Abstract
The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.
Highlights
The microtubule (MT) cytoskeleton provides structural support for vital functions such as cell motility, cell division, and material flow in eukaryotic cells
TgICMAP1 is likely to have a high affinity for MTs, as heavy eGFP-TgICMAP1 coating correlated with much weaker anti-tubulin staining when compared with that of uncoated MT segments, suggesting that perhaps TgICMAP1 binding blocks the accessibility of tubulin antibody to the MTs (Figure 2, insets)
To determine if TgICMAP1 homologues are present in other protozoan parasites species, the TgICMAP1 protein sequence was blasted against the available apicomplexan parasite genomic sequences, including those of Plasmodium spp, Cryptosporidium spp, Theileria spp, and Neospora caninum
Summary
The microtubule (MT) cytoskeleton provides structural support for vital functions such as cell motility, cell division, and material flow in eukaryotic cells. Toxoplasma gondii is a fantastic cell biological model organism for studying the biogenesis of tubulin containing structures It has a strictly reproducible shape, defined by a highly ordered cytoskeleton maintained precisely through generations. Intra-conoid MTs, and cortical MTs, MTs are assembled into two other structures: spindle poles, which organize the intra-nucleus spindle during parasite replication, and centrioles, which contain a central single MT and nine outer single MTs [4] This MT organization of the centrioles is distinct from the centrioles of higher eukaryotes that are composed of two central MTs and nine triplet peripheral MTs. The parasite contains another novel cytoskeletal structure, termed the basal complex, at its basal end [6,8]. The basal complex contains a number of putative MT binding proteins such as dynein light chain and centrin 2, but no tubulin based structures have been observed in the basal complex
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