Biology subjects

Johnsson, K.

Publications and source records attributed to Johnsson, K..

34 records · Page 2Linked to original sources

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

Fluorescent and bioluminescent calcium indicators with tuneable colors and affinities

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum (ER). Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput-compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/468356v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@b7aa92org.highwire.dtl.DTLVardef@1f7141org.highwire.dtl.DTLVardef@447f30org.highwire.dtl.DTLVardef@fa34c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

cell biology↗

bioRxiv2021

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

{beta}-cells produce, store and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of {beta}-cell function is mostly derived from studies of ex vivo isolated islets and/or rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

cell biology↗

bioRxiv2021

Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag and CLIP-tag

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study on the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rates with certain rhodamine substrates, which are more than two orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rates however are less affected by the structure of the label than those of HaloTag7, which vary over six orders of magnitude for commonly employed substrates. Solving the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

biochemistry↗

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↗

bioRxiv2020

Receptor-linked environment-sensitive probe monitors the local membrane environment surrounding the insulin receptor

Functional membrane proteins in the plasma membrane are suggested to have specific membrane environments that play important roles to maintain and regulate the function of proteins. However, the local membrane environments of membrane proteins remain largely unexplored due to the lack of techniques allowing to monitor them in living cells. We have developed a method to probe the local membrane environment surrounding a membrane protein in the plasma membrane by covalently tethering a solvatochromic, environment-sensitive dye, Nile red, to a membrane protein via a flexible linker. Our direct imaging reported on the spatio-temporal properties of membrane fluidity of the local environment surrounding the insulin receptor. The local environment was distinct from the average plasma membrane fluidity and was quite dynamic and heterogeneous. Upon addition of insulin, the local membrane environment surrounding the receptor increased in fluidity in an insulin receptor-kinase dependent manner. This new technology should allow researchers to examine changes in membrane properties caused by receptor activation and devise ways to address the role of these changes in physiological processes.

cell biology↗

bioRxiv2020

Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. The competence of secretory granules for exocytosis depends on spatio-temporal variables such as proximity to the plasma membrane and age, with newly-generated granules being preferentially released. Despite recent advances, we still lack a comprehensive view about the molecular composition of insulin granules and how this may change over the lifetime of the organelles. Here we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtained intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time along with the physical properties of the membrane.

cell biology↗

bioRxiv2019

A general strategy to develop cell permeable and fluorogenic probes for multi-colour nanoscopy

Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from low cell permeability and unspecific background signal. We report a general strategy to transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signal. The strategy is based on the conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide. This conversion does not affect the spectroscopic properties of the fluorophore but permits it to exist in a dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell permeable spirolactam. Probes based on such fluorophores generally are fluorogenic as the equilibrium shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1000-fold. Using this simple design principle we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy. The work establishes a general strategy to develop fluorogenic probes for live-cell bioimaging.

biochemistry↗

bioRxiv2019

Chemogenetic control of nanobodies

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows to study their role in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

synthetic biology↗

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↗

bioRxiv2019

LUXendins reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and STED-compatible LUXendin651 we describe islet GLP1R expression patterns, reveal higher-order GLP1R organization including the existence of membrane nanodomains, and track single receptor subpopulations. We furthermore show that different fluorophores can confer agonistic behavior on the LUXendin backbone, with implications for the design of stabilized incretin-mimetics. Thus, our labeling probes possess divergent activation modes, allow visualization of endogenous GLP1R, and provide new insight into class B GPCR distribution and dynamics.

cell biology↗

bioRxiv2018

A chemogenetic approach for optical monitoring of voltage in neurons

Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for interrogation of the cellular and molecular logic of the nervous system. Here we describe a Semisynthetic Tethered Voltage Indicator (STeVI1) based upon Nile Red that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and subthreshold activity in neurons.

neuroscience↗

bioRxiv2018

SNAP-tagged nanobodies enable reversible optical control of a G protein-coupled receptor via a remotely tethered photoswitchable ligand

G protein-coupled receptors (GPCRs) mediate the transduction of extracellular signals into complex intracellular responses. Despite their ubiquitous roles in physiological processes and as drug targets for a wide range of disorders, the precise mechanisms of GPCR function at the molecular, cellular, and systems levels remain partially understood. In order to dissect the function of individual receptors subtypes with high spatiotemporal precision, various optogenetic and photopharmacological approaches have been reported that use the power of light for receptor activation and deactivation. Here, we introduce a novel and, to date, most remote way of applying photoswitchable orthogonally remotely-tethered ligands (PORTLs) by using a SNAP-tag fused nanobody. Our nanobody-photoswitch conjugates (NPCs) can be used to target a GFP-fused metabotropic glutamate receptor by either gene-free application of purified complexes or co-expression of genetically encoded nanobodies to yield robust, reversible control of agonist binding and subsequent downstream activation. By harboring and combining the selectivity and flexibility of both nanobodies and self-labelling enzymes, we set the stage for targeting endogenous receptors in vivo.

pharmacology and toxicology↗

bioRxiv2017

Control of mechanical pain hypersensitivity through ligand-targeted photoablation of TrkB positive sensory neurons

SummaryMechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both necessary and sufficient for producing pain from light touch after nerve injury. Mice in which TrkB-Cre expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.\n\nHighlightsO_LITrkB+ neurons detect light touch under basal conditions\nC_LIO_LITrkB+ neurons convey mechanical allodynia in neuropathic pain states\nC_LIO_LIA photosensitizing derivative of BDNF allows for photoablation of TrkB+ neurons\nC_LIO_LIBDNF-guided photoablation reverses allodynia in multiple types of neuropathic pain\nC_LI

neuroscience↗