Actin Polymerization 1 N Entamoeba

The mechanism by which trophozoites of the protozoan parasite, Entamoeba histolytica destroy mammalian cells is not clear. A variety of amoeba-associated properties have been correlated with virulence (reviewed in 1 and 2). In vitro studies (3-6) have shown that contact of the trophozoite with a target cell is required. Contact killing has been described (6) as occurring in the sequence: adherence, cytolysis, then phagocytosis (possibly) of the target or its remnants. Dependence of in vivo virulence upon the phagocytic competence of the amoeba has also been reported (7). All steps of the sequence are inhibited by cytochalasins (6, 8, 9), indicating that dynamic participation of the amoeba actin cytoskeleton is required. However, the role of actin in the attack process has not been defined. By fluorescence microscopy of glutaraldehyde-fixed and rhodamine-phal-loidin-stained (10) trophozoites, we have revealed the organization and distribution of polymerized actin in Entamoeba involved in a variety of motility-related activities (11, 12), including target cell interactions. Furthermore, by methanol extraction and spectrofluorometric measurement of the bound fluorescence, we have been able to quantitate polymerized actin in trophozoites. In this report, we describe this latter procedure and studies of the organization and quantity of polymerized actin in E. histolytica trophozoites before and immediately after challenge with human red blood cells (RBC) 1. Within 5 s after challenge, polymerized actin appeared at the contact interface with many adherent RBC. A net increase in amoeba polymerized actin content was detectable 1 rnin after the challenge, and reached a maximum of approximately twice the value in unchallenged cells within 4 min. Latex beads, which were phagocytized by E. histolytica trophozoites, neither stimulated an actin response nor were able to diminish the interaction with RBC. RBC, on the other hand, inhibited uptake of latex beads. These results indicate that the initial interaction of E. histolytica trophozoites with target cells is a recognition-specific process that triggers rapid polymeriza-tion of amoeba actin at the site of target contact. This actin appears to be involved in phagocytosis of the target cells. The procedure developed to quan-i Abbreviations used in this paper: BSA, bovine serum albumin; PBS, phosphate-buffered saline; RBC, red blood cell.