Biology subjects

Gregor, C.

Publications and source records attributed to Gregor, C..

6 recordsLinked to original sources

bioRxiv2024

Increased autonomous bioluminescence emission from mammalian cells by enhanced cofactor synthesis

The bacterial bioluminescence system has been successfully implemented in mammalian cell lines, enabling substrate-free luminescence imaging of living cells. One of the major limitations of the system is its comparatively low brightness. To improve light emission, we aimed at increasing the cellular production of FMNH2 and NADPH which serve as cosubstrates in the bacterial bioluminescence reaction. We coexpressed different proteins involved in the synthesis of these two cofactors together with the proteins of the bacterial bioluminescence system in different mammalian cell lines. Combined expression of a riboflavin kinase (RFK) and a constitutively active Akt2 variant (Akt2CA) that participate in the cellular production of FMN and NADP+, respectively, increased bioluminescence emission up to 2.4-fold. The improved brightness allows autonomous bioluminescence imaging of mammalian cells at a higher signal-to-noise ratio and enhanced spatiotemporal resolution.

biochemistry↗

bioRxiv2024

Autonomous bioluminescence emission from transgenic mice

The bacterial bioluminescence system can be applied to produce autonomous bioluminescence in mammalian cells. Until now, the system has only been stably inserted into cells cultured in vitro. Here we report the generation of an autobioluminescent transgenic mouse line constitutively expressing the genes of the bacterial bioluminescence system, enabling substrate-free in vivo luminescence imaging of a living mammal.

bioengineering↗

bioRxiv2022

Two-color live-cell STED nanoscopy by click labeling with cell-permeable fluorophores

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are required. In addition, the fluorophores should preferentially emit in the red spectral range to reduce potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. Here we demonstrate two-color STED imaging of living cells using various pairs of dyes that fulfill all of the above requirements. To this end, we combine click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, enabling long-term STED imaging of different target structures in living specimens.

biophysics↗

bioRxiv2021

Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2

The bacterial bioluminescence system enables light production in living cells without an external luciferin. Due to its relatively low levels of light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with 7-fold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

biochemistry↗

bioRxiv2020

Environmental enrichment enhances precision and remodeling speed of synaptic nanoarchitecture revealed by crosstalk free two-color in vivo STED nanoscopy

Synaptic plasticity underlies long-lasting structural and functional changes to brain circuitry and its experience-dependent remodeling can be fundamentally enhanced by environmental enrichment. It is unknown, however, whether and how environmental enrichment alters the morphology and dynamics of individual synapses. Here, we present a virtually crosstalk free, two-color in vivo STED microscope to simultaneously superresolve the dynamics of endogenous PSD95 of the post-synaptic density and spine geometry. With environmental enrichment, the size distributions of PSD95 and spine head sizes were sharper than in controls, indicating that synaptic strength is set more precisely with environmental enrichment. Spine head geometry and PSD95 assemblies were highly dynamic but their changes correlated only mildly. With environmental enrichment, the topography of the PSD95 nanoorganization was more dynamic; changes in size were smaller than in mice housed in standard cages and depended linearly on their original size. Thus, two-color in vivo time-lapse imaging of synaptic nanoorganization uncovers a unique synaptic nanoplasticity associated with the enhanced learning capabilities under environmental enrichment.

neuroscience↗

bioRxiv2019

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

Bioluminescence based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has so far not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.\n\nSignificance statementBioluminescence is generated by luciferases that oxidize a specific luciferin. The enzymes involved in the synthesis of the luciferin from widespread cellular metabolites have so far been identified for only two bioluminescence systems, those of bacteria and fungi. In these cases, the complete reaction cascade is genetically encodable, meaning that heterologous expression of the corresponding genes can potentially produce autonomous bioluminescence in cell types other than the bacterial or fungal host cells. However, the light levels achieved in mammalian cells so far are not sufficient for single-cell applications. Here we present, for the first time, autonomous bioluminescence images of single mammalian cells by coexpression of the genes encoding the six enzymes from the bacterial bioluminescence system.

biochemistry↗