the pH-sensitive fluorescence of FITC-dextran. Furthermore, the hypothesis proposing a defective acidification of the endosomal pathway was supported by the measurement of ATP-dependent vesicular acidification with acridine orange, and by in vivo 31P NMR spectroscopy with aminomethylphosphonate as a pH probe.
Key words: Dictyostelium, amoeba, methylene diphosphonate, lysosome, endocytosis, acidification
Submitted on September 2, 1991
Accepted on October 9, 1991
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