Actin-mediated plasma membrane plasticity of the intracellular parasite Theileria annulata Kathrin Kühni-Boghenbor, 1‡ Min Ma, 2†‡ Leandro Lemgruber, 3‡ Marek Cyrklaff, 3 Friedrich Frischknecht, 3 Véronique Gaschen, 1 Michael Stoffel 1 and Martin Baumgartner 2 * † 1 University of Bern, Division of Veterinary Anatomy, Bern, Switzerland. 2 University of Bern, Division of Molecular Pathobiology, Bern, Switzerland. 3 University of Heidelberg Medical School, Department of Infectious Diseases, Parasitology, Heidelberg, Germany. Summary Pathogen–host interactions are modulated at mul- tiple levels by both the pathogen and the host cell. Modulation of host cell functions is particularly intriguing in the case of the intracellular Theileria parasite, which resides as a multinucleated sch- izont free in the cytosol of the host cell. Direct contact between the schizont plasma membrane and the cytoplasm enables the parasite to affect the function of host cell proteins through direct interaction or through the secretion of regulators. Structure and dynamics of the schizont plasma membrane are poorly understood and whether schizont membrane dynamics contribute to para- site propagation is not known. Here we show that the intracellular Theileria schizont can dynamically change its shape by actively extending filamen- tous membrane protrusions. We found that iso- lated schizonts bound monomeric tubulin and in vitro polymerized microtubules, and monomeric tubulin polymerized into dense assemblies at the parasite surface. However, we established that iso- lated Theileria schizonts free of host cell micro- tubules maintained a lobular morphology and extended filamentous protrusions, demonstrating that host microtubules are dispensable both for the maintenance of lobular schizont morphology and for the generation of membrane protrusions. These protrusions resemble nanotubes and extend in an actin polymerization-dependent manner; using cryo-electron tomography, we detected thin actin filaments beneath these protrusions, indicat- ing that their extension is driven by schizont actin polymerization. Thus the membrane of the schizont and its underlying actin cytoskeleton possess intrinsic activity for shape control and likely function as a peri-organelle to interact with and manipulate host cell components. Introduction To understand the intricate relationship of intracellular pathogens with their host cells requires many different angles of observation. These include pathogen-dependent gene and protein expression profiling, biochemical pathway analysis and the description of immune reactions to intracellular parasitism. To complement these quantita- tive approaches, we examined surface structure and mem- brane dynamics of the apicomplexan parasite Theileria annulata by different microscopy methods inside its host cell and isolated from it. Theileria are important intracellular parasites of rumi- nants in tropical and subtropical regions. Two species – T. parva and T. annulata – induce the lympho- and myeloproliferative disorders East Coast Fever and tropi- cal Theileriosis in cattle, and thereby constitute the two most pathogenic variants (Mehlhorn et al., 1994). Patho- genesis is caused by massive, parasite-dependent amplification of infected lymphoid and myeloid cells and of their concomitant spread inside the infected host animal. Inside the cell, the parasite resides free as a syncytium (schizont) within the host cell cytoplasm, not surrounded by a parasitophorous vacuole like some other better-known apicomplexan parasites, such as Toxoplasma or Plasmodium (Mehlhorn et al., 1994; Dob- belaere and Baumgartner, 2009) but similar to Babesia (Lobo et al., 2012). Theileria parasites transform their host cells. This transformation results in continuous pro- liferation of the infected cells and in protection against the induction of apoptosis. The amplification of the para- site population depends on partition of syncytia onto the two daughter cells. This process is ensured by the physi- Received 24 February, 2012; revised 22 June, 2012, 6 August, 2012; accepted 7 August, 2012. *For correspondence. E-mail martin. baumgartner@kispi.uzh.ch; Tel. (+41) 44 634 88 51; Fax (+41) 44 634 88 59. † Present address: University of Zürich, University Children’s Hospital, Department of Infectious Diseases and Cancer Research, August- Forel Strasse 1, CH-8008 Zürich, Switzerland. ‡ Equal contribution. Cellular Microbiology (2012) 14(12), 1867–1879 doi:10.1111/cmi.12006 First published online 4 September 2012 © 2012 Blackwell Publishing Ltd cellular microbiology