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The proper intracellular trafficking of the photopigment receptor rhodopsin (Rho) is essential for normal rod homeostasis, as Rho mislocalization precedes rod cell death in inherited retinal disorders such as retinitis pigmentosa. Despite its importance, the molecular mechanisms of Rho trafficking in mammalian rods remain largely undefined. In this study, we combined multiple Rho-labeling strategies with super-resolution microscopy to investigate the subcellular organization of Rho in the Golgi complex of mammalian rods. Using stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM) super-resolution imaging modalities, we mapped the localization of Golgi proteins with Rho in mouse and macaque rods and found that Rho specifically colocalizes with Rab6a in the trans-Golgi. To test the functional significance of this interaction, we utilized a dominant-negative Rab6a mutant in both HEK293T cells and mouse rods. We demonstrated that the dominant negative Rab6a significantly inhibits Rho secretion in cell culture, causing intracellular retention. In mouse rods, we found that this mutant similarly causes significant Rho retention in the trans-Golgi. However, surprisingly, a majority of Rho protein still escaped the Golgi and reached the outer segment. Together, these findings uncover critical new subcellular details about Rho organization at the Golgi and establish a role for Rab6a as a regulator of Rho protein release from the trans-Golgi in mammalian rods. Our results provide critical insight into the protein trafficking mechanisms that must be sustained and regulated in mammalian rods for long-term retinal health.<\/jats:p>","DOI":"10.1101\/2025.09.24.678292","type":"posted-content","created":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T15:18:18Z","timestamp":1758813498000},"source":"Crossref","is-referenced-by-count":0,"title":["Super-resolution microscopy reveals a Rab6a-dependent trafficking hub for rhodopsin at the mammalian rod photoreceptor Golgi"],"prefix":"10.64898","author":[{"given":"Maryam","family":"Hekmatara","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0756-5421","authenticated-orcid":false,"given":"Samantha L.","family":"Thompson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-2512-7451","authenticated-orcid":false,"given":"Kristen N.","family":"Haggerty","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sydney","family":"Hagen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5846-804X","authenticated-orcid":false,"given":"Brooke A.","family":"Brothers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bali","family":"Daniels","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guillaume","family":"Luxardi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ala","family":"Moshiri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9357-0656","authenticated-orcid":false,"given":"Wen-Tao","family":"Deng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2559-4912","authenticated-orcid":false,"given":"Michael A.","family":"Robichaux","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025092707550772000_2025.09.24.678292v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.preteyeres.2016.06.002"},{"key":"2025092707550772000_2025.09.24.678292v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.preteyeres.2017.10.002"},{"key":"2025092707550772000_2025.09.24.678292v1.3","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0010904"},{"issue":"20","key":"2025092707550772000_2025.09.24.678292v1.4","doi-asserted-by":"crossref","first-page":"e145256","DOI":"10.1172\/jci.insight.145256","article-title":"Super-resolution microscopy reveals photoreceptor-specific subciliary location and function of ciliopathy-associated protein CEP290","volume":"6","year":"2021","journal-title":"JCI Insight"},{"key":"2025092707550772000_2025.09.24.678292v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2011.08.005"},{"key":"2025092707550772000_2025.09.24.678292v1.6","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1096-9861(19971013)387:1<148::AID-CNE12>3.0.CO;2-Q"},{"key":"2025092707550772000_2025.09.24.678292v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.preteyeres.2004.10.004"},{"key":"2025092707550772000_2025.09.24.678292v1.8","doi-asserted-by":"publisher","DOI":"10.1002\/aja.1001070103"},{"key":"2025092707550772000_2025.09.24.678292v1.9","doi-asserted-by":"publisher","DOI":"10.1007\/BF01258247"},{"key":"2025092707550772000_2025.09.24.678292v1.10","doi-asserted-by":"publisher","DOI":"10.1080\/13816810902962405"},{"key":"2025092707550772000_2025.09.24.678292v1.11","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.2609-20.2021"},{"issue":"17","key":"2025092707550772000_2025.09.24.678292v1.12","doi-asserted-by":"crossref","first-page":"8201","DOI":"10.1016\/S0021-9258(19)86876-X","article-title":"Rhodopsin carbohydrate. 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