HOST-CELL INVASION BY TRYPANOSOMES REQUIRES LYSOSOMES AND MICROTUBULEKINESIN-MEDIATED TRANSPORT/

Invasion of mammalian cells by the protozoan parasite Trypanosoma cruz i occurs by an actin-independent mechanism distinct from phagocytosis. Clusters of host lysosomes are observed at the site of parasite attac hment, and lysosomal markers are detected in the vacuolar membrane at early stages of the entry process. These observations led to the hypot hesis that the trypanosomes recruit host lysosomes to their attachment site, and that lysosomal fusion serves as a source of membrane to for m the parasitophorous vacuole. Here we directly demonstrate directiona l migration of lysosomes to the parasite entry site, using time-lapse video-enhanced microscopy of L(6)E(9) myoblasts exposed to T. cruzi tr ypomastigotes. BSA-gold-loaded lysosomes moved towards the cell periph ery, in the direction of the parasite attachment site, but only when t heir original position was less than 11-12 mu m from the invasion site . Lysosomes more distant from the invasion area exhibited only the sho rt multi-directional saltatory movements previously described for lyso somes, regardless of their proximity to the cell margins. Specific dep letion of peripheral lysosomes was obtained by microinjection of NRK c ells with antibodies against the cytoplasmic domain of Igp 120, a trea tment that aggregated lysosomes in the perinuclear area and inhibited T. cruzi entry. The microtubule-binding drugs nocodazole, colchicine, vinblastine, and taxol also inhibited invasion, in both NRK and L(6)E( 9) cells. Furthermore, microinjection of antibodies to the heavy chain of kinesin blocked the acidification-induced, microtubule-dependent r edistribution of lysosomes to the host cell periphery, and reduced try pomastigote entry. Our results therefore demonstrate that during T. cr uzi invasion of host cells lysosomes are mobilized from the immediatel y surrounding area, and that availability of lysosomes at the cell per iphery and microtubule/kinesin-mediated transport are requirements for parasite entry.