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Here, we compared the result of sodium dodecyl sulfate\u2013treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)\/eisosome, especially in the curved MCC\/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1\u03b4-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells.<\/jats:p>","DOI":"10.1083\/jcb.202311067","type":"journal-article","created":{"date-parts":[[2024,10,17]],"date-time":"2024-10-17T02:21:13Z","timestamp":1729131673000},"update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":7,"title":["Definition of phosphatidylinositol 4,5-bisphosphate distribution by freeze-fracture replica labeling"],"prefix":"10.1083","volume":"224","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9638-4974","authenticated-orcid":true,"given":"Takuma","family":"Tsuji","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01692sz90","id-type":"ROR","asserted-by":"publisher"}],"name":"Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine 1 , Tokyo,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1216-7468","authenticated-orcid":true,"given":"Junya","family":"Hasegawa","sequence":"additional","affiliation":[{"name":"Medical Research Institute, Tokyo Medical and Dental University 2 , Tokyo,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1837-3748","authenticated-orcid":true,"given":"Takehiko","family":"Sasaki","sequence":"additional","affiliation":[{"name":"Medical Research Institute, Tokyo Medical and Dental University 2 , Tokyo,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3601-7977","authenticated-orcid":true,"given":"Toyoshi","family":"Fujimoto","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01692sz90","id-type":"ROR","asserted-by":"publisher"}],"name":"Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine 1 , Tokyo,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2024,11,4]]},"reference":[{"key":"2024110414414686900_bib1","doi-asserted-by":"publisher","first-page":"17346","DOI":"10.1074\/jbc.M413307200","article-title":"The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters","volume":"280","author":"Aoyagi","year":"2005","journal-title":"J. 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