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In\n                      <jats:italic toggle=\"yes\">Toxoplasma gondii<\/jats:italic>\n                      , this specialized cytoskeleton is involved in all aspects of the disease-causing lytic cycle and notably acts as a scaffold for parasite offspring in the internal budding process. Despite advances in our understanding of the architecture and molecular composition, insights pertaining to the coordinated assembly of the scaffold are still largely elusive. Here,\n                      <jats:italic toggle=\"yes\">T. gondii<\/jats:italic>\n                      tachyzoites were dissected by advanced, iterative expansion microscopy (pan-expansion microscopy), revealing new insights into the very early sequential formation steps of the tubulin and IMC scaffold. A comparative study of the related parasite\n                      <jats:italic toggle=\"yes\">Sarcocystis neurona<\/jats:italic>\n                      revealed that different MT bundling organizations of the nascent SPMTs correlate with the number of central and basal alveolar vesicles. In the absence of a so-far identified MT nucleation mechanism, we genetically dissected\n                      <jats:italic toggle=\"yes\">T. gondii<\/jats:italic>\n                      \u03b3-tubulin and \u03b3-tubulin complex proteins 4, 5, and 6 (GCP4\/5\/6). While \u03b3-tubulin depletion abolished the formation of the tubulin scaffold, a set of MTs still formed that suggests SPMTs are nucleated at the outer core of the centrosome. Depletion of GCP4\/5\/6 interfered with the correct assembly of nascent SPMTs into the forming daughter buds, further indicating that the parasite utilizes the \u03b3-tubulin ring complex in tubulin scaffold formation.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Apicomplexan protozoan parasites rely on their specialized cytoskeleton to form offspring. The cytoskeleton serves as an essential scaffold for the emerging daughter cells and is formed by the inner membrane complex (IMC) and underlying subpellicular microtubules (SPMTs). In\n                        <jats:italic toggle=\"yes\">Toxoplasma gondii<\/jats:italic>\n                        , the IMC is composed of several membranous sacks and supported by 22 SPMTs, the latter are evenly spaced around the apical end of mature parasites. Although many advances have been made, little is known about the earliest steps of scaffold formation. Here, we gain unprecedented insights into IMC and SPMT establishment via iterative expansion microscopy and comparative cell biology. We show that at the onset of division, SPMTs are grouped and reveal that the number of groups determines the number of IMC sacks that are assembled. We further dissect the parasite\u2019s \u03b3-tubulin ring complex and show that it is critically involved in scaffold formation.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01389-25","type":"journal-article","created":{"date-parts":[[2025,8,13]],"date-time":"2025-08-13T13:00:12Z","timestamp":1755090012000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":13,"title":["Co-dependent formation of the\n                    <i>Toxoplasma gondii<\/i>\n                    subpellicular microtubules and inner membrane skeleton"],"prefix":"10.1128","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1948-5496","authenticated-orcid":true,"given":"Klemens","family":"Engelberg","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02n2fzt79","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, Boston College","place":["Chestnut Hill, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ciara","family":"Bauwens","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02n2fzt79","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, Boston College","place":["Chestnut Hill, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David J. P.","family":"Ferguson","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04v2twj65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological and Medical Sciences, Oxford Brookes University, and NDCLS, Oxford University","place":["Oxford, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2769-8600","authenticated-orcid":true,"given":"Marc-Jan","family":"Gubbels","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02n2fzt79","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, Boston College","place":["Chestnut Hill, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2020.00269"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.pt.2020.09.001"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1111\/cmi.12285"},{"key":"e_1_3_3_5_2","first-page":"743","volume-title":"The Toxoplasma cytoskeleton: structures, proteins, and processes, in Toxoplasma gondii: the model apicomplexan - perspective and methods","author":"Gubbels MJ","year":"2020","unstructured":"Gubbels MJ, Morrissette NS. 2020. 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