TY - JOUR
T1 - Stability and function of a putative microtubule-organizing center in the human parasite Toxoplasma gondii
AU - Leung, Jacqueline M.
AU - He, Yudou
AU - Zhang, Fangliang
AU - Hwang, Yu Chen
AU - Nagayasu, Eiji
AU - Liu, Jun
AU - Murray, John M.
AU - Hu, Ke
N1 - Funding Information:
We thank Con Beckers (University of North Carolina, Chapel Hill, NA) for the rabbit anti-TgIMC1 antibody, Peter Bradley (University of California, Los Angeles, CA) for the mouse anti-TgISP1 antibody, Vern Carruthers (University of Michigan, Ann Arbor, MI) for the RHΔku80Δhx parasites and mouse monoclonal 6D10 anti-TgMIC2 antibody, Richard Day (Indiana University School of Medicine, Indianapolis, IN) for the pmNeonGreenFP-N1 and pmAppleFP-C1 plasmids, Sebastian Lourido (Whitehead Institute, Boston, MA) for the pU6-Universal plasmid, sharing the ProtoMatch v1.0 program, and advice on the CRISPR/Cas9 system, and Naomi Morrissette (University of California, Irvine, CA) for the rabbit anti-Tgβ2-tubulin antibody and RNG1-mCherry-LIC-DHFR plasmid. We thank Christiane Hassel of the Indiana University Bloomington Flow Cytometry Core Facility for assistance with flow cytometry, Jim Powers of the Indiana University Bloomington Light Microscopy Imaging Center for assistance and support with light microscopy, and David Morgan and Barry Stein of the Indiana University Bloomington Electron Microscopy Center for advice and support with electron microscopy. We also thank Amanda Rollins and Tiffany Fortney for technical support. This study was supported by an American Heart Association Postdoctoral Fellowship (16POST31330004) awarded to J.M.L., funding from the March of Dimes (6-FY15-198) and the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R01-AI098686) awarded to K.H., and facility funding from the Indiana Clinical and Translational Sciences Institute to K.H., funded in part by Grant UL1 TR001108 from a National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Publisher Copyright:
© 2017 Leung et al.
PY - 2017/5/15
Y1 - 2017/5/15
N2 - The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and show that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and invade host cells, as well as decreased secretion of effectors important for these processes. Together the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.
AB - The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and show that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and invade host cells, as well as decreased secretion of effectors important for these processes. Together the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.
UR - http://www.scopus.com/inward/record.url?scp=85019678703&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85019678703&partnerID=8YFLogxK
U2 - 10.1091/mbc.E17-01-0045
DO - 10.1091/mbc.E17-01-0045
M3 - Article
C2 - 28331073
AN - SCOPUS:85019678703
SN - 1059-1524
VL - 28
SP - 1361
EP - 1378
JO - Molecular biology of the cell
JF - Molecular biology of the cell
IS - 10
ER -