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Our study utilized stochastic optical reconstruction microscopy (STORM) and single-molecule analysis to investigate the distribution of CD47 on the human erythrocyte membrane. Contrary to previous findings in mouse erythrocytes, we discovered that CD47 exists in randomly distributed monomers rather than in clusters across the human erythrocyte membrane. Using secondary antibody-induced crosslinking, we found that CD47 aggregates into stable clusters within minutes. By comparing these STORM results with those of the fully mobile protein CD59 and the cytoskeleton-bound membrane protein glycophorin C under similar conditions, as well as devising two-color STORM co-labeling and co-clustering experiments, we further quantitatively revealed an intermediate, self-limiting clustering behavior of CD47, elucidating its fractional (\u223c14%) attachment to the cytoskeleton. Moreover, we report reductions in both the amount of CD47 and its clustering capability in aged erythrocytes, providing new insight into erythrocyte senescence. Together, the combination of STORM and secondary antibody-based crosslinking unveils the unique self-limiting clustering behavior of CD47 due to its fractional cytoskeleton attachment.<\/jats:p>","DOI":"10.1093\/jmcb\/mjae041","type":"journal-article","created":{"date-parts":[[2024,10,5]],"date-time":"2024-10-05T03:36:08Z","timestamp":1728099368000},"source":"Crossref","is-referenced-by-count":10,"title":["Super-resolution microscopy unveils the nanoscale organization and self-limiting clustering of CD47 in human erythrocytes"],"prefix":"10.1093","volume":"16","author":[{"given":"Jianyu","family":"Yang","sequence":"first","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fulin","family":"Xing","sequence":"additional","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fen","family":"Hu","sequence":"additional","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengdi","family":"Hou","sequence":"additional","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Dong","sequence":"additional","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayu","family":"Cheng","sequence":"additional","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wan","family":"Li","sequence":"additional","affiliation":[{"name":"University of California Department of Chemistry, , Berkeley, CA 94720 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Yan","sequence":"additional","affiliation":[{"name":"University of California Department of Chemistry, , Berkeley, CA 94720 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingjun","family":"Xu","sequence":"additional","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]},{"name":"Shenzhen Research Institute of Nankai University , Shenzhen 518083 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Xu","sequence":"additional","affiliation":[{"name":"University of California Department of Chemistry, , Berkeley, CA 94720 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leiting","family":"Pan","sequence":"additional","affiliation":[{"name":"The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University , Tianjin 300071 ,","place":["China"]},{"name":"Shenzhen Research Institute of Nankai University , Shenzhen 518083 ,","place":["China"]},{"name":"State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Responses, College of Life Sciences, Nankai University , Tianjin 300071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,10,4]]},"reference":[{"key":"2025042904014144900_bib1","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/S1473-0502(02)00070-8","article-title":"Expression of CD47 (integrin-associated protein) decreases on red blood cells during storage","volume":"27","author":"Anniss","year":"2002","journal-title":"Transfus. 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