No abstract

Background: The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actinbased motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp.

Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.

Apicomplexan parasites, such as Toxoplasma gondii, rely on actin-based motility for cell invasion, yet conventional actin does not appear to be required for cell division in these parasites. Apicomplexans also contain a variety of actin-related proteins (Arps); however, most of these not directly orthologous to Arps in well-studied systems. We recently identified an apicomplexanspecific member of this family called Actin-Like Protein 1, (ALP1), which plays a role in the assembly of vesicular components recruited to the inner membrane complex (IMC) of daughter cells during cell division. In addition to its enrichment at daughter cell membranes, ALP1 is localized throughout the cytoplasm both diffusely distributed and concentrated in clusters that are detected by fluorescence microscopy, suggesting it forms complexes. Using quantitative optical imaging methods, including fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we demonstrated that ALP1 is a component of a large complex, and that it readily exchanges between diffusible and complex-bound forms. Sedimentation and density gradient analyses revealed that ALP1 is found in a freely soluble state as well as high molecular weight complexes. During cell division, ALP1 was dynamically associated with the IMC, suggesting it rapidly cycles between freely diffusible and complex forms during daughter cell assembly.

Leishmania are important protozoan pathogens of humans in temperate and tropical regions. The study of gene expression during the infectious cycle, in mutants or after environmental or chemical stimuli, is a powerful approach towards understanding parasite virulence and the development of control measures. Like other trypanosomatids, Leishmania gene expression is mediated by a polycistronic transcriptional process that places increased emphasis on post-transcriptional regulatory mechanisms including RNA processing and protein translation. With the impending completion of the Leishmania genome, global approaches surveying mRNA and protein expression are now feasible. Our laboratory has developed the Drosophila transposon mariner as a tool for trapping Leishmania genes and studying their regulation in the form of protein fusions; a classic approach in other microbes that can be termed 'proteogenomics'. Similarly, we have developed reagents and approaches for the creation of DNA microarrays, which permit the measurement of RNA abundance across the parasite genome. Progress in these areas promises to greatly increase our understanding of global mechanisms of gene regulation at both mRNA and protein levels, and to lead to the identification of many candidate genes involved in virulence.