{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:03:12Z","timestamp":1785420192035,"version":"3.56.0"},"reference-count":48,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T00:00:00Z","timestamp":1776384000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001459","name":"Ministry of Education","doi-asserted-by":"publisher","award":["MOE-T2EP30223-0042"],"award-info":[{"award-number":["MOE-T2EP30223-0042"]}],"id":[{"id":"10.13039\/501100001459","id-type":"DOI","asserted-by":"publisher"}]},{"award":["MOE-T2EP30223-0042"],"award-info":[{"award-number":["MOE-T2EP30223-0042"]}],"id":[{"id":"https:\/\/ror.org\/01kcva023","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001352","name":"National University of Singapore","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100001352","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/01tgyzw49","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>The lateral organization of the plasma membrane (PM) is vital for cellular signaling, yet the specific mechanisms by which the internal cortical actin meshwork templates the organization of the external lipid leaflet remain poorly understood. While established models like the \u2018picket-fence\u2019 emphasize physical barriers to diffusion, recent observations of fiber-like \u201cghost\u201d structures in the distribution of glycosylphosphatidylinositol-anchored proteins (GPI-APs) suggest a more intricate mode of spatial coordination. In this study, we utilize imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) and variable-angle TIRF to resolve whether these filamentous patterns represent genuine membrane-proximal features or optical artifacts of cytosolic transport. Our results demonstrate that these fiber-like tracks are strictly confined to the immediate PM interface and disappear as the evanescent field probes deeper into the cytosol. While the spatial distribution of GPI-APs is templated by the underlying actin meshwork, quantitative diffusion mapping shows that the lateral dynamics of the probe remains largely uniform and is not significantly modulated by these filamentous patterns. By pharmacologically perturbing the actin scaffold and membrane cholesterol, we show that this transbilayer coupling is contingent upon a cholesterol-dependent cytoskeletal pinning mechanism. These findings demonstrate a decoupling of spatial organization and molecular dynamics, providing evidence for how the actin scaffold patterns nanoscale membrane organization without imposing long-range barriers to diffusion.<\/jats:p>","DOI":"10.3390\/biom16040596","type":"journal-article","created":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T10:11:04Z","timestamp":1776420664000},"page":"596","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Ghosts on the Membrane: Cytoskeletal Pinning Influences Nanoscale Cell Membrane Organization"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8304-4150","authenticated-orcid":false,"given":"Shambhavi","family":"Pandey","sequence":"first","affiliation":[{"name":"Centre for Bio-Imaging Sciences, National University of Singapore, Singapore 117546, Singapore"},{"name":"Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0148-4321","authenticated-orcid":false,"given":"Thorsten","family":"Wohland","sequence":"additional","affiliation":[{"name":"Centre for Bio-Imaging Sciences, National University of Singapore, Singapore 117546, Singapore"},{"name":"Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore"},{"name":"Department of Chemistry, National University of Singapore, Singapore 117543, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1038\/42408","article-title":"Functional rafts in cell membranes","volume":"387","author":"Simons","year":"1997","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1126\/science.1174621","article-title":"Lipid rafts as a membrane-organizing principle","volume":"327","author":"Lingwood","year":"2010","journal-title":"Science"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"a041394","DOI":"10.1101\/cshperspect.a041394","article-title":"A survey of models of cell membranes: Toward a new understanding of membrane organization","volume":"15","author":"Mayor","year":"2023","journal-title":"Cold Spring Harb. 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