TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii Aoife T. Heaslip1, Stephanie C. Ems-McClung 2 , Ke Hu 1 * 1 Department of Biology, Indiana University, Bloomington, Indiana, United States of America, 2 Medical Science Program, Indiana University, Bloomington, Indiana, United States of America 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. Citation: Heaslip AT, Ems-McClung SC, Hu K (2009) TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii. PLoS ONE 4(10): e7406. doi:10.1371/ journal.pone.0007406 Editor: Dana Davis, University of Minnesota, United States of America Received July 20, 2009; Accepted September 16, 2009; Published October 12, 2009 Copyright: 2009 Heaslip et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Beckman Young Investigator Award provided to KH by Arnold and Mabel Beckman Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kehu@indiana.edu Introduction The microtubule (MT) cytoskeleton provides structural support for vital functions such as cell motility, cell division, and material flow in eukaryotic cells. A MT is a polymer with distinct polarity, where each MT subunit, i.e. alpha and beta tubulin heterodimer, is placed in the same orientation in the polymer, resulting in two ends (minus and plus ends) that have different structure and assembly dynamics. Higher order structures formed by MT and MT-binding proteins perform drastically different functions such as flagella beating, chromosome alignment and segregation, and vesicular transport. Understanding how MT-containing assemblies are built in a spatially and temporally controlled manner is fundamental to understanding cell physiology. 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 cytoskel- eton maintained precisely through generations. The ultrastructure of T. gondii cytoskeleton is very well characterized [1–7]. The cytoskeleton of every single parasite in a population contains exactly 22 cortical MTs, one basal complex, and one apical complex made of three ring structures, 14 filaments of a novel tubulin polymer (collectively termed ‘‘the conoid’’) [7], and two intra-conoid MTs. There are at least five distinct tubulin- containing structures within T. gondii (Figure 1). Besides the conoid, intra-conoid MTs, and cortical MTs, MTs are also assembled into two other structures: spindle poles, which organize the intra-nucleus spindle during parasite replication, and centri- oles, 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 also 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. How T. gondii constructs and maintains the five structurally and functionally distinct sets of tubulin containing structures in the same cell is an intriguing problem. For example, both the intra- conoid MTs and cortical MTs are canonical MTs with 13 protofilaments; however, their localization and length are very different. The 22 cortical MTs originate from a ring like structure at the distal end of the apical complex, and extend for ,5 mm, or ,2/3 of the parasite body [1,3,4,7]. The two intra-conoid MTs, however, do not have an obvious organizing center. They are positioned close to the center of the apical complex, and only about 0.4 mm in length. CryoEM studies show that both sets of MTs are heavily decorated with MT binding proteins with quite different association patterns [1,7]. The identification and characterization of MT binding proteins, therefore, is key to understanding the construction and functions of these various MT-containing assemblies. Finally, any new knowledge concern- ing T. gondii cytoskeleton is particularly welcome, as T. gondii is PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7406