Alessandro Sardini

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.

In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.

ClC-3 expression enhances etoposide resistance by increasing acidification of the late endocytic compartment

Resistance to anticancer drugs and consequent failure of chemotherapy is a complex problem severely limiting therapeutic options in metastatic cancer. Many studies have shown a role for drug efflux pumps of the ATP-binding cassette transporters family in the development of drug resistance. ClC-3, a member of the CLC family of chloride channels and transporters, is expressed in intracellular compartments of neuronal cells and involved in vesicular acidification. It has previously been suggested that acidification of intracellular organelles can promote drug resistance by increasing drug sequestration. Therefore, we hypothesized a role for ClC-3 in drug resistance. Here, we show that ClC-3 is expressed in neuroendocrine tumor cell lines, such as BON, LCC-18, and QGP-1, and localized in intracellular vesicles colabeled with the late endosomal/lysosomal marker LAMP-1. ClC-3 overexpression increased the acidity of intracellular vesicles, as assessed by acridine orange staining, and enhanced resistance to the chemotherapeutic drug etoposide by almost doubling the IC50 in either BON or HEK293 cell lines. Prevention of organellar acidification, by inhibition of the vacuolar H+-ATPase, reduced etoposide resistance. No expression of common multidrug resistance transporters, such as P-glycoprotein or multidrug-related protein-1, was detected in either the BON parental cell line or the derivative clone overexpressing ClC-3. The probable mechanism of enhanced etoposide resistance can be attributed to the increase of vesicular acidification as consequence of ClC-3 overexpression. This study therefore provides first evidence for a role of intracellular CLC proteins in the modulation of cancer drug resistance. [Mol Cancer Ther 2007;6(3):979–86]

The human ClC-4 protein, a member of the CLC chloride channel/transporter family, is localized to the endoplasmic reticulum by its N-terminus

Despite considerable similarity in their amino acid sequences and structural features, the mammalian members of the CLC chloride channel/transporter family have different subcellular locations. The subcellular location and function of one of these members, hClC‐4, is controversial. To characterize its cellular function, we investigated its tissue distribution and subcellular location. Expression was high in excitable tissues such as the nervous system and skeletal muscle. When heterologously expressed in HEK293 cells and in skeletal muscle fibers, hClC‐4 localizes to the endoplasmic/sarcoplasmic reticulum (ER/SR) membranes, in contrast to hClC‐3, which localizes to vesicular structures. This location was confirmed by identification of endogenous ClC‐4 in membrane fractions from mouse brain homogenate enriched for the sarco‐endoplasmic reticulum ATPase SERCA2, an ER/SR marker. To identify the motif responsible for ER localization of hClC‐4, we generated hClC‐4 truncations and chimeras between hClC‐4 and hClC‐3 or the unrelated plasma membrane protein Ly49E. A stretch of amino acids, residues 14–63, at the N‐terminus constitutes a novel motif both necessary and sufficient for targeting hClC‐4 and other membrane proteins to the ER.—Okkenhaug, H., Weylandt, K.‐H., Carmena, D., Wells, D. J., Higgins, C. F., Sardini, A. The human ClC‐4 protein, a member of the CLC chloride channel/ transporter family, is localized to the endoplasmic reticulum by its N‐terminus. FASEB J. 20, E1716–E1731 (2006)

Muscarinic receptor activation of AMP-activated protein kinase inhibits orexigenic neuropeptide mRNA expression.

AMP-activated protein kinase (AMPK) plays a crucial role in both cellular and whole body energy homeostasis. Here we demonstrate that the muscarinic receptor agonist carbachol activates AMPKα1-containing complexes in the human SH-SY5Y cell line via a mechanism specific for the AMPK upstream kinase, Ca2+/calmodulin-dependent protein kinase kinase β. Activation of AMPK inhibits mRNA expression of the orexigenic neuropeptides Agouti-related peptide and melanin-concentrating hormone but surprisingly has no effect on neuropeptide Y mRNA, a neuropeptide previously shown to be regulated by AMPK. Rather than restoring mRNA levels to baseline, pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase kinase β or AMPK greatly increases Agouti-related peptide and melanin-concentrating hormone mRNA expression. These data support a hypothesis that modulating basal AMPK activity in the hypothalamus is essential for maintaining tight regulation of pathways contributing to food intake.

Fluorescence lifetime tomography of live cells expressing enhanced green fluorescent protein embedded in a scattering medium exhibiting background autofluorescence

We present a novel fluorescence lifetime tomography system applied to a highly scattering autofluorescent phantom containing live cells expressing the fluorophore enhanced green fluorescent protein (EGFP). The fluorescence signal was excited using a fiber-laser-pumped supercontinuum source and detected using wide-field time gating imaging. To facilitate rapid 3D reconstruction of the fluorescence lifetime distribution, the time-resolved data were Fourier-transformed in time to give complex functions that formed a data set for the Fourier domain reconstruction. Initially the presence of an unspecified background autofluorescence signal impeded reconstruction of the lifetime distribution, but we show that this problem can be addressed using a simple iterative technique.

Fluorescence lifetime readouts of Troponin-C-based calcium FRET sensors: a quantitative comparison of CFP and mTFP1 as donor fluorophores.

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.

Lifespan regulation of conventional protein kinase C isotypes

Plasma membrane translocation, following allosteric binding of second messengers, initiates the signal transduction process mediated by cPKC [conventional PKC (protein kinase C)] isotypes. Mechanisms regulating the lifespan of the active enzyme such as its phosphorylation, internalization, dephosphorylation and degradation are key elements of the signalling network. The understanding of such mechanisms is essential for the design of therapeutic strategies targeting PKC isoenzymes.

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults

Summary Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.

Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.