Publications by Kai Johnsson | EPFL Graph Search

Low temperature and mTOR inhibition favor stem cell maintenance in human keratinocyte cultures

Fahd Azzabi Zouraq, Yann Barrandon, Thomas Michael Braschler, Jean-Baptiste Bureau, Kai Johnsson, Mako Kamiya, Johannes Alexander Mosig, Ariane Rochat

Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single-cell analysis that long-term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications.

WILEY2023

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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 nonfluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, and an adjustment of the equilibrium 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 one to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell-stimulated emission depletion (STED) microscopy or 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.

AMER CHEMICAL SOC2021

A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy

Kai Johnsson, Lu Wang, Lin Xue

It is difficult to develop suitable fluorescent probes for live-cell nanoscopy, but a general strategy is now reported that can transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signals. Using this approach, probes in a variety of colours were developed for different cellular targets and used for wash-free, multicolour, live-cell confocal and STED microscopy. 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 a low cell permeability and an unspecific background signal. Here we report a general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal. Conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide does not affect the spectroscopic properties of the fluorophore, but allows us to rationally tune the dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell-permeable spirolactam. Furthermore, the equilibrium generally shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1,000-fold. Using this simple design principle, we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy.

NATURE PUBLISHING GROUP2020

Activatable fluorescent probes for hydrolase enzymes based on coumarin-hemicyanine hybrid fluorophores with large Stokes shifts

Kai Johnsson, Mako Kamiya

We show that the equilibrium of intramolecular spirocyclization of coumarin-hemicyanine hybrid fluorophores can be finely tuned by means of chemical modifications. We used this scaffold to develop activatable fluorescent probes with large Stokes shifts for gamma-glutamyltranspeptidase and esterase.

ROYAL SOC CHEMISTRY2020

Chemogenetic Control of Nanobodies

Helen Rebecca Ostlund Farrants, Julien Hiblot, Kai Johnsson

Engineered nanobody allows reversible control of activity in cells through the binding of small molecules. 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 interrogation of their roles 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.

NATURE PUBLISHING GROUP2020

SLC25A51 is a mammalian mitochondrial NAD(+) transporter

Kai Johnsson, Liang Wang

Mitochondria require nicotinamide adenine dinucleotide (NAD(+)) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD(+) transporters have been identified in yeast and plants(1,2), but their existence in mammals remains controversial(3-5). Here we demonstrate that mammalian mitochondria can take up intact NAD(+), and identify SLC25A51 (also known as MCART1)-an essential(6,7) mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD(+) transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD(+) content, impairs mitochondrial respiration, and blocks the uptake of NAD(+) into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD(+) levels and restores NAD(+) uptake into yeast mitochondria lacking endogenous NAD(+) transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD(+) into mitochondria.

NATURE RESEARCH2020

A biosensor for measuring NAD(+) levels at the point of care

Johan Auwerx, Kai Johnsson, Elena Katsyuba, Luc Patiny, Lin Xue, Qiuliyang Yu

The cellular level of nicotinamide adenine dinucleotide (NAD(+)), through its different functions, affects cellular metabolism and signalling(1-3). A decrease in the NAD(+) content has been associated with various pathologies and physiological aging(4,5), while strategies to boost cellular NAD(+) levels have been shown to be effective against age-related diseases in many animal models(6). The link between decreased NAD(+) levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD(+), ideally applicable at the point of care. Here, we introduce a bioluminescent biosensor for the rapid quantification of NAD(+) levels in biological samples, which can be used either in laboratories or at the point of care. The biosensor is a semisynthetic, light-emitting sensor protein that changes the colour of emitted light from blue to red on binding of NAD(+). This NAD(+)-dependent colour change enables the use of the biosensor in paper-based assays in which NAD(+) is quantified by measuring the colour of the emitted light by using either a simple digital camera or a plate reader. We used the approach to quantify NAD(+) levels in cell culture, tissue and blood samples, yielding results that agreed with those from standard testing methods. The same biosensor furthermore allows the quantification of NAD(+)-dependent enzymatic activities in blood samples, thus expanding its utility as a tool for point-of-care diagnostics.

NATURE PUBLISHING GROUP2019

A ligand-based system for receptor-specific delivery of proteins

Kai Johnsson, Luc Reymond

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand's cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

Springer2019

Photoactivation of silicon rhodamines via a light-induced protonation

Michelle Susanne Frei, Kai Johnsson, Luc Reymond

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent 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 create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

2019

Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy

Michelle Susanne Frei, Kai Johnsson, Aleksandar Salim, Lu Wang

Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell super resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super resolution microscopy are given.

AMER CHEMICAL SOC2019