Biology subjects

Kompa, J.

Publications and source records attributed to Kompa, J..

9 recordsLinked to original sources

bioRxiv2026

Fast and Luminous: CLIP-tag2

CLIP-tag is a self-labeling protein tag used for the specific fluorescence labeling of proteins. However, its low labeling speed and the poor cell permeability of its substrates result in low labeling efficiencies in live-cell applications. Here, we introduce a substrate optimized for live-cell applications, as well as an engineered CLIP-tag variant, CLIP-tag2, which reacts with the new substrate almost 1000-fold faster than the original CLIP-tag-substrate pair. CLIP-tag2 fusion proteins can be specifically and efficiently fluorescently labeled in cells within minutes at nanomolar substrate concentrations, and can be multiplexed with other self-labeling tags such as SNAP-tag2 and HaloTag7. These advances establish CLIP-tag2 as a powerful tagging platform for high-performance live-cell bioimaging.

biochemistry↗

bioRxiv2025

Fast, Bright and Reversible Rhodamine Tags for Live-Cell Imaging

We present Rho-tag and SiR-tag, engineered protein tags derived from bacterial multidrug-resistance proteins that bind unsubstituted (silicon-) rhodamines with nanomolar affinity, enabling fast, reversible, and fluorogenic protein labeling. In live cells, Rho-tag labeling occurs within seconds -- faster than HaloTag7 -- and the tags are compatible with super-resolution methods like STED, SMLM, and MINFLUX. The high specificity of Rho-tag and SiR-tag for unsubstituted rhodamines allows their use alongside HaloTag7 and SNAP-tag. In vivo applications are demonstrated by efficient neuronal labeling in zebrafish larvae.

bioengineering↗

bioRxiv2025

A high-affinity split-HaloTag for live-cell protein labeling

We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHalo{Delta}3). Hpep binds to cpHalo{Delta}3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHalo{Delta}3 and in fixed cells through incubation with cpHalo{Delta}3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.

biochemistry↗

bioRxiv2025

SiR-XActin: A fluorescent probe for imaging actin dynamics in live cells

Imaging actin-dependent processes in live cells is important for understanding numerous biological processes. However, currently used natural-product based fluorescent probes for actin filaments affect the dynamics of actin polymerization and can induce undesired cellular phenotypes. Here, we introduce SiR-XActin, a simplified jasplakinolide-based, far-red fluorescent probe that enables bright and photostable staining in various cell types without requiring genetic modifications. Due to its relatively weak binding affinity, the probe exhibits minimal cytotoxicity and labels actin filaments without significantly altering actin dynamics. Furthermore, SiR-XActin is suitable for time-resolved, live-cell super-resolution STED microscopy. Exchanging the SiR fluorophore in SiR-XActin for other fluorophores yields probes in different colors. All these properties make SiR-XActin and its analogs powerful tools for studying actin dynamics using live-cell fluorescence microscopy.

biochemistry↗

bioRxiv2024

SNAP-tag2: faster and brighter protein labeling

SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here we introduce new labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 107 s-1 M-1, a 100-fold improvement over the corresponding SNAP-tag-substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a 5-fold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translates into a greatly improved performance in various live-cell (super-resolution) imaging applications.

biochemistry↗

bioRxiv2024

Long-term single-molecule tracking in living cells using weak-affinity protein labeling

Single-particle tracking (SPT) has become a powerful tool to monitor the dynamics of membrane proteins in living cells. However, permanent labeling strategies for SPT suffer from photobleaching as a major limitation, restricting observation times, and obstructing the study of long-term cellular processes within single living cells. Here, we use exchangeable HaloTag Ligands (xHTLs) as an easy-to-apply labeling approach for live-cell SPT and demonstrate extended observation times of individual live cells of up to 30 minutes. Using the xHTL/HT7 labeling system, we measure the ligand-induced activation kinetics of the epidermal growth factor receptor (EGFR) in single living cells. Furthermore, we generate spatial maps of receptor diffusion in cells, report non-uniform distributions of receptor activation, and the formation of spatially confined hot spots of EGFR activation. This approach represents a general strategy to monitor protein dynamics in a functional context and for extended observation times in single living cells.

biophysics↗

bioRxiv2024

Gentle rhodamines for live-cell fluorescence microscopy

Rhodamines have been continuously optimized in brightness, biocompatibility, and colors to fulfill the demands of modern bioimaging. However, the problem of phototoxicity caused by the excited fluorophore under long-term illumination has been largely neglected, hampering their use in time-lapse imaging. Here we introduce cyclooctatetraene (COT) conjugated rhodamines that span the visible spectrum and exhibit significantly reduced phototoxicity. We identified a general strategy for the generation of Gentle Rhodamines, which preserved their outstanding spectroscopic properties and cell permeability while showing an efficient reduction of singlet-oxygen formation and diminished cellular photodamage. Paradoxically, their photobleaching kinetics do not go hand in hand with reduced phototoxicity. By combining COT-conjugated spirocyclization motifs with targeting moieties, these gentle rhodamines compose a toolkit for time-lapse imaging of mitochondria, DNA, and actin and synergize with covalent and exchangeable HaloTag labeling of cellular proteins with less photodamage than their commonly used precursors. Taken together, the Gentle Rhodamines generally offer alleviated phototoxicity and allow advanced video recording applications, including voltage imaging.

cell biology↗

bioRxiv2022

Synergizing exchangeable fluorophore labels for multi-target STED microscopy

Investigating the interplay of cellular proteins with optical microscopy requires multi-target labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Non-covalent, weak-affinity labels bypass this "spectral barrier" through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate 6-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching, facilitate long acquisition times and multi-color live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increase the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.

biophysics↗

bioRxiv2022

Exchangeable HaloTag Ligands (xHTLs) for multi-modal super-resolution fluorescence microscopy

We introduce exchangeable ligands for fluorescence labeling of HaloTag7 as an alternative to covalently bound probes. The exchangeable ligands open up new possibilities in imaging for a widely used labeling approach, including applications in points accumulation for imaging in nanoscale topography (PAINT), MINFLUX and live-cell, multi-frame stimulated emission depletion (STED) microscopy. We furthermore introduce orthogonal pairs of exchangeable ligands and HaloTags for dual-color PAINT and STED microscopy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/496706v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1e7d689org.highwire.dtl.DTLVardef@1b5db18org.highwire.dtl.DTLVardef@433efaorg.highwire.dtl.DTLVardef@dbbd61_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗