Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process.

Scanning and transmission electron microscopy were used to determine the morphological changes in the egg plasma membrane associated with sperm binding, fusion and incorporation in Xenopus laevis. Sperm incorporation in Xenopus is rapid, occurring within 3-5 min following addition of sperm. Images have been obtained of both early sperm-egg interactions and fertilisation bodies. Additionally, two drugs that specifically alter F-actin dynamics, latrunculin and jasplakinolide, were used to determine whether sperm incorporation is a microfilament-dependent process. Jasplakinolide did not prevent sperm incorporation, cortical granule exocytosis or cortical contraction, suggesting these events can occur without depolymerisation of existing, stabilised filaments. Latrunculin A, which competes with thymosin β4 in ooplasm for binding actin monomer, did not inhibit cortical granule exocytosis, but blocked cortical contraction in 100% of eggs at a concentration of 5 μM. Although a single penetrating sperm was found on an egg pretreated in latrunculin, fertilisation bodies were never observed. At <5 μM latrunculin, many eggs did undergo cortical contraction with some exhibiting severe distortions of the plasma membrane and abnormal accumulations of pigment granules. Preincubation of eggs in jasplakinolide before latrunculin mitigated both these effects to some degree. However, eggs incubated in latrunculin either prior to or after insemination never progressed through first cleavage.

Phenolic compounds, packaged within vesicles known as physodes, are a major cytoplasmic constituent of eggs and zygotes of the Australasian fucoid Hormosira banksii. Physode movement in early life stages of Hormosira is described. We have used the cytoskeletal inhibitors cytochalasin B, latrunculin B and nocodazole added during different stages of development to investigate the role of the cytoskeleton in movement of these vesicles to regions of active wall formation in the growing rhizoid tip and the plane of cytokinesis prior to cell division. Actin inhibition prevented polarization, cross-wall formation and physode movement. When microtubules were disrupted polarization was still possible, but cross-wall formation and physode movement were prevented. Both actin microfilaments and microtubules are necessary to move physodes to regions of accumulation and active wall formation.