Quantitative Lipid Imaging Reveals a New Signaling Function of Phosphatidylinositol-3,4-Bisphophate: Isoform- and Site-Specific Activation of Akt.

Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP3) and phosphatidylinositol-3,4-bisphophate (PI34P2), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quantitative imaging of PIP3 and PI34P2 in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P2 selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP3 selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling molecules. Collectively, our studies show that different spatiotemporal dynamics of PIP3 and PI34P2 and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.

Targeting lipid-protein interaction to treat Syk-mediated acute myeloid leukemia.

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid-SH2 domain interactions. Here we report development of new nonlipidic small molecule inhibitors of the lipid-SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid-SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity, but also its noncatalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid-protein interaction is a powerful approach to developing new small molecule drugs.

Accurate Cancer Screening and Prediction of PD-L1-Guided Immunotherapy Efficacy Using Quantum Dot Nanosphere Self-Assembly and Machine Learning.

Accurate quantification of exosomal PD-L1 protein in tumors is closely linked to the response to immunotherapy, but robust methods to achieve high-precision quantitative detection of PD-L1 expression on the surface of circulating exosomes are still lacking. In this work, we developed a signal amplification approach based on aptamer recognition and DNA scaffold hybridization-triggered assembly of quantum dot nanospheres, which enables bicolor phenotyping of exosomes to accurately screen for cancers and predict PD-L1-guided immunotherapeutic effects through machine learning. Through DNA-mediated assembly, we utilized two aptamers for simultaneous ultrasensitive detection of exosomal antigens, which have synergistic roles in tumor diagnosis and treatment prediction, and thus, we achieved better sample classification and prediction through machine-learning algorithms. With a drop of blood, we can distinguish between different cancer patients and healthy individuals and predict the outcome of immunotherapy. This approach provides valuable insights into the development of personalized diagnostics and precision medicine.

Electrochemiluminescence-Based Single-Particle Tracking of the Biomolecules Moving along Intercellular Membrane Nanotubes between Live Cells.

Electrochemiluminescence (ECL) imaging, a rapidly evolving technology, has attracted significant attention in the field of cellular imaging. However, its primary limitation lies in its inability to analyze the motion behaviors of individual particles in live cellular environments. In this study, we leveraged the exceptional ECL properties of quantum dots (QDs) and the excellent electrochemical properties of carbon dots (CDs) to develop a high-brightness ECL nanoprobe (CDs-QDs) for real-time ECL imaging between living cells. This nanoprobe has excellent signal-to-noise ratio imaging capabilities for the single-particle tracking (SPT) of biomolecules. Our finding elucidated the enhanced ECL mechanism of CDs-QDs in the presence of reactive oxygen species through photoluminescence, electrochemistry, and ECL techniques. We further tracked the movement of single particles on membrane nanotubes between live cells and confirmed that the ECL-based SPT technique using CD-QD nanoparticles is an effective approach for monitoring the transport behaviors of biomolecules on membrane nanotubes between live cells. This opens a promising avenue for the advancement of ECL-based single-particle detection and the dynamic quantitative imaging of biomolecules.

In Situ Quantitative Imaging of Plasma Membrane Stiffness in Live Cells Using a Genetically Encoded FRET Sensor.

Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

Ratiometric Fluorescence Probes for In Situ Imaging of Membrane Tension in Live Cells.

Membrane tension is an important physical parameter of describing cellular homeostasis, and it is widely used in the study of cellular processes involving membrane deformation and reorganization, such as cell migration, cell spreading, and cell division. Despite the importance of membrane tension, direct measurement remains difficult. In this work, we developed a ratiometric fluorescent probe sensitive to membrane tension by adjusting the carbon chain structure based on polarity-sensitive fluorophores. The probe is sensitive to changes in membrane tension after cells were subjected to physical or chemical stimuli, such as osmotic shock, lipid peroxidation, and mechanical stress. When the polarity of the plasma membrane increases (the green/red ratio decreases) and the membrane tension increases, the relative magnitude of the membrane tension can be quantitatively calculated by fluorescence ratio imaging. Thus, the probe proved to be an efficient and sensitive membrane tension probe.

Spatiotemporal Quantification of Endosomal Acidification on the Viral Journey.

Many enveloped viruses utilize endocytic pathways and vesicle trafficking to infect host cells, where the acidification of virus-containing endosomes triggers the virus-endosome fusion events. Therefore, simultaneous correlation of intracellular location, local pH, and individual virus dynamics is important for gaining insight into viral infection mechanisms. Here, an imaging approach is developed for spatiotemporal quantification of endosomal acidification on the viral journey in host cells using a fluorescence resonance energy transfer based ratiometric pH sensor consisting of a photostable and high-brightness QD, pH-sensitive fluorescent dyes, and virus-binding proteins. Ratiometric analysis of sensor-based single-virus tracking data enables to dissect a two-step endosomal acidification process during the infection of influenza viruses and elucidates the occurrence of the fission and sorting of virus-containing endosomes to recycling endosomes after initial acidification. This technique should serve as a robust approach for in situ quantification of endosomal acidification on the viral journey.

In Situ Quantification of Lipids in Live Cells by Using Lipid-Binding Domain-Based Biosensors.

Lipid molecules contribute to a large extent to the regulation of cellular signaling, as cellular signals are generated primarily through the selective interaction of various cellular proteins with lipids in the plasma membrane. Hence the location, concentration, and duration of lipids on the cell membrane are critical for the selection of proteins and the initiation of signaling. To monitor the concentration and location of lipid molecules on the cell membrane, researchers have developed a variety of lipid biosensors that allow quantitative in situ visualization of lipid molecules in living cells based on lipid-binding domains with high specificity, sensitivity, and biocompatibility, providing a powerful tool for the study of cellular signaling mechanisms involving lipid molecules. In this review, we first introduced the emergence of lipid-binding domains and then focused on the practical considerations on how to implement the lipid sensor, including probe selection, modification, characterization, and imaging techniques. We then described experimental observables and the relevant physicochemical parameters in the context of single-molecule studies in cells. Finally, we presented our views on the future development of lipid sensors and methods for lipid quantification.

Real-Time Dissecting the Dynamics of Drug Transportation in the Live Brain.

Brain diseases are becoming a more and more serious threat to human health. Many critical properties of the transport mechanisms of drugs in live brains remain poorly understood. In this work, single-particle tracking was used to dissect the transport dynamics of wheat germ agglutinin (WGA) in live brain and characterize the geometry and rheology of the extracellular space (ECS). The results revealed that the movements of WGA were influenced by the specific-binding molecules and the nature of the ECS. We further analyzed the mobility behaviors of WGA globally and quantitatively and found that movement of WGA in brain cells of acute slices was an active transport process associated with actin filaments and microtubules. This work paves the way for studies aiming at characterizing the biophysics of drug transport in the context of live brains, which may contribute to developing potential new therapeutic applications for brain diseases.

Accurate and Efficient Lipoprotein Detection Based on the HCR-DNAzyme Platform.

Cardiovascular disease is one of the main causes of death in the world, which is closely associated with dyslipidemia. Dyslipidaemia is usually manifested as a relatively higher level of low-density lipoprotein (LDL) and lower level of high-density lipoprotein (HDL). Thus, the quantitative detection of the LDL and HDL particles is of great importance to predict the risk of cardiovascular diseases. However, the traditional methods can only indirectly reflect the HDL/LDL particle concentrations by detecting the cholesterol or proteins in HDL/LDL particles and are always laborious and time-consuming. Thus, the accurate and efficient approach for the detection of intact HDL and LDL particles is still lacking so far. We developed an enzyme- and isolation-free method to measure the concentration of HDL and LDL based on DNAzyme and hybridization chain reaction (HCR)-based signal amplification. This method can be used to directly and accurately detect the concentration of "actual" HDL and LDL particles instead of the cholesterol in HDL and LDL, with limits of detection of 10 and 30 mg/dL, respectively, which also satisfied the lipoprotein analysis in clinical samples. Therefore, this HCR-DNAzyme platform has great potential in clinical applications and health management.

Quantitative single-virus tracking for revealing the dynamics of SARS-CoV-2 fusion with plasma membrane.

Viral envelope fusion with the host plasma membrane (PM) for genome release is a hallmark step in the life cycle of many enveloped viruses. This process is regulated by a complex network of biomolecules on the PM, but robust tools to precisely elucidate the dynamic mechanisms of virus-PM fusion events are still lacking. Here, we developed a quantitative single-virus tracking approach based on highly efficient dual-color labelling of viruses and batch trajectory analysis to achieve the spatiotemporal quantification of fusion events. This approach allows us to comprehensively analyze the membrane fusion mechanism utilized by pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the single-virus level and precisely elucidate how the relevant biomolecules synergistically regulate the fusion process. Our results revealed that SARS-CoV-2 may promote the formation of supersaturated clusters of cholesterol to facilitate the initiation of the membrane fusion process and accelerate the viral genome release.

Chronic sleep fragmentation impairs brain interstitial clearance in young wildtype mice.

Accumulating evidence shows that most chronic neurological diseases have a link with sleep disturbances, and that patients with chronically poor sleep undergo an accelerated cognitive decline. Indeed, a single-night of sleep deprivation may increase metabolic waste levels in cerebrospinal fluid. However, it remains unknown how chronic sleep disturbances in isolation from an underlying neurological disease may affect the glymphatic system. Clearance of brain interstitial waste by the glymphatic system occurs primarily during sleep, driven by multiple oscillators including arterial pulsatility, and vasomotion. Herein, we induced sleep fragmentation in young wildtype mice and assessed the effects on glymphatic activity and cognitive functions. Chronic sleep fragmentation reduced glymphatic function and impaired cognitive functions in healthy mice. A mechanistic analysis showed that the chronic sleep fragmentation suppressed slow vasomotion, without altering cardiac-driven pulsations. Taken together, results of this study document that chronic sleep fragmentation suppresses brain metabolite clearance and impairs cognition, even in the absence of disease.

In-situ synthesis of quantum dots in the nucleus of live cells.

The cell nucleus is the main site for the storage and replication of genetic material, and the synthesis of substances in the nucleus is rhythmic, regular and strictly regulated by physiological processes. However, whether exogenous substances, such as nanoparticles, can be synthesized in situ in the nucleus of live cells has not been reported. Here, we have achieved in-situ synthesis of CdSxSe1-x quantum dots (QDs) in the nucleus by regulation of the glutathione (GSH) metabolic pathway. High enrichment of GSH in the nucleus can be accomplished by the addition of GSH with the help of the Bcl-2 protein. Then, high-valence Se is reduced to low-valence Se by glutathione-reductase-catalyzed GSH, and interacts with the Cd precursor formed through Cd and thiol-rich proteins, eventually generating QDs in the nucleus. Our work contributes to a new understanding of the syntheses of substances in the cell nucleus and will pave the way for the development of advanced 'supercells'.

In-situ quantification of lipids in live cells through imaging approaches.

Lipids are important molecules that are widely distributed within the cell, and they play a crucial role in several biological processes such as cell membrane formation, signaling, cell motility and division. Monitoring the spatiotemporal dynamics of cellular lipids in real-time and quantifying their concentrations in situ is crucial since the local concentration of lipids initiates various signaling pathways that regulate cellular processes. In this review, we first introduced the historical background of lipid quantification methods. We then delve into the current state of the art of in situ lipid quantification, including the establishment and utility of fluorescence imaging techniques based on sensors of lipid-binding domains labeled with organic dyes or fluorescent proteins, and Raman and magnetic resonance imaging (MRI) techniques that do not require lipid labeling. Next, we highlighted the biological applications of live-cell lipid quantification techniques in the study of in situ lipid distribution, lipid transformation, and lipid-mediated signaling pathways. Finally, we discussed the technical challenges and prospects for the development of lipid quantification in live cells, with the aim of promoting the development of in situ lipid quantification in live cells, which may have a profound impact on the biological and medical fields.

PI(3,5)P 2 Controls the Signaling Activity of Class I PI3K.

3'-Phosphoinositides are ubiquitous cellular lipids that play pivotal regulatory roles in health and disease. Generation of 3'-phosphoinositides are driven by three families of phosphoinositide 3-kinases (PI3K) but the mechanisms underlying their regulation and cross-talk are not fully understood. Among 3'-phosphoinositides, phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) remains the least understood species in terms of its spatiotemporal dynamics and physiological function due to the lack of specific probes. By means of spatiotemporally resolved in situ quantitative imaging of PI(3,5)P 2 using a newly developed ratiometric PI(3,5)P 2 sensor we demonstrate that a special pool of PI(3,5)P 2 is generated on lysosomes and late endosomes in response to growth factor stimulation. This PI(3,5)P 2 pool, the formation of which is mediated by Class II PI3KC2ß and PIKFyve, plays a crucial role in terminating the activity of growth factor-stimulated Class I PI3K, one of the most frequently mutated proteins in cancer, via specific interaction with its regulatory p85 subunit. Cancer-causing mutations of Class I PI3K inhibit the p85-PI(3,5)P 2 interaction and thereby induce sustained activation of Class I PI3K. Our results unravel a hitherto unknown tight regulatory interplay between Class I and II PI3Ks mediated by PI(3,5)P 2 , which may be important for controlling the strength of PI3K-mediated growth factor signaling. These results also suggest a new therapeutic possibility of treating cancer patients with p85 mutations.

A visualization pipeline for in vivo two-photon volumetric astrocytic calcium imaging.

Astrocytes, the multi-functional glial cells with the most abundant population in the brain, integrate information across their territories to regulate neuronal synaptic and cerebrovascular activities. Astrocytic calcium (Ca 2+) signaling is the major readout of cellular functional state of astrocytes. The conventional two-photon in vivo imaging usually focuses on a single horizontal focal plane to capture the astrocytic Ca 2+ signals, which leaves >80% spatial information undetected. To fully probe the Ca 2+ activity across the whole astrocytic territory, we developed a pipeline for imaging and visualizing volumetric astrocytic Ca 2+ time-lapse images. With the pipeline, we discovered a new signal distribution pattern from three-dimensional (3D) astrocytic Ca 2+ imaging data of mice under isoflurane anesthetic states. The tools developed in this study enable a better understanding of the spatiotemporal patterns of astrocytic activity in 3D space.

High-Throughput Fluorometric Assay for Membrane-Protein Interaction.

Membrane-protein interaction plays key roles in a wide variety of biological processes. To facilitate rapid and sensitive measurement of membrane binding of soluble proteins, we developed a fluorescence-based quantitative assay that is universally applicable to all proteins. This fluorescence-quenching assay employs fluorescence protein (FP)-tagged proteins whose fluorescence intensity is greatly decreased when they bind vesicles containing synthetic lipid dark quenchers, such as N-dimethylaminoazobenzenesulfonylphosphatidylethanolamine (dabsyl-PE). This simple assay can be performed with either a spectrofluorometer or a plate reader and optimized for different proteins with various combinations of FPs and quenching lipids. The assay allows rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains and proteins, and also high-throughput screening of small molecules that modulate membrane binding of proteins.