Biology subjects

Aprile-Garcia, F.

Publications and source records attributed to Aprile-Garcia, F..

4 recordsLinked to original sources

bioRxiv2023

Single-molecule FRET and tracking of transfected biomolecules: multi-dimensional protein dynamics in living cells

Proteins and DNA in cells exhibit different conformational states, which are influenced by dynamic interactions with other biomolecules. All these interactions are affected by the molecules localization within the cell, i.e., their compartmentalization. Such, in cellula, compartment-specific dynamics is difficult to measure, because of limitations in instrumentation, autofluorescence of cells, and the necessity to track diffusing molecules. Here, we present a bottom-up engineering approach, which allows us to track transfected proteins in cellula and to analyze time-resolved single-molecule FRET efficiencies. This has been achieved by alternating laser excitation (ALEX) based three-channel (donor, acceptor and FRET) tracking with a live-cell HILO microscope. We validate our strategy by characterizing long-term static-FRET traces of customized DNA with known dye positions. We utilize two different transfection strategies, namely a biological (Streptolysin-O toxin protein) and a physical one (Microinjection). By comparing in vitro and in cellula measurements we show that the cellular environment in this case changes the FRET efficiency by about 25%. In addition, we evaluate single-molecule FRET traces for the heat shock protein Hsp90 in cellula. The obtained FRET efficiency distribution is largely consistent with known Hsp90 structures and in vitro distributions, but also shows some clear differences. Altogether, we show that FRET-TTB (Forster Resonance Energy Transfer-Tracking of Transfected Biomolecules) opens the path to study protein state changes of transfected biomolecules in cellula, including time-resolved cellular localization. SignificanceInside cells, proteins and DNA can change shape depending on their environment and their interactions. Studying these changes for single biomolecules is challenging because they move around in the cell and the cell environment makes it hard to see them clearly. Here, we demonstrate a new technique called FRET-TTB that enables us to track individual proteins inside living cells over time using single-molecule fluorescence microscopy. We validate the approach with custom DNA and apply it to study the heat shock protein Hsp90. Our results show that this method can reveal changes in protein conformation and location within living cells.

biophysics↗

bioRxiv2023

HSP70 binds to specific non-coding RNA and regulates human RNA Polymerase III

Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone Heat-Shock Protein 70 (HSP70) binds to non-coding RNA transcribed by RNA Polymerase III (Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by Pol III. Detailed biochemical analyses showed that HSP70 facilitates transcription of its target non-coding RNA by binding to Pol III. Thus our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in neurodegeneration and cancer.

biochemistry↗

bioRxiv2022

An optimized and validated workflow for developing stable producer cell lines with >99.99% assurance of clonality and high clone recovery

There is a constant pressure to reduce timelines in mammalian cell line development (CLD) for biotherapeutic protein production. Demonstration of clonal derivation of the generated cell lines is key for health authorities approval. To meet these regulatory and process-oriented demands, single-cell dispensers have become vital instruments for single-cell cloning. We conducted validation experiments with the UP.SIGHT (CYTENA GmbH) to determine this instruments single-cell dispensing efficiency (SCDE) and probability of clonal derivation (p(clonal)). Process optimization to maximize clone recovery with several cell lines was also performed, focusing on cloning media and plate type. With a SCDE >97%, p(clonal) >99.99% and clone recovery values of up to 80%, the data reported here support the notion that the UP.SIGHT covers all steps in the single-cell dispensing process with assurance of clonality and colony tracking, leading to faster and more efficient CLD workflows. This work also serves as a guideline for instrument validation and guidance towards process optimization.

bioengineering↗

bioRxiv2022

Quantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation

The formation of biomolecular condensates underpins many cellular processes; however, our current understanding of condensate formation within cells is largely based on observing the final near-equilibrium condensate state. It is less clear how proteins behave before condensates form or at concentrations at which condensation does not occur in cells. Here, we use a combination of fluorescence microscopy and photobleaching analysis to quantify phase separation of negative elongation factor (NELF) in living and stressed cells. We use the recently reported system of stress-induced condensation of NELF in human nuclei as a model to study the behaviour of proteins before condensation. We find that pre-condensate heterogeneous clusters both grow and shrink and are not freely diffusing. Unexpectedly, we also find such small dynamic clusters in unstressed cells in which condensates do not form. We provide a categorisation of small and large clusters based on their dynamics and their response to p38 kinase inhibition. Overall, our data are best explained as non-classical nucleation with a flat free-energy landscape for clusters of a range of sizes and an inhibition of condensation.

biophysics↗