Biology subjects

Frei, M. S.

Publications and source records attributed to Frei, M. S..

11 recordsLinked to original sources

bioRxiv2026

A chemigenetic fluorescence lifetime-based biosensor for imaging of ERK activity

Extracellular signal-regulated kinase (ERK) governs many cellular processes, and its spatiotemporal activity dynamics are important for downstream signaling specificity. Existing fluorescent ERK activity biosensors, however, fall short in enabling spectrally multiplexed, spatially resolved, or deep tissue measurements. Here, we introduce a chemigenetic, single-fluorophore biosensor, WHaloEKAR, that reports ERK activities through changes in fluorescence lifetime. WHaloEKAR combines large lifetime changes (-0.81 ns) with the spectral versatility of the self-labeling protein HaloTag giving access to orange, red, and far-red ERK biosensors. The latter enabled the investigation of heterogeneous ERK activity at distinct subcellular locations in cultured cell lines and primary cortical rat neurons via fluorescence lifetime imaging microscopy (FLIM). Moreover, multiplexed FLIM measurements allowed to study ERK activity in parallel to the second messengers calcium and cyclic adenosine monophosphate. Finally, WHaloEKAR enabled the investigation of ERK activity in 3D samples as well as in live animals.

cell biology↗

bioRxiv2026

Nanobodies equipped with HaloTag variants enable rapid and straightforward one-step immunofluorescence lifetime multiplexing

Multiplexed fluorescence imaging is limited by spectral overlap, whereas fluorescence lifetime provides an orthogonal encoding dimension. We replace genetically fused HaloTags with recombinant nanobody-HaloTag constructs applied as immunofluorescence reagents and determine a lifetime palette. Integrating lifetime and spectral encoding enables up to eight targets in a single acquisition and supports multiplexed imaging in cells and tissue using a simple OneStep labeling strategy compatible with fluorescent proteins, STED nanoscopy, and any standard laboratory antibody.

cell biology↗

bioRxiv2026

Highly Sensitive Chemigenetic FRET-Based Kinase Biosensors

Fluorescent protein-based biosensors have transformed the study of cell physiology and pathology by enabling direct, live-cell measurements of biochemical activities with spatiotemporal precision. FRET-based biosensors offer a quantitative and well-defined readout mechanism popular among researchers, but have struggled to break free of characteristically low dynamic ranges and overall dependence on the cyan-yellow spectral region. Chemigenetic approaches that combine synthetic fluorophores with self-labeling protein tags represent an attractive solution to these longstanding constraints. Here, we pair different fluorescent protein donors with a HaloTag acceptor conjugated to a far-red fluorophore to obtain a suite of highly sensitive, chemigenetic FRET-based kinase activity biosensors with red-shifted emission and unprecedented dynamic range. We demonstrate the generalizability of this chemigenetic platform by developing biosensors for multiple kinases, as well as small GTPases and second messengers, all while maintaining high sensitivity. The high sensitivity and spectral tunability of these chemigenetic tools enabled us to perform robust multiplexed activity imaging of receptor-mediated signaling networks to quantitatively map isoform-specific coupling by GPCRs, as well as clear visualization of kinase activity in acute brain slices via two-photon fluorescence lifetime imaging. Our chemigenetic sensor toolkit thus provides the sensitivity and dimensionality needed to illuminate the spatiotemporal regulation of signaling networks in cells and tissues.

cell biology↗

bioRxiv2026

Sensitive and Selective Next-Generation FRET-based PKA Biosensors

The cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway regulates diverse cellular processes through precise spatiotemporal control across subcellular compartments. Forster resonance energy transfer (FRET)-based A-kinase activity reporters (AKARs) have enabled live-cell visualization of PKA activity, but their limited dynamic range constrains detection of subtle or compartment-specific signaling events. Here, we present a suite of sixth-generation cyan/yellow FRET-based PKA sensors (the AKAR6 series) with substantially enhanced sensitivity and improved selectivity. Systematic optimization of the FRET donor-acceptor pair and sensor backbone yields superior performance versus previous best-in-class FRET-based AKARs, enabling robust detection of subtle PKA activity changes across diverse experimental modalities, including flow cytometry, fluorescence lifetime-based FRET, and two-photon imaging in brain slices. We further leverage kinome atlas data to engineer a variant with improved selectivity for more accurate visualization of nuclear PKA activity. Using the AKAR6 toolkit, we showed that in contrast to strong GPCR-induced PKA activities across all tested compartments in PC12 cells, growth factors stimulated a significant PKA activity at the trans-Golgi network but no detectable activity at cis-Golgi, signifying highly compartmentalized PKA signaling at the sub-organelle level. Furthermore, NGF and EGF induced sustained and transient PKA activity, respectively, across various intracellular compartments, including the nucleus, suggesting that growth factor-specific temporal controls are maintained across subcellular compartments. Together, the AKAR6 toolkit provides a sensitive, selective, and versatile platform for dissecting compartmentalized PKA signaling across cells and tissues.

bioengineering↗

bioRxiv2024

Far-red chemigenetic biosensors for multi-dimensional and super-resolved kinase activity imaging

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

bioengineering↗

bioRxiv2023

A modular chemigenetic calcium indicator enables in vivo functional imaging with near-infrared light

Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7x increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM).

neuroscience↗

bioRxiv2022

A general method for the development of multicolor biosensors with large dynamic ranges

Fluorescent biosensors enable to study cell physiology with spatiotemporal resolution, yet most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Forster Resonance Energy Transfer (FRET) pairs with near quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by either changing the fluorescent protein or the synthetic fluorophore, which enabled to monitor simultaneously free NAD+ in different subcellular compartments upon genotoxic stress. Minimal modifications furthermore allow the readout of these biosensors to be switched to fluorescence intensity, lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/518186v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@9ae8b9org.highwire.dtl.DTLVardef@250477org.highwire.dtl.DTLVardef@482d03org.highwire.dtl.DTLVardef@833397_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗

bioRxiv2022

Live-cell fluorescence lifetime multiplexing using synthetic fluorescent probes

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here we demonstrate that from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image up to six different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.

cell biology↗

bioRxiv2021

HaloTag8: an engineered protein tag to improve fluorophore performance

HaloTag9 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag9 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag9 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

biochemistry↗

bioRxiv2019

Photoactivation of silicon rhodamines via a light-induced protonation

We present a new type of photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent isomerization followed by protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we created probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophores outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

biophysics↗