@article {70169, title = {Optimizing multifunctional fluorescent ligands for intracellular labeling.}, journal = {Proc Natl Acad Sci U S A}, volume = {122}, year = {2025}, month = {2025 Nov 04}, pages = {e2510046122}, abstract = {

Enzyme-based self-labeling tags enable the covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing cell-permeable multifunctional ligands that contain fluorophores in combination with affinity tags or pharmacological agents. This is challenging since attachment of additional chemical moieties onto fluorescent ligands can adversely affect membrane permeability. To address this problem, we examined the chemical properties of rhodamine-based self-labeling tag ligands through the lens of medicinal chemistry. We found that the lactone-zwitterion equilibrium constant () of rhodamines inversely correlates with their distribution coefficients (log), suggesting that ligands based on dyes exhibiting low and high log values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 within the nucleus. We found that translocation of BRD4 to constitutive heterochromatin in cells leads to apparent increases in transcriptional activity. These fluorescent reagents enable affinity capture and translocation of intracellular proteins in living cells, and our general design concepts will facilitate the design of multifunctional chemical tools for biology.

Preprint:\ https://doi.org/10.1101/2022.07.02.498544
Preprint:\ https://doi.org/10.32388/0xcyuc

}, keywords = {Bromodomain Containing Proteins, Cell Cycle Proteins, Fluorescent Dyes, Humans, Ligands, Mitochondria, Nuclear Proteins, Rhodamines, Staining and Labeling, Transcription Factors}, issn = {1091-6490}, doi = {10.1073/pnas.2510046122}, url = {https://doi.org/10.1073/pnas.2510046122}, author = {Kumar, Pratik and Vevea, Jason D and Tkachuk, Ariana N and Campbell, Kirby R and Watson, Emma T and Ayala, Anthony X and Grimm, Jonathan B and Chapman, Edwin R and Solecki, David J and Lavis, Luke D} } @article {65281, title = {Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.}, journal = {Science}, volume = {367}, year = {2020}, month = {2020 Jan 17}, abstract = {

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

}, issn = {1095-9203}, doi = {10.1126/science.aaz5357}, author = {Hoffman, David P and Shtengel, Gleb and Xu, C Shan and Campbell, Kirby R and Freeman, Melanie and Wang, Lei and Milkie, Daniel E and Pasolli, H Amalia and Iyer, Nirmala and Bogovic, John A and Stabley, Daniel R and Shirinifard, Abbas and Pang, Song and Peale, David and Schaefer, Kathy and Pomp, Wim and Chang, Chi-Lun and Lippincott-Schwartz, Jennifer and Kirchhausen, Tom and Solecki, David J and Betzig, Eric and Hess, Harald F} }