Multiplexed bioluminescence microscopy via phasor analysis.

Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase-luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time and over multiple length scales.

Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging.

Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids.

Conformational response to charge clustering in synthetic intrinsically disordered proteins.

BACKGROUND: Recent theoretical and computational studies have shown that the charge content and, most importantly, the linear distribution of opposite charges are major determinants of conformational properties of intrinsically disordered proteins (IDPs). Charge segregation in a sequence can be measured through κ, which represents a normalized measure of charge asymmetry. A strong inverse correlation between κ and radius of gyration has been previously demonstrated for two independent sets of permutated IDP sequences. METHODS: We used two well-characterized IDPs, namely measles virus NTAIL and Hendra virus PNT4, sharing a very similar fraction of charged residues and net charge per residue, but differing in proline (Pro) content. For each protein, we have rationally designed a low- and a high-κ variant endowed with the highest and the lowest κ values compatible with their natural amino acid composition. Then, the conformational properties of wild-type and κ-variants have been assessed by biochemical and biophysical techniques. RESULTS: We confirmed a direct correlation between κ and protein compaction. The analysis of our original data along with those available from the literature suggests that Pro content may affects the responsiveness to charge clustering. CONCLUSIONS: Charge clustering promotes IDP compaction, but the extent of its effects depends on the sequence context. Proline residues seem to play a role contrasting compaction. GENERAL SIGNIFICANCE: These results contribute to the identification of sequence determinants of IDP conformational properties. They may also serve as an asset for rational design of non-natural IDPs with tunable degree of compactness.

Aggregation properties of a disordered protein are tunable by pH and depend on its net charge per residue.

Intrinsically disordered proteins (IDPs) possess a peculiar amino acid composition that makes them very soluble. Nevertheless, they can encounter aggregation in physiological and pathological contexts. In this work, we addressed the issue of how electrostatic charges can influence aggregation propensity by using the N-terminus moiety of the measles virus phosphoprotein, PNT, as a model IDP. Taking advantage of the high sequence designability of IDPs, we have produced an array of PNT variants sharing the same hydrophobicity, but differing in net charges per residue and isoelectric points (pI). The solubility and conformational properties of these proteins were analysed through biochemical and biophysical techniques in a wide range of pH values and compared with those of the green fluorescence protein (GFP), a globular protein with lower net charge per residue, but similar hydrophobicity. Tested proteins showed a solubility minimum close to their pI, as expected, but the pH-dependent decrease of solubility was not uniform and driven by the net charge per residue of each variant. A parallel behaviour was observed also in fusion proteins between PNT variants and GFP, which minimally contributes to the solubility of chimeras. Our data suggest that the overall solubility of a protein can be dictated by protein regions endowed with higher net charge per residue and, hence, prompter to respond to pH changes. This finding could be exploited for biotechnical purposes, such as the design of solubility/aggregation tags, and in studies aimed to clarify the pathological and physiological behaviour of IDPs.

Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DNA end resection.

Homologous recombination requires nucleolytic degradation (resection) of DNA double-strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long-range resection is coordinated.

Multimodal Phasor Approach to study breast cancer cells invasion in 3D spheroid model.

We implemented a multimodal set of functional imaging techniques optimized for deep-tissue imaging to investigate how cancer cells invade surrounding tissues and how their physiological properties change in the process. As a model for cancer invasion of the extracellular matrix, we created 3D spheroids from triple-negative breast cancer cells (MDA-MB-231) and non-tumorigenic breast epithelial cells (MCF-10A). We analyzed multiple hallmarks of cancer within the same spheroid by combining a number of imaging techniques, such as metabolic imaging of NADH by Fluorescence Lifetime Imaging Microscopy (NADH-FLIM), hyperspectral imaging of a solvatochromic lipophilic dye (Nile Red) and extracellular matrix imaging by Second Harmonic Generation (SHG). We included phasor-based bioimage analysis of spheroids at three different time points, tracking both morphological and biological properties, including cellular metabolism, fatty acids storage, and collagen organization. Employing this multimodal deep-imaging framework, we observed and quantified cancer cell plasticity in response to changes in the environment composition.

Monitoring macrophage polarization with gene expression reporters and bioluminescence phasor analysis.

Macrophages exhibit a spectrum of behaviors upon activation and are generally classified as one of two types: inflammatory (M1) or anti-inflammatory (M2). Tracking these phenotypes in living cells can provide insight into immune function, but remains a challenging pursuit. Existing methods are mostly limited to static readouts or difficult to employ for multiplexed imaging in complex 3D environments while maintaining cellular resolution. We aimed to fill this void using bioluminescent technologies. Here we report genetically engineered luciferase reporters for long-term monitoring of macrophage polarization via spectral phasor analysis. M1- and M2- specific promoters were used to drive the expression of bioluminescent enzymes in macrophage cell lines. The readouts were multiplexed and discernable in both 2D and 3D formats with single cell resolution in living samples. Collectively, this work expands the toolbox of methods for monitoring macrophage polarization and provides a blueprint for monitoring other multifaceted networks in heterogeneous environments.

The supramolecular processing of liposomal doxorubicin hinders its therapeutic efficacy in cells.

The successful trajectory of liposome-encapsulated doxorubicin (e.g., Doxil, which has been approved by the U.S. Food and Drug Administration) as an anticancer nanodrug in clinical applications is contradicted by in vitro cell viability data that highlight its reduced efficacy in promoting cell death compared with non-encapsulated doxorubicin. No reports to date have provided a mechanistic explanation for this apparently discordant evidence. Taking advantage of doxorubicin intrinsic fluorescence and time-resolved optical microscopy, we analyze the uptake and intracellular processing of liposome-encapsulated doxorubicin (L-DOX) in several in vitro cellular models. Cell entry of L-DOX was found to lead to a rapid (seconds to minutes), energy- and temperature-independent release of crystallized doxorubicin nanorods into the cell cytoplasm, which then disassemble into a pool of fibril-shaped derivatives capable of crossing the cellular membrane while simultaneously releasing active drug monomers. Thus, a steady state is rapidly established in which the continuous supply of crystal nanorods from incoming liposomes is counteracted by a concentration-guided efflux in the extracellular medium of fibril-shaped derivatives and active drug monomers. These results demonstrate that liposome-mediated delivery is constitutively less efficient than isolated drug in establishing favorable conditions for drug retention in the cell. In addition to explaining previous contradictory evidence, present results impose careful rethinking of the synthetic identity of encapsulated anticancer drugs.

Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry.

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with ß subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

Synapsins are expressed at neuronal and non-neuronal locations in Octopus vulgaris.

Synapsins are a family of phosphoproteins fundamental to the regulation of neurotransmitter release. They are typically neuron-specific, although recent evidence pointed to their expression in non-neuronal cells where they play a role in exocytosis and vesicle trafficking. In this work, we characterized synapsin transcripts in the invertebrate mollusk Octopus vulgaris and present evidence of their expression not only in the brain but also in male and female reproductive organs. We identified three synapsin isoforms phylogenetically correlated to that of other invertebrates and with a modular structure characteristic of mammalian synapsins with a central, highly conserved C domain, important for the protein functions, and less conserved A, B and E domains. Our molecular modeling analysis further provided a solid background for predicting synapsin functional binding to ATP, actin filaments and secretory vesicles. Interestingly, we found that synapsin expression in ovary and testis increased during sexual maturation in cells with a known secretory role, potentially matching the occurrence of a secretion process. This might indicate that its secretory role has evolved across animals according to cell activity in spite of cell identity. We believe that this study may yield insights into the convergent evolution of ubiquitously expressed proteins between vertebrates and invertebrates.