Ticagrelor increases its own potency at the P2Y12 receptor by directly changing the plasma membrane lipid order in platelets.

BACKGROUND AND PURPOSE: Although the amphiphilic nature of the widely used antithrombotic drug Ticagrelor is well known, it was never considered as a membranotropic agent capable of interacting with the lipid bilayer in a receptor-independent way. In this study, we investigated the influence of Ticagrelor on plasma membrane lipid order in platelets and if this modulates the potency of Ticagrelor at the P2Y12 receptor. EXPERIMENTAL APPROACH: We combined fluorescent in situ, in vitro and in silico approaches to probe the interactions between the plasma membrane of platelets and Ticagrelor. The influence of Ticagrelor on the lipid order of the platelet plasma membrane and large unilamellar vesicles was studied using the advanced fluorescent probe NR12S. Furthermore, the properties of model lipid bilayers in the presence of Ticagrelor were characterized by molecular dynamics simulations. Finally, the influence of an increased lipid order on the dose-response of platelets to Ticagrelor was studied. KEY RESULTS: Ticagrelor incorporates spontaneously into lipid bilayers and affects the lipid order of the membranes of model vesicles and isolated platelets, in a nontrivial composition and concentration-dependent manner. We showed that higher plasma membrane lipid order in platelets leads to a lower IC50 value for Ticagrelor. It is shown that membrane incorporation of Ticagrelor increases its potency at the P2Y12 receptor, by increasing the order of the platelet plasma membrane. CONCLUSION AND IMPLICATIONS: A novel dual mechanism of Ticagrelor action is suggested that combines direct binding to P2Y12 receptor with simultaneous modulation of receptor-lipid microenvironment.

Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct.

The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli.

TRPV4 expression in the renal tubule is necessary for maintaining whole body K+ homeostasis.

The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency.

TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium.

Mechanosensitive TRPV4 channel plays a dominant role in maintaining [Ca2+ ]i homeostasis and flow-sensitive [Ca2+ ]i signaling in the renal tubule. Polycystic kidney disease (PKD) manifests as progressive cyst growth due to cAMP-dependent fluid secretion along with deficient mechanosensitivity and impaired TRPV4 activity. Here, we tested how regulation of renal TRPV4 function by dietary K+ intake modulates the rate of cystogenesis and mechanosensitive [Ca2+ ]i signaling in cystic cells of PCK453 rats, a homologous model of human autosomal recessive PKD (ARPKD). One month treatment with both high KCl (5% K+ ) and KB/C (5% K+ with bicarbonate/citrate) diets significantly increased TRPV4 levels when compared to control (0.9% K+ ). High KCl diet caused an increased TRPV4-dependent Ca2+ influx, and partial restoration of mechanosensitivity in freshly isolated monolayers of cystic cells. Unexpectedly, high KB/C diet induced an opposite effect by reducing TRPV4 activity and worsening [Ca2+ ]i homeostasis. Importantly, high KCl diet decreased cAMP, whereas high KB/C diet further increased cAMP levels in cystic cells (assessed as AQP2 distribution). At the systemic level, high KCl diet fed PCK453 rats had significantly lower kidney-to-bodyweight ratio and reduced cystic area. These beneficial effects were negated by a concomitant administration of an orally active TRPV4 antagonist, GSK2193874, resulting in greater kidney weight, accelerated cystogenesis, and augmented renal injury. High KB/C diet also exacerbated renal manifestations of ARPKD, consistent with deficient TRPV4 activity in cystic cells. Overall, we demonstrate that TRPV4 channel activity negatively regulates cAMP levels in cystic cells thus attenuating (high activity) or accelerating (low activity) ARPKD progression.

Modus operandi of ClC-K2 Cl- Channel in the Collecting Duct Intercalated Cells.

The renal collecting duct is known to play a critical role in many physiological processes, including systemic water-electrolyte homeostasis, acid-base balance, and the salt sensitivity of blood pressure. ClC-K2 (ClC-Kb in humans) is a Cl--permeable channel expressed on the basolateral membrane of several segments of the renal tubule, including the collecting duct intercalated cells. ClC-Kb mutations are causative for Bartters' syndrome type 3 manifested as hypotension, urinary salt wasting, and metabolic alkalosis. However, little is known about the significance of the channel in the collecting duct with respect to the normal physiology and pathology of Bartters' syndrome. In this review, we summarize the available experimental evidence about the signaling determinants of ClC-K2 function and the regulation by systemic and local factors as well as critically discuss the recent advances in understanding the collecting-duct-specific roles of ClC-K2 in adaptations to changes in dietary Cl- intake and maintaining systemic acid-base homeostasis.

Plasma membrane lipid bilayer is druggable: Selective delivery of gemcitabine-squalene nano-medicine to cancer cells.

Up to now the lipid bilayers were rarely considered as targets in cancer therapy despite pronounced differences in lipid composition between plasma membranes of benign and malignant cells. In this study we demonstrate that the lipid bilayer of the plasma membrane is druggable and suitable for facilitating selective delivery of amphiphilic gemcitabine-squalene nanomedicines to cancer cells. Data from radioactive assays, fluorescent membrane probes and molecular dynamics simulations provide evidence of selective accumulation of gemcitabine-squalene in the plasma membranes with disrupted lipid asymmetry and its subsequent preferential uptake by malignant cells. This causes pronounced cytotoxicity on cancer cells in comparison to their benign counterparts originating from the same tissue.

Evolving concepts of TRPV4 in controlling flow-sensitivity of the renal nephron.

Kidneys are central for whole body water and electrolyte balance by first filtering plasma at the glomeruli and then processing the filtrate along the renal nephron until the final urine is produced. Renal nephron epithelial cells mediate transport of water and solutes which is under the control of systemic hormones as well as local mechanical stimuli arising from alterations in fluid flow. TRPV4 is a mechanosensitive Ca2+ channel abundantly expressed in different segments of the renal nephron. The accumulated evidence suggests a critical role for TRPV4 in sensing variations in flow rates. In turn, TRPV4 activation triggers numerous downstream cellular responses stimulated by elevated intracellular Ca2+ concentrations [Ca2+]i. In this review, we discuss the recent concepts in flow-mediated regulation of solute homeostasis by TRPV4 in different segments of renal nephron. Specifically, we summarize the evidence for TRPV4 involvement in endocytosis-mediated albumin uptake in the proximal tubule, reactive oxygen species (ROS) generation in the ascending loop of Henle, and maintaining K+ homeostasis in the connecting tubule/collecting duct. Finally, we outline the function and significance of TRPV4 in the setting of polycystic kidney disease.

Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission.

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[14C] glutamate and [3H] GABA, and also [3H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.

ClC-K2 Cl- channel allows identification of A- and B-type of intercalated cells in split-opened collecting ducts.

The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid-secreting A- and base secreting B-type of intercalated cells cannot be easily separated in functional studies. We used BCECF-sensitive intracellular pH (pHi ) measurements in split-opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell-specific ClC-K2-/- mice to demonstrate that ClC-K2 inhibition enables to distinguish signals from A- and B-intercalated cells. We show that ClC-K2 Cl- channel is expressed on the basolateral side of intercalated cells, where it governs Cl- -dependent H+ /HCO3- transport. ClC-K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC-K2-/- mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pHi in AE1-positive A-type and decreased pHi in pendrin-positive B-type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H+ secretion in A-type and decreased proton transport in B-type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance. Overall, we show that inhibition of ClC-K2 can be employed to discriminate between A- and B-type of intercalated cells in split-opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A- and B-type in response to systemic acid/base stimuli.

Epac1-/- and Epac2-/- mice exhibit deficient epithelial Na+ channel regulation and impaired urinary Na+ conservation.

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1-/- and Epac2-/- mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.

TTAPE-Me dye is not selective to cardiolipin and binds to common anionic phospholipids nonspecifically.

Identification, visualization, and quantitation of cardiolipin (CL) in biological membranes is of great interest because of the important structural and physiological roles of this lipid. Selective fluorescent detection of CL using noncovalently bound fluorophore 1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)-phenylethene (TTAPE-Me) has been recently proposed. However, this dye was only tested on wild-type mitochondria or liposomes containing negligible amounts of other anionic lipids, such as phosphatidylglycerol (PG) and phosphatidylserine (PS). No clear preference of TTAPE-Me for binding to CL compared to PG and PS was found in our experiments on artificial liposomes, Escherichia coli inside-out vesicles, or Saccharomyces cerevisiae mitochondria in vitro or in situ, respectively. The shapes of the emission spectra for these anionic phospholipids were also found to be indistinguishable. Thus, TTAPE-Me is not suitable for detection, visualization, and localization of CL in the presence of other anionic lipids present in substantial physiological amounts. Our experiments and complementary molecular dynamics simulations suggest that fluorescence intensity of TTAPE-Me is regulated by dynamic equilibrium between emitting dye aggregates, stabilized by unspecific but thermodynamically favorable electrostatic interactions with anionic lipids, and nonemitting dye monomers. These results should be taken into consideration when interpreting past and future results of CL detection and localization studies with this probe in vitro and in vivo. Provided methodology emphasizes minimal experimental requirements, which should be considered as a guideline during the development of novel lipid-specific probes.

With-No-Lysine Kinase 1 (WNK1) Augments TRPV4 Function in the Aldosterone-Sensitive Distal Nephron.

Kidneys play a central role in regulation of potassium homeostasis and maintenance of plasma K+ levels within a narrow physiological range. With-no-lysine (WNK) kinases, specifically WNK1 and WNK4, have been recognized to regulate K+ balance, in part, by orchestrating maxi K+ channel (BK)-dependent K+ secretion in the aldosterone-sensitive distal nephron (ASDN), which includes the connecting tubule and collecting duct. We recently demonstrated that the Ca2+-permeable TRPV4 channel is essential for BK activation in the ASDN. Furthermore, high K+ diet increases TRPV4 activity and expression largely in an aldosterone-dependent manner. In the current study, we aimed to test whether WNK kinases contribute to regulation of TRPV4 activity and its stimulation by aldosterone. Systemic inhibition of WNK with WNK463 (1 mg/kgBW for 3 days) markedly decreased TRPV4-dependent Ca2+ influx in freshly isolated split-opened collecting ducts. Aldosterone greatly increased TRPV4 activity and expression in cultured mpkCCDc14 cells and this effect was abolished in the presence of WNK463. Selective inhibition of WNK1 with WNK-in-11 (400 nM, 24 h) recapitulated the effects of WNK463 on TRPV4-dependent Ca2+ influx. Interestingly, WNK-in-11 did not interfere with up-regulation of TRPV4 expression by aldosterone, but prevented translocation of the channel to the apical plasma membrane. Furthermore, co-expression of TRPV4 and WNK1 into Chinese hamster ovary (CHO) cells increased the macroscopic TRPV4-dependent cation currents. In contrast, over-expression of TRPV4 with a dominant negative WNK1 variant (K233M) decreased the whole-cell currents, suggesting both stimulatory and permissive roles of WNK1 in regulation of TRPV4 activity. Overall, we show that WNK1 is essential for setting functional TRPV4 expression in the ASDN at the baseline and in response to aldosterone. We propose that this new mechanism contributes to regulation of K+ secretion and, by extension, urinary K+ levels to maintain systemic potassium homeostasis.

Angiotensin II increases activity of the ClC-K2 Cl- channel in collecting duct intercalated cells by stimulating production of reactive oxygen species.

The renal collecting duct plays a critical role in setting urinary volume and composition, with principal cells transporting Na+ and K+ and intercalated cells mediating Cl- reabsorption. Published evidence implies Angiotensin II (Ang II) is a potent regulator of the collecting duct apical transport systems in response to systemic volume depletion. However, virtually nothing is known about Ang II actions on the basolateral conductance of principal and intercalated cells. Here, we combined macroscopic and single channel patch clamp recordings from freshly isolated mouse collecting ducts with biochemical and fluorescence methods to demonstrate an acute stimulation of the basolateral Cl- conductance and specifically the ClC-K2 Cl- channel by nanomolar Ang II concentrations in intercalated cells. In contrast, Ang II did not exhibit measurable effects on the basolateral conductance and on Kir4.1/5.1 potassium channel activity in principal cells. Although both Ang II receptors AT1 and AT2 are expressed in collecting duct cells, we show that AT1 receptors were essential for stimulatory actions of Ang II on ClC-K2. Moreover, AT1R-/- mice had decreased renal ClC-K2 expression. We further demonstrated that activation of NADPH oxidases is the major signaling pathway downstream of Ang II-AT1R that leads to stimulation of ClC-K2. Treatment of freshly isolated collecting ducts with Ang II led to production of reactive oxygen species on the same timescale as single channel ClC-K2 activation. Overall, we propose that Ang II-dependent regulation of ClC-K2 in intercalated cells is instrumental for stimulation of Cl- reabsorption by the collecting duct, particularly during hypovolemic states.

Phospholipid distribution in the cytoplasmic membrane of Gram-negative bacteria is highly asymmetric, dynamic, and cell shape-dependent.

The distribution of phospholipids across the inner membrane (IM) of Gram-negative bacteria is unknown. We demonstrate that the IMs of Escherichia coli and Yersinia pseudotuberculosis are asymmetric, with a 75%/25% (cytoplasmic/periplasmic leaflet) distribution of phosphatidylethanolamine (PE) in rod-shaped cells and an opposite distribution in E. coli filamentous cells. In initially filamentous PE-lacking E. coli cells, nascent PE appears first in the periplasmic leaflet. As the total PE content increases from nearly zero to 75%, cells progressively adopt a rod shape and PE appears in the cytoplasmic leaflet of the IM. The redistribution of PE influences the distribution of the other lipids between the leaflets. This correlates with the tendency of PE and cardiolipin to regulate antagonistically lipid order of the bilayer. The results suggest that PE asymmetry is metabolically controlled to balance temporally the net rates of synthesis and translocation, satisfy envelope growth capacity, and adjust bilayer chemical and physical properties.

Fluorescence Probes Exhibit Photoinduced Structural Planarization: Sensing In Vitro and In Vivo Microscopic Dynamics of Viscosity Free from Polarity Interference.

We demonstrate the construction of wavelength λ-ratiometric images that allow visualizing the distribution of microscopic dynamics within living cells and tissues by using the newly developed principle of fluorescence response. The bent-to-planar motion in the excited state of incorporated fluorescence probes leads to elongation of the π-delocalization, resulting in microviscosity-dependent but polarity-insensitive interplay between well-separated blue and red bands in emission spectra. This allows constructing the exceptionally contrasted images of cellular dynamics. Moreover, the application of probes with increased affinity toward biological membranes allowed detecting the differences in dynamics between the plasma membrane and intracellular membrane structures. Such λ-ratiometric microviscosity imaging was extended for mapping the living tissues and observing their inflammation-dependent changes.

Double-exponential kinetics of binding and redistribution of the fluorescent dyes in cell membranes witness for the existence of lipid microdomains.

New technique of detecting lateral heterogeneity of the plasma membrane of living cells by means of membrane-binding fluorescent dyes is proposed. The kinetics of dye incorporation into the membrane or its lateral diffusion inside the membrane is measured and decomposed into exponential components by means of the Maximum Entropy Method. Two distinct exponential components are obtained consistently in all cases for several fluorescent dyes, two different cell lines and in different types of experiments including spectroscopy, flow cytometry and fluorescence recovery after photobleaching. These components are attributed to the liquid-ordered and disordered phases in the plasma membrane of studied cells in their dynamic equilibrium.

Apoptosis and eryptosis: Striking differences on biomembrane level.

The cell plasma membrane plays an essential role in programmed cell death of nucleated cells (apoptosis) and erythrocytes (eryptosis), and its changes due to loss of transmembrane asymmetry are quite similar. However, nucleated cells possess the network of intracellular membranes, which are missing in erythrocytes. Providing comparative studies with series of molecular probes, we observe dramatic differences in membrane lipid order in the course of apoptosis and eryptosis. In contrast to nucleated cells, in which a significant drop of the lipid order in the plasma membrane is observed, the erythrocyte membrane retains the relatively high level of the lipid order. Observation in nucleated cells of significant differences between inner and plasma membranes and detection of apoptotic bodies with different organization suggest that the decrease in the lipid order of their plasma membrane could be at least partially explained by the phospholipid and/or cholesterol exchange between membranes. Such features are absent in erythrocytes.

Caspase-3 activation decreases lipid order in the outer plasma membrane leaflet during apoptosis: A fluorescent probe study.

In this research we investigate the connection between the cytoplasmic machinery of apoptosis and the plasma membrane organization by studying the coupling of caspase-3 activation and inhibition with PS exposure and the change of lipid order in plasma membrane sensed by a fluorescent membrane probe NR12S. First, we performed in silico molecular dynamics simulations, which suggest that the mechanism of response of NR12S to lipid order may combine both sensitivity to membrane polarity/hydration and change in the fluorophore orientation. Second, cellular studies revealed that upon triggering apoptosis with IPA-3 and camptothecin the NR12S response is similar to that observed after decrease of lipid order induced by cholesterol depletion, 7-ketocholesterol enrichment or sphingomyelin hydrolysis. NR12S response can be influenced by a caspase-3 inhibitor Z-DEVD-FMK. Flow cytometry data further indicate that the NR12S response correlates with the response of FITC-labeled DEVD-FMK peptide and GFP-labeled Annexin V on the whole time scale (0-24h) of apoptosis induction by camptothecin. We conclude that fine changes in lipid order observed by NR12S are coupled with early steps of cellular events in apoptosis.

Visualization and detection of live and apoptotic cells with fluorescent carbon nanoparticles.

Apoptosis is a genetically encoded cell death program that involves different processes occurring on molecular and sub-cellular levels. Here we report on its new features--the increased accumulation of fluorescent carbon nanoparticles (CDots) in cells and their changed distribution within cell interior, which can witness on altered mechanisms of their translocation through the membrane. The comparative studies of living (intact) and apoptotic cells were provided with two cell lines (HeLa, Vero) using two types of fluorescent nanoparticles ("violet" and "blue" CDots). In all studied cases the images of living and apoptotic cells were different; the apoptotic cells incorporated larger number of CDots resulting in their much brighter images. These nanoparticles are distributed in cell cytoplasm, however, when the cells are fixed and treated with detergent, their nucleus is also labeled. Flow cytometry allows distinguishing the sub-populations of living and apoptotic cells in their cultures and suggests a very cheap and easy way to characterize them.

Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research.