The Microtubule-Stabilizing Protein CLASP1 Associates with the Theileria annulata Schizont Surface via Its Kinetochore-Binding Domain

Abstract

T. annulata, the only eukaryote known to be capable of transforming another eukaryote, is a widespread parasite of veterinary importance that puts 250 million cattle at risk worldwide and limits livestock development for some of the poorest people in the world. Crucial to the pathology of Theileria is its ability to interact with host microtubules and the mitotic spindle of the infected cell. This study builds on our previous work in investigating the host and parasite molecules involved in mediating this interaction. Because it is not possible to genetically manipulate Theileria schizonts, identifying protein interaction partners is critical to understanding the function of parasite proteins. By identifying two Theileria surface proteins that are involved in the interaction between CLASP1 and the parasite, we provide important insights into the molecular basis of Theileria persistence within a dividing cell.

Identification and characterisation of aTheileria annulataproline-rich microtubule and SH3 domain-interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface

“…Among the proteins found were several MAPs, including CLASP1 itself, CLASP2, CAP‐Gly domain‐containing linker protein 1 (CLIP‐170), and Janus kinase and MT‐interacting protein 1 (Jakmip1). The presence of endogenous CLASP1 and CLASP2 validated our approach, as we have already shown that both proteins associate with the schizont surface (Huber et al, ). Jakmip1 interacts with both MTs and members of the Jak family (Jak1 and Tyk2; Steindler et al, ).…”

“…TA03615 encodes a hypothetical protein, which is predicted to be secreted and also contains an SxIP‐motif and one FAINT (“frequently associated in Theileria ”) domain. We recently confirmed that, like Ta‐p104, TA03615 is expressed on the schizont surface and that both TA03615 and Ta‐p104 interact (directly or indirectly) with CLASP1 (Huber et al, ).…”

“…We also show that proteins involved in nuclear transport and constituents of the nuclear pore complex (NPCs) including RanGAP1, RanBP2, Importin B1, Nup214, and Nup160 accumulate close to defined parts of the schizont surface. We show that the schizont membrane protein Ta‐p104, which we have previously described to interact with both EB1 and CLASP1 (Huber et al, ; Woods et al, ), is a part of the CD2AP/CLASP1 complex, together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain‐interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface where it interacts with bovine CD2AP via conserved SH3‐binding Px(P/A)xPR motifs.…”

“…These properties make CD2AP the second host cell protein that we identified which exclusively localises to the Theileria schizont surface throughout the host cell cycle. CLASP1 shows a very similar behaviour (Huber et al, ), whereas the interaction of other host cell proteins with the schizont varies throughout the cell cycle (e.g., PLK1, Schubert et al, , and EB1, Woods et al, ), or depend on the presence of MTs (e.g., CLASP2, Huber et al, ). CIN85 shares significant structural similarity with CD2AP.…”

“…The transforming schizont remains intracellular and achieves its persistence within the continually dividing host cell by interacting closely with host MTs and the mitotic apparatus, ensuring its distribution to both daughter cells (Hulliger, Wilde, Brown, & Turner, ; Schubert et al, ). Considering the importance of parasite–cytoskeleton interactions in maintaining the transformed phenotype, we recently showed that the host cell MT‐associated proteins (MAPs) EB1 (Woods et al, ) and CLASP1 (Huber et al, ) are recruited to the schizont and speculated on their possible role in recruiting and stabilising MTs on the parasite surface. We discovered that the MT‐stabilising protein CLASP1 is sequestered almost completely at the parasite surface, where it remains throughout the entire host cell cycle, and we showed that the kinetochore‐binding domain of CLASP1 is necessary and sufficient for parasite interaction.…”