{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T19:02:47Z","timestamp":1790622167399,"version":"4.1.0"},"posted":{"date-parts":[[2026,9,24]]},"group-title":"Biochemistry","reference-count":33,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["2247939"],"award-info":[{"award-number":["2247939"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["2247938"],"award-info":[{"award-number":["2247938"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"name":"National Institutes of Health","award":["R01NS140594"],"award-info":[{"award-number":["R01NS140594"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,24]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Robo receptors are conserved across bilaterians and best known for their ability to mediate axonal repulsion in response to Slit family ligands. In mammals, this applies to Robo1 and Robo2, but mammalian Robo3 binds NELL proteins instead of Slits. The evolutionary origin of NELL-Robo interactions and the possible existence of dual-ligand responsiveness across species remain unknown. Here, we systematically analyzed Robo and NELL homologs across bilaterians and found that NELL-Robo binding is conserved among chordate Robos, but not in protostomes. We show that cephalochordate Robo and NELL can mediate axon repulsion\n                  <jats:italic>in vitro<\/jats:italic>\n                  , suggesting conserved functionality. We observed that conformational masking of the NELL-binding site is prevalent among chordate Robos, modulating NELL-Robo affinity. We also demonstrate that NELL\u2013Robo complexes undergo liquid\u2013liquid phase separation\n                  <jats:italic>in vitro<\/jats:italic>\n                  , a property preserved from cephalochordates to mammals. Our findings support a model in which an ancestral chordate Robo receptor was dual-responsive to Slit and NELL, still the case for some extant Robos, and vertebrate paralogs subfunctionalized, with full ligand specialization emerging in mammals.\n                <\/jats:p>","DOI":"10.64898\/2026.09.23.753883","type":"posted-content","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T19:05:11Z","timestamp":1790276711000},"source":"Crossref","is-referenced-by-count":0,"title":["Evolution of NELL binding by dual-ligand-responsive axon guidance receptor Robo"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2004-9015","authenticated-orcid":false,"given":"Joseph S.","family":"Pak","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA"},{"name":"Institute for Neuroscience, The University of Chicago, Chicago, Illinois, USA"},{"name":"Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8605-4805","authenticated-orcid":false,"given":"Lakshmi","family":"Prakash","sequence":"additional","affiliation":[{"name":"Department of Neuroscience, Brown University, Providence, Rhode Island, USA"},{"name":"Robert J. and Nancy D. Carney Institute for Brain Science, Brown University, Providence, Rhode Island, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3665-8002","authenticated-orcid":false,"given":"Yeonwoo","family":"Park","sequence":"additional","affiliation":[{"name":"Committee on Genetics, Genomics, and Systems Biology, The University of Chicago, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5019-7196","authenticated-orcid":false,"given":"Wioletta I.","family":"Nawrocka","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA"},{"name":"Institute for Neuroscience, The University of Chicago, Chicago, Illinois, USA"},{"name":"Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-7215-4513","authenticated-orcid":false,"given":"Raunak","family":"Kundagrami","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9589-6994","authenticated-orcid":false,"given":"Joseph W.","family":"Thornton","sequence":"additional","affiliation":[{"name":"Committee on Genetics, Genomics, and Systems Biology, The University of Chicago, Chicago, Illinois, USA"},{"name":"Department of Human Genetics, The University of Chicago, Chicago, Illinois, USA"},{"name":"Department of Ecology and Evolution, The University of Chicago, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4596-5450","authenticated-orcid":false,"given":"Alexander","family":"Jaworski","sequence":"additional","affiliation":[{"name":"Department of Neuroscience, Brown University, Providence, Rhode Island, USA"},{"name":"Robert J. and Nancy D. Carney Institute for Brain Science, Brown University, Providence, Rhode Island, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0263-6729","authenticated-orcid":false,"given":"Engin","family":"\u00d6zkan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA"},{"name":"Institute for Neuroscience, The University of Chicago, Chicago, Illinois, USA"},{"name":"Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026092811300716000_2026.09.23.753883v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2017.12.003"},{"key":"2026092811300716000_2026.09.23.753883v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2019.02.004"},{"key":"2026092811300716000_2026.09.23.753883v1.3","doi-asserted-by":"publisher","DOI":"10.1242\/dev.132829"},{"key":"2026092811300716000_2026.09.23.753883v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80590-5"},{"key":"2026092811300716000_2026.09.23.753883v1.5","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.21-05-01548.2001"},{"key":"2026092811300716000_2026.09.23.753883v1.6","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0078-08.2008"},{"key":"2026092811300716000_2026.09.23.753883v1.7","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.cellbio.21.090704.151234"},{"key":"2026092811300716000_2026.09.23.753883v1.8","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-5-113"},{"key":"2026092811300716000_2026.09.23.753883v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.conb.2009.12.006"},{"key":"2026092811300716000_2026.09.23.753883v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2019.02.080"},{"key":"2026092811300716000_2026.09.23.753883v1.11","doi-asserted-by":"publisher","DOI":"10.1093\/sysbio\/syq010"},{"key":"2026092811300716000_2026.09.23.753883v1.12","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.45530"},{"key":"2026092811300716000_2026.09.23.753883v1.13","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.6290-09.2010"},{"key":"2026092811300716000_2026.09.23.753883v1.14","doi-asserted-by":"publisher","DOI":"10.1126\/science.aad2615"},{"key":"2026092811300716000_2026.09.23.753883v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(02)00901-7"},{"key":"2026092811300716000_2026.09.23.753883v1.16","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80589-9"},{"key":"2026092811300716000_2026.09.23.753883v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80915-0"},{"key":"2026092811300716000_2026.09.23.753883v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/S0896-6273(00)80431-6"},{"key":"2026092811300716000_2026.09.23.753883v1.19","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a001727"},{"key":"2026092811300716000_2026.09.23.753883v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/j.mcn.2004.01.002"},{"key":"2026092811300716000_2026.09.23.753883v1.21","doi-asserted-by":"publisher","DOI":"10.1002\/dvdy.20324"},{"key":"2026092811300716000_2026.09.23.753883v1.22","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2004.06.018"},{"key":"2026092811300716000_2026.09.23.753883v1.23","doi-asserted-by":"crossref","first-page":"167777","DOI":"10.1016\/j.jmb.2022.167777","article-title":"Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1\/2 Binding","volume":"434","year":"2022","journal-title":"J Mol Biol"},{"key":"2026092811300716000_2026.09.23.753883v1.24","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0705310104"},{"key":"2026092811300716000_2026.09.23.753883v1.25","doi-asserted-by":"crossref","first-page":"101161","DOI":"10.1016\/j.xgen.2026.101161","article-title":"Nematode extracellular protein interactome expands connections between signaling pathways","volume":"6","year":"2026","journal-title":"Cell Genom"},{"key":"2026092811300716000_2026.09.23.753883v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15211-1"},{"key":"2026092811300716000_2026.09.23.753883v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(04)00303-4"},{"key":"2026092811300716000_2026.09.23.753883v1.28","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btu033"},{"key":"2026092811300716000_2026.09.23.753883v1.29","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1111\/jse.12587","article-title":"Evolution and diversity of axon guidance Robo receptor family genes","volume":"59","year":"2021","journal-title":"J Syst Evol"},{"key":"2026092811300716000_2026.09.23.753883v1.30","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.005819"},{"key":"2026092811300716000_2026.09.23.753883v1.31","doi-asserted-by":"crossref","first-page":"e94970","DOI":"10.1371\/journal.pone.0094970","article-title":"Coevolution of axon guidance molecule Slit and its receptor Robo","volume":"9","year":"2014","journal-title":"PLoS ONE"},{"key":"2026092811300716000_2026.09.23.753883v1.32","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1999.9371"},{"key":"2026092811300716000_2026.09.23.753883v1.33","doi-asserted-by":"crossref","first-page":"1258","DOI":"10.1016\/j.neuron.2014.11.004","article-title":"Signaling Switch of the Axon Guidance Receptor Robo3 during Vertebrate Evolution","volume":"84","year":"2014","journal-title":"Neuron"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.09.23.753883","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T18:30:16Z","timestamp":1790620216000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.09.23.753883"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,24]]},"references-count":33,"URL":"https:\/\/doi.org\/10.64898\/2026.09.23.753883","relation":{},"subject":[],"published":{"date-parts":[[2026,9,24]]},"subtype":"preprint"}}