Automated Screen of in Vivo Molecular Interactions using Fluorescence Cross-Correlation Spectroscopy (FCCS)

ABSTRACTThe ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence based methods like two-color fluorescence cross-correlation spectroscopy can provide this information but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross-talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photo-stability, bleaching and fluorescence brightness can have an impact.Here we take advantage of lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross-talk. To establish sc-FLCCS we first engineered and tested multiple GFP-like fluorescent proteins for their suitability. This identified a novel GFP variant termed slmGFP (short lifetime monomeric GFP) with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and slmGFP were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color(dc)-FCCS measurements.STATEMENT OF SIGNIFICANCEThe quantification of protein concentrations and protein-protein interactions in vivo is a crucial information needed for the understanding of complex processes inside cells. Determination of such information is unfortunately still challenging. Fluorescence-based method like fluorescence cross-correlation spectroscopy (FCCS) is the only method which provides this information in vivo and almost in the real time, however it suffers from limitations caused by experimental setup and biological origin of fluorescent proteins. We present single-color fluorescence lifetime cross-correlation spectroscopy as an alternative to FCCS, which uses the information of fluorescence lifetime to overcome some of these limitations. We challenged the method and determined its advantages and limitations and demonstrated the applicability of the method on the proteins of yeast proteasome.

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In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

Single Molecule Spectroscopy in Chemistry, Physics and Biology - Springer Series in Chemical Physics ◽

10.1007/978-3-642-02597-6_7 ◽

2009 ◽

pp. 139-154 ◽

Cited By ~ 1

Author(s):

Jörg Mütze ◽

Thomas Ohrt ◽

Zdeněk Petrášek ◽

Petra Schwille

Keyword(s):

Cross Correlation ◽

Correlation Spectroscopy ◽

Fluorescence Correlation ◽

In Vivo Fluorescence

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Single-Color Fluorescence Lifetime Cross-Correlation Spectroscopy In Vivo

Biophysical Journal ◽

10.1016/j.bpj.2020.06.039 ◽

2020 ◽

Vol 119 (7) ◽

pp. 1359-1370 ◽

Cited By ~ 1

Author(s):

Martin Štefl ◽

Konrad Herbst ◽

Marc Rübsam ◽

Aleš Benda ◽

Michael Knop

Keyword(s):

Fluorescence Lifetime ◽

Cross Correlation ◽

Correlation Spectroscopy

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Two-Hybrid Fluorescence Cross-Correlation Spectroscopy Detects Protein-Protein Interactions In Vivo

ChemPhysChem ◽

10.1002/cphc.200400639 ◽

2005 ◽

Vol 6 (5) ◽

pp. 984-990 ◽

Cited By ~ 75

Author(s):

Nina Baudendistel ◽

Gabriele Müller ◽

Waldemar Waldeck ◽

Peter Angel ◽

Jörg Langowski

Keyword(s):

Protein Interactions ◽

Cross Correlation ◽

Correlation Spectroscopy ◽

Protein Protein Interactions ◽

Two Hybrid

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Degradation of Drug Delivery Nanocarriers and Payload Release: A Review of Physical Methods for Tracing Nanocarrier Biological Fate

Pharmaceutics ◽

10.3390/pharmaceutics13060770 ◽

2021 ◽

Vol 13 (6) ◽

pp. 770

Author(s):

Patrick M. Perrigue ◽

Richard A. Murray ◽

Angelika Mielcarek ◽

Agata Henschke ◽

Sergio E. Moya

Keyword(s):

Drug Delivery ◽

Laser Scanning ◽

Single Photon ◽

Photon Emission ◽

Correlation Spectroscopy ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Emission Computed Tomography ◽

Systemic Delivery

Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, usually through nanocarrier degradation. A premature degradation, or the loss of the nanocarrier coating, may prevent the drug’s delivery to the targeted tissue. Despite their importance, stability and degradation of nanocarriers in biological environments are largely not studied in the literature. Here we review techniques for tracing the fate of nanocarriers, focusing on nanocarrier degradation and drug release both intracellularly and in vivo. Intracellularly, we will discuss different fluorescence techniques: confocal laser scanning microscopy, fluorescence correlation spectroscopy, lifetime imaging, flow cytometry, etc. We also consider confocal Raman microscopy as a label-free technique to trace colocalization of nanocarriers and drugs. In vivo we will consider fluorescence and nuclear imaging for tracing nanocarriers. Positron emission tomography and single-photon emission computed tomography are used for a quantitative assessment of nanocarrier and payload biodistribution. Strategies for dual radiolabelling of the nanocarriers and the payload for tracing carrier degradation, as well as the efficacy of the payload delivery in vivo, are also discussed.

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Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

Pharmaceuticals ◽

10.3390/ph14080757 ◽

2021 ◽

Vol 14 (8) ◽

pp. 757

Author(s):

Iga Jakobowska ◽

Frank Becker ◽

Stefano Minguzzi ◽

Kerrin Hansen ◽

Björn Henke ◽

...

Keyword(s):

Rate Constants ◽

Cross Correlation ◽

Fluorescent Protein ◽

Correlation Spectroscopy ◽

Confocal Imaging ◽

Binding Kinetics ◽

Acceptor Site ◽

Lactate Transport ◽

Entry Site ◽

Hydrogen Bond Acceptor

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.

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