{"doi":"10.1101/2024.09.20.614087","title":"A one-step protocol to generate impermeable fluorescent HaloTag substrates for <i>in situ</i> live cell application and super-resolution imaging","abstract":"Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ('dye shuttle') conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":491925,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9488,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1337820,"name":"Siqi Sun","orcid":"0009-0002-8291-3358","position":1,"is_corresponding":false},{"id":1337821,"name":"Christina Holmboe Olesen","orcid":"0000-0002-0575-1597","position":2,"is_corresponding":false},{"id":1338248,"name":"Maria Kowald","orcid":null,"position":3,"is_corresponding":false},{"id":1338249,"name":"E. Tapp","orcid":null,"position":4,"is_corresponding":false},{"id":1338250,"name":"Ulrich Pabst","orcid":null,"position":5,"is_corresponding":false},{"id":1338251,"name":"Marie Bieck","orcid":null,"position":6,"is_corresponding":false},{"id":821973,"name":"Ramona Birke","orcid":null,"position":7,"is_corresponding":false},{"id":987452,"name":"Brenda C. Shields","orcid":"0000-0001-9036-2686","position":8,"is_corresponding":false},{"id":774677,"name":"Pyeonghwa Jeong","orcid":"0009-0000-3796-7627","position":9,"is_corresponding":false},{"id":494203,"name":"Jiyong Hong","orcid":"0000-0002-5253-0949","position":10,"is_corresponding":false},{"id":950945,"name":"Michael R. Tadross","orcid":"0000-0002-7752-6380","position":11,"is_corresponding":false},{"id":577472,"name":"Joshua Levitz","orcid":"0000-0002-8169-6323","position":12,"is_corresponding":false},{"id":344263,"name":"Martin Lehmann","orcid":"0000-0002-8370-6353","position":13,"is_corresponding":false},{"id":425765,"name":"Noa Lipstein","orcid":"0000-0002-0755-5899","position":14,"is_corresponding":false},{"id":260273,"name":"Johannes Broichhagen","orcid":"0000-0003-3084-6595","position":15,"is_corresponding":false},{"id":821419,"name":"Kilian Roßmann","orcid":"0000-0003-4965-9669","position":0,"is_corresponding":true}],"reference_count":44,"raw_metadata":null,"created_at":"2026-07-19T02:08:49.768795Z","pmid":"39386703","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}