Li Av Tia Segev Zarko Zarko

All Publications

• Cryo-electron tomography with mixed-scale dense neural networks reveals key steps in deployment of Toxoplasma invasion machinery. PNAS nexus Segev-Zarko, L. A., Dahlberg, P. D., Sun, S. Y., Pelt, D. M., Kim, C. Y., Egan, E. S., Sethian, J. A., Chiu, W., Boothroyd, J. C. 2022; 1 (4): pgac183

  Abstract

  Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of mixed-scale dense networks for image processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3-D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization.

  View details for DOI 10.1093/pnasnexus/pgac183

  View details for PubMedID 36329726

  View details for PubMedCentralID PMC9615128

• Cryo-ET of Toxoplasma parasites gives subnanometer insight into tubulin-based structures. Proceedings of the National Academy of Sciences of the United States of America Sun, S. Y., Segev-Zarko, L., Chen, M., Pintilie, G. D., Schmid, M. F., Ludtke, S. J., Boothroyd, J. C., Chiu, W. 2022; 119 (6)

  Abstract

  Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.

  View details for DOI 10.1073/pnas.2111661119

  View details for PubMedID 35121661

• Characterizing the distribution of myosin H in the apical complex of conoid protruded and conoid retracted Toxoplasma gondii Balaji, A., Dahlberg, P. D., Segev-Zarko, L., Sun, S., Chiu, W., Boothroyd, J., Moerner, W. E. CELL PRESS. 2022: 409A

  View details for Web of Science ID 000759523002535

• NANOSCALE ELUCIDATION OF THE INVASION APPARATUS OF APICOMPLEXAN PARASITES Segev-Zarko, L., Sun, S. Y., Dahlberg, P. D., Pelt, D., Chen, J., Schmid, M. F., Galaz-Montoya, J., Moerner, W. E., Larabell, C., Sethian, J., Chiu, W., Boothroyd, J. AMER SOC TROP MED & HYGIENE. 2019: 620

  View details for Web of Science ID 000507364505127