{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T08:04:20Z","timestamp":1774685060251,"version":"3.50.1"},"reference-count":38,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T00:00:00Z","timestamp":1774656000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T00:00:00Z","timestamp":1774656000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["1553798"],"award-info":[{"award-number":["1553798"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"publisher","award":["F32NS117672"],"award-info":[{"award-number":["F32NS117672"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cytoskeleton"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Eukaryotic cells depend on dynamic microtubule arrays to execute a wide range of functions vital for life. These microtubule filaments are formed through the polymerization of\n                    <jats:italic>\u03b1<\/jats:italic>\n                    \/\n                    <jats:italic>\u03b2<\/jats:italic>\n                    \u2010tubulin proteins, which can be generated from numerous tubulin genes, or isotypes. In the neuron, microtubule networks are responsible for a myriad of specialized functions, including neuronal migration, neurite extension, and long\u2010range cargo transport. Pathogenic mutations in tubulin genes can result in a wide range of neurodevelopmental defects, collectively termed tubulinopathies. How distinct mutations within different tubulin genes lead to functional defects remains an area of active investigation. Neuronal microtubule networks are significantly challenging to visualize due to the dense, spatially confined microenvironments within the neuron. Further, the conserved nature of tubulin proteins makes the development of isotype\u2010specific antibodies challenging. These factors pose a critical limitation to tubulinopathy research, making visualization of specific tubulin isotypes or mutant tubulin proteins inherently difficult. To address the current technological limitations, we describe a method for immunocytochemical labeling of a single \u0251\u2010tubulin isotype harboring distinct pathogenic mutations. This approach utilizes a novel method for cellular tubulin visualization by inserting a hexahistidine (His6) epitope into a previously identified internal loop in the \u0251\u2010tubulin protein. This modification acts as a visualization handle, providing direct evidence of the incorporation of ectopically expressed\n                    <jats:italic>\u03b1<\/jats:italic>\n                    \u2010tubulin\n                    <jats:italic>TUBA1A<\/jats:italic>\n                    proteins into the cellular microtubule network using available 6\u00d7\u2010His antibodies without disrupting tubulin function. We validate and describe this approach in human iNeurons and COS\u20107 cells\u2014large, flat, fibroblast\u2010like primate cells ideal for visualizing microtubule networks. This protocol highlights the efficacy of the His6\u2010tag in wild\u2010type TUBA1A, polymerization\u2010defective mutant TUBA1A\u2010N102D, and a highly polymer\u2010stable mutant TUBA1A\u2010E254A. This methodological pipeline can be harnessed to study the molecular and cellular phenotypes of numerous tubulin isotypes and pathogenic mutations.\n                  <\/jats:p>","DOI":"10.1002\/cm.70127","type":"journal-article","created":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T07:40:49Z","timestamp":1774683649000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Visualizing Specific Tubulin Isotypes and Pathogenic Variants in Cellular Microtubule Arrays"],"prefix":"10.1002","author":[{"given":"Sareen","family":"Fiaz","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences Colorado State University  Fort Collins Colorado USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benjamin","family":"Powers","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences Colorado State University  Fort Collins Colorado USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6084-602X","authenticated-orcid":false,"given":"Jayne","family":"Aiken","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences Colorado State University  Fort Collins Colorado USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2026,3,28]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/cm.21567"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/jdb5030008"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2021.02.024"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddy416"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.12.12.3973"},{"key":"e_1_2_11_7_1","volume-title":"Tubulinopathies Overview","author":"Bahi\u2010Buisson N.","year":"2016"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1093\/brain\/awu082"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2012.11.017"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.119.4.867"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.3389\/fcell.2021.789438"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1523\/ENEURO.0045\u201020.2020"},{"issue":"6","key":"e_1_2_11_13_1","doi-asserted-by":"crossref","first-page":"1022","DOI":"10.1016\/j.acthis.2014.04.006","article-title":"Immunocytochemical Staining of Endogenous Nuclear Proteins With HIS\u20101\u2010Anti\u2010Poly\u2010Histidine Monoclonal Antibody: A Potential Source of Error in His\u2010Tagged Protein Detection","volume":"116","author":"Chilumuri A.","year":"2014","journal-title":"Acta Histochemica"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1093\/brain\/aws338"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2022.101915"},{"key":"e_1_2_11_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2024.103556"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpcb.51"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1093\/gbe\/evu187"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ydbio.2015.11.022"},{"key":"e_1_2_11_20_1","doi-asserted-by":"publisher","DOI":"10.3389\/fcell.2022.898076"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/dneu.22745"},{"key":"e_1_2_11_22_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13023\u2010019\u20101020\u2010x"},{"key":"e_1_2_11_23_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.76189"},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.05.046"},{"key":"e_1_2_11_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2006.12.017"},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ygeno.2007.04.008"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.brainresbull.2016.06.016"},{"key":"e_1_2_11_28_1","unstructured":"Nikon.2013.\u201cStorm Protocol\u2010Sample Preparation.\u201d"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.259539"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.201603050"},{"issue":"12","key":"e_1_2_11_31_1","doi-asserted-by":"crossref","first-page":"1685","DOI":"10.1016\/j.stem.2022.11.004","article-title":"A Reference Human Induced Pluripotent Stem Cell Line for Large\u2010Scale Collaborative Studies","volume":"29","author":"Pantazis C. 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