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Mass Spectrometry
Publications
2022
Geraci I., Autour A., Pietruschka G., Shiian A., Borisova M., Mayer C., Ryckelynck M., Mayer G.
Fluorogenic RNA-Based Biosensor to Sense the Glycolytic Flux in Mammalian Cells Journal Article
In: ACS Chem Biol, vol. 17, iss. 5, pp. 1164-1173, 2022, ISBN: 35427113, (1554-8937 (Electronic) 1554-8929 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Labex, RYCKELYNCK, Unité ARN
@article{nokey,
title = {Fluorogenic RNA-Based Biosensor to Sense the Glycolytic Flux in Mammalian Cells},
author = {I. Geraci and A. Autour and G. Pietruschka and A. Shiian and M. Borisova and C. Mayer and M. Ryckelynck and G. Mayer},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35427113},
doi = {10.1021/acschembio.2c00100},
isbn = {35427113},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {ACS Chem Biol},
volume = {17},
issue = {5},
pages = {1164-1173},
abstract = {The visualization of metabolic flux in real time requires sensor molecules that transduce variations of metabolite concentrations into an appropriate output signal. In this regard, fluorogenic RNA-based biosensors are promising molecular tools as they fluoresce only upon binding to another molecule. However, to date no such sensor is available that enables the direct observation of key metabolites in mammalian cells. Toward this direction, we selected and characterized an RNA light-up sensor designed to respond to fructose 1,6-bisphosphate and applied it to probe glycolytic flux variation in mammal cells.},
note = {1554-8937 (Electronic)
1554-8929 (Linking)
Journal Article},
keywords = {Labex, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The visualization of metabolic flux in real time requires sensor molecules that transduce variations of metabolite concentrations into an appropriate output signal. In this regard, fluorogenic RNA-based biosensors are promising molecular tools as they fluoresce only upon binding to another molecule. However, to date no such sensor is available that enables the direct observation of key metabolites in mammalian cells. Toward this direction, we selected and characterized an RNA light-up sensor designed to respond to fructose 1,6-bisphosphate and applied it to probe glycolytic flux variation in mammal cells.
Fam K. T., Pelletier R., Bouhedda F., Ryckelynck M., Collot M., Klymchenko A. S.
Rational Design of Self-Quenched Rhodamine Dimers as Fluorogenic Aptamer Probes for Live-Cell RNA Imaging Journal Article
In: Anal Chem, vol. 94, iss. 18, pp. 6657-6664, 2022, ISBN: 35486532, (1520-6882 (Electronic) 0003-2700 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Labex, RYCKELYNCK, Unité ARN
@article{nokey,
title = {Rational Design of Self-Quenched Rhodamine Dimers as Fluorogenic Aptamer Probes for Live-Cell RNA Imaging},
author = {K. T. Fam and R. Pelletier and F. Bouhedda and M. Ryckelynck and M. Collot and A. S. Klymchenko},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35486532},
doi = {10.1021/acs.analchem.1c04556},
isbn = {35486532},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Anal Chem},
volume = {94},
issue = {18},
pages = {6657-6664},
abstract = {With the growing interest in the understanding of the importance of RNAs in health and disease, detection of RNAs in living cells is of high importance. Fluorogenic dyes that light up specifically selected RNA aptamers constitute an attractive direction in the design of RNA imaging probes. In this work, based on our recently proposed concept of a fluorogenic dimer, we aim to develop a robust molecular tool for intracellular RNA imaging. We rationally designed a fluorogenic self-quenched dimer (orange Gemini, o-Gemini) based on rhodamine and evaluated its capacity to light up its cognate aptamer o-Coral in solution and live cells. We found that the removal of biotin from the dimer slightly improved the fluorogenic response without losing the affinity to the cognate aptamer (o-Coral). On the other hand, replacing sulforhodamine with a carboxyrhodamine produced drastic improvement of the affinity and the turn-on response to o-Coral and, thus, a better limit of detection. In live cells expressing o-Coral-tagged RNAs, the carboxyrhodamine analogue of o-Gemini without a biotin unit displayed a higher signal as well as faster internalization into the cells. We suppose that less hydrophilic carboxyrhodamine compared to sulforhodamine can more readily penetrate through the cell plasma membrane and, together with its higher affinity to o-Coral, provide the observed improvement in the imaging experiments. The promiscuity of the o-Coral RNA aptamer to the fluorogenic dimer allowed us to tune a fluorogen chemical structure and thus drastically improve the fluorescence response of the probe to o-Coral-tagged RNAs.},
note = {1520-6882 (Electronic)
0003-2700 (Linking)
Journal Article},
keywords = {Labex, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
With the growing interest in the understanding of the importance of RNAs in health and disease, detection of RNAs in living cells is of high importance. Fluorogenic dyes that light up specifically selected RNA aptamers constitute an attractive direction in the design of RNA imaging probes. In this work, based on our recently proposed concept of a fluorogenic dimer, we aim to develop a robust molecular tool for intracellular RNA imaging. We rationally designed a fluorogenic self-quenched dimer (orange Gemini, o-Gemini) based on rhodamine and evaluated its capacity to light up its cognate aptamer o-Coral in solution and live cells. We found that the removal of biotin from the dimer slightly improved the fluorogenic response without losing the affinity to the cognate aptamer (o-Coral). On the other hand, replacing sulforhodamine with a carboxyrhodamine produced drastic improvement of the affinity and the turn-on response to o-Coral and, thus, a better limit of detection. In live cells expressing o-Coral-tagged RNAs, the carboxyrhodamine analogue of o-Gemini without a biotin unit displayed a higher signal as well as faster internalization into the cells. We suppose that less hydrophilic carboxyrhodamine compared to sulforhodamine can more readily penetrate through the cell plasma membrane and, together with its higher affinity to o-Coral, provide the observed improvement in the imaging experiments. The promiscuity of the o-Coral RNA aptamer to the fluorogenic dimer allowed us to tune a fluorogen chemical structure and thus drastically improve the fluorescence response of the probe to o-Coral-tagged RNAs.
Geersens E., Vuilleumier S., Ryckelynck M.
Growth-Associated Droplet Shrinkage for Bacterial Quantification, Growth Monitoring, and Separation by Ultrahigh-Throughput Microfluidics Journal Article
In: ACS Omega, vol. 7, no. 14, pp. 12039-12047, 2022, ISBN: 35449964, (2470-1343 (Electronic) 2470-1343 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Labex, RYCKELYNCK, Unité ARN
@article{nokey,
title = {Growth-Associated Droplet Shrinkage for Bacterial Quantification, Growth Monitoring, and Separation by Ultrahigh-Throughput Microfluidics},
author = {E. Geersens and S. Vuilleumier and M. Ryckelynck},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35449964},
doi = {10.1021/acsomega.2c00248},
isbn = {35449964},
year = {2022},
date = {2022-01-01},
journal = {ACS Omega},
volume = {7},
number = {14},
pages = {12039-12047},
abstract = {Microbiology still relies on en masse cultivation for selection, isolation, and characterization of microorganisms of interest. This constrains the diversity of microbial types and metabolisms that can be investigated in the laboratory also because of intercellular competition during cultivation. Cell individualization by droplet-based microfluidics prior to experimental analysis provides an attractive alternative to access a larger fraction of the microbial biosphere, miniaturizing the required equipment and minimizing reagent use for increased and more efficient analytical throughput. Here, we show that cultivation of a model two-strain bacterial community in droplets significantly reduces representation bias in the grown culture compared to batch cultivation. Further, and based on the droplet shrinkage observed upon cell proliferation, we provide proof-of-concept for a simple strategy that allows absolute quantification of microbial cells in a sample as well as selective recovery of microorganisms of interest for subsequent experimental characterization.},
note = {2470-1343 (Electronic)
2470-1343 (Linking)
Journal Article},
keywords = {Labex, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Microbiology still relies on en masse cultivation for selection, isolation, and characterization of microorganisms of interest. This constrains the diversity of microbial types and metabolisms that can be investigated in the laboratory also because of intercellular competition during cultivation. Cell individualization by droplet-based microfluidics prior to experimental analysis provides an attractive alternative to access a larger fraction of the microbial biosphere, miniaturizing the required equipment and minimizing reagent use for increased and more efficient analytical throughput. Here, we show that cultivation of a model two-strain bacterial community in droplets significantly reduces representation bias in the grown culture compared to batch cultivation. Further, and based on the droplet shrinkage observed upon cell proliferation, we provide proof-of-concept for a simple strategy that allows absolute quantification of microbial cells in a sample as well as selective recovery of microorganisms of interest for subsequent experimental characterization.
Ponce J. R. Jaramillo, Kapps D., Paulus C., Chicher J., Frugier M.
Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein Journal Article
In: J Biol Chem, vol. 298, iss. 6, pp. 101987, 2022, ISBN: 35487244, (1083-351X (Electronic) 0021-9258 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: FRUGIER, Labex, PPSE, Unité ARN
@article{nokey,
title = {Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein},
author = {J. R. Jaramillo Ponce and D. Kapps and C. Paulus and J. Chicher and M. Frugier},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35487244},
doi = {0.1016/j.jbc.2022.101987},
isbn = {35487244},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {J Biol Chem},
volume = {298},
issue = {6},
pages = {101987},
abstract = {Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multi-synthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that has been shown to participate in tRNA trafficking; here, we show that tRip also functions as an AIMP. We identified three aaRSs, namely the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases, which were specifically co-immunoprecipitated with tRip in P. berghei blood stage parasites. All four proteins contain an N-terminal GST-like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip:ERS:QRS) and the M-complex (tRip:ERS:MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties, and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together our results demonstrate that neither the singular homodimerization of tRip, nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host-pathogen interaction is discussed.},
note = {1083-351X (Electronic)
0021-9258 (Linking)
Journal Article},
keywords = {FRUGIER, Labex, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multi-synthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that has been shown to participate in tRNA trafficking; here, we show that tRip also functions as an AIMP. We identified three aaRSs, namely the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases, which were specifically co-immunoprecipitated with tRip in P. berghei blood stage parasites. All four proteins contain an N-terminal GST-like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip:ERS:QRS) and the M-complex (tRip:ERS:MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties, and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together our results demonstrate that neither the singular homodimerization of tRip, nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host-pathogen interaction is discussed.