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However, permanent labeling strategies for SPT suffer from photobleaching as a major limitation, restricting observation times, and obstructing the study of long\u2010term cellular processes within single living cells. Here, we use exchangeable HaloTag Ligands (xHTLs) as an easy\u2010to\u2010apply labeling approach for live\u2010cell SPT and demonstrate extended observation times of individual living cells of up to 30\u2005minutes. Using the xHTL\/HaloTag7 labeling system, we measure the ligand\u2010induced activation kinetics of the epidermal growth factor receptor (EGFR) in single living cells. We generate spatial maps of receptor diffusion in cells, report non\u2010uniform distributions of receptor mobility, and the formation of spatially confined \u2018hot spots\u2019 of EGFR activation. Furthermore, we measured the mobility of an ER\u2010luminal protein in living cells and found diffusion coefficients that correlated with the ER nano\u2010structure. This approach represents a general strategy to monitor protein mobility in a functional context and for extended observation times in single living cells.<\/jats:p>","DOI":"10.1002\/anie.202413117","type":"journal-article","created":{"date-parts":[[2024,11,15]],"date-time":"2024-11-15T05:20:32Z","timestamp":1731648032000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Long\u2010Term Single\u2010Molecule Tracking in Living Cells using Weak\u2010Affinity Protein Labeling"],"prefix":"10.1002","volume":"64","author":[{"given":"Claudia","family":"Catapano","sequence":"first","affiliation":[{"name":"Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt  Max-von-Laue-Str.\u20057 60438 Frankfurt Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0504-1824","authenticated-orcid":false,"given":"Marina S.","family":"Dietz","sequence":"additional","affiliation":[{"name":"Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt  Max-von-Laue-Str.\u20057 60438 Frankfurt Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4479-5429","authenticated-orcid":false,"given":"Julian","family":"Kompa","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology Max Planck Institute for Medical Research  Jahnstr.\u200529 69120 Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9719-5446","authenticated-orcid":false,"given":"Soohyen","family":"Jang","sequence":"additional","affiliation":[{"name":"Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt  Max-von-Laue-Str.\u20057 60438 Frankfurt Germany"},{"name":"Institute of Physical and Theoretical Chemistry IMPRS on Cellular Biophysics  Max-von-Laue-Str.\u20057 60438 Frankfurt Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petra","family":"Freund","sequence":"additional","affiliation":[{"name":"Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt  Max-von-Laue-Str.\u20057 60438 Frankfurt Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8002-1981","authenticated-orcid":false,"given":"Kai","family":"Johnsson","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology Max Planck Institute for Medical Research  Jahnstr.\u200529 69120 Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9821-3578","authenticated-orcid":false,"given":"Mike","family":"Heilemann","sequence":"additional","affiliation":[{"name":"Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt  Max-von-Laue-Str.\u20057 60438 Frankfurt Germany"},{"name":"Institute of Physical and Theoretical Chemistry IMPRS on Cellular Biophysics  Max-von-Laue-Str.\u20057 60438 Frankfurt Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,11,25]]},"reference":[{"key":"e_1_2_8_1_1","volume-title":"The Membranes of Cells","author":"Yeagle P. 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