Phagosome maturation and fusion with lysosomes in relation to surface property and size of the phagocytic particle.
Phagosomes with pathogenic mycobacteria, or with hydrophobic polystyrene beads of 1 micron in size, do not mature, but remain fusogenic towards early endosomes and do not fuse with lysosomes (de Chastellier et al. Eur. J. Cell Biol. 68, 167-182 (1995)). Both types of phagocytic particles display a close apposition to the phagosome membrane. We have postulated that due to the absence of tubule formation of the phagosomal membrane, efficient recycling of hypothetical fusion-mediating factors is impaired thus keeping the phagosome fusogenic to early endosomes and unable to fuse with lysosomes. To test this hypothesis, we now analyzed phagosome maturation for particles which were expected to display a less close particle-membrane apposition, in addition to confirming our previous results for non-maturing phagosomes as a direct comparison. In contrast to hydrophobic latex beads as before, we now used beads with a more hydrophilic surface, being carboxylated, with and without additional coating by protein (horseradish peroxidase, HRP; or bovine serum albumin, BSA). In addition, we used hydrophobic beads of smaller sizes (0.5, 0.3, 0.1 micron), in order to determine the limiting size at which the particle no longer determined the size and the fate of the phagosome. As predicted, all the above particles displayed a less tight interaction with the phagosome membrane. Tubule formation was observed to a similar extent as for early endosomes. Morphological evidence showed that phagosomes rapidly lost their ability to fuse with early endosomes, after which they could be seen fusing with lysosomes labeled with gold-conjugated BSA. Functional evidence for the formation of phagolysosomes was based on the kinetic observation that subsequently endocytosed contents marker (HRP) was acquired by phagosomes only after a lag of about 5 min as is typical for lysosomes. The present observations could be explained in terms of a model which suggests that mycobacteria can prevent phagosome maturation and, therefore, fusion with lysosomes, by a tight interaction with constituents of the phagosomal membrane. Furthermore, these results show that it is important to choose artificial phagocytic particles according to the appropriate surface properties when using them as a model system to study phagosome processing.
de Chastellier
C
C
INSERM U 411, Laboratoire de Microbiologie, UFR de Médecine Necker-Enfants Malades, Paris, France.
Thilo
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L
D000818:Animals; D002417:Cattle; D006735:Horseradish Peroxidase; D008247:Lysosomes / Q000648:ultrastructure*; D008561:Membrane Fusion / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008863:Microspheres; D010587:Phagocytosis; D010588:Phagosomes / Q000648:ultrastructure*; D012710:Serum Albumin, Bovine; D013499:Surface Properties
D012710:Serum Albumin, Bovine; D006735:Horseradish Peroxidase