BIOGENESIS OF PHAGOLYSOSOMES PROCEEDS THROUGH A SEQUENTIAL SERIES OF INTERACTIONS WITH THE ENDOCYTIC APPARATUS

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of lo w density latex beads as markers of phagosomes (latex bead compartment s, LBC) allowed the isolation of these organelles by flotation on a si mple sucrose gradient. Two-dimensional gel electrophoresis, immunocyto chemistry, and biochemical assays have been used to characterize the c omposition of LBC at different time points after their formation, as w ell as their interactions with the organelles of the endocytic pathway . Our results show that LBC acquire and lose various markers during th eir transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. T he transfer to the LBC of lamp 2, a membrane protein associated with l ate endocytic structures, was shown to be microtubule dependent. Video microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Colle ctively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisi tion of markers from endocytic organelles.