Membrane Labeling Research Articles

Peptide-Conjugated Aggregation-Induced Emission Fluorogen: Precise and Firm Cell Membrane Labeling by Multiple Weak Interactions

Cell membrane is a vital barrier to protect the cell from outside damage and involved in many biochemical processes. It is of great significance to label the cell membrane to explore its function. ...

Buccal Mucosa Cells as a Potential Diagnostic Tool to Study Onset and Progression of Arrhythmogenic Cardiomyopathy

In arrhythmogenic cardiomyopathy (ACM) pathogenic variants are found in genes encoding desmosomal proteins and in non-desmosomal genes, such as phospholamban (PLN, p.Arg14del variant). Previous research showed that plakoglobin protein levels and localization in the cardiac tissue of ACM patients, and PLN p.Arg14del patients diagnosed with an ACM phenotype, are disturbed. Moreover, the effects of pathogenic variants in desmosomal genes are reflected in non-cardiac tissues like buccal mucosa cells (BMC) which could serve as a promising new and non-invasive tool to support diagnosis. We collected the BMC of 33 ACM patients, 17 PLN p.Arg14del patients and 34 controls, labelled the BMC with anti-plakoglobin antibodies at different concentrations, and scored their membrane labelling. We found that plakoglobin protein levels were significantly reduced in BMC obtained from diagnosed ACM patients and preclinical variant carriers when compared to controls. This effect was independent from age and sex. Moderate to strong correlations were found with the revised 2010 Task Force Criteria score which is commonly used for ACM diagnosis (rs = −0.67, n = 64, p < 0.0001 and rs = −0.71, n = 64, p < 0.0001). In contrast, plakoglobin scores in PLN p.Arg14del patients were comparable to controls (p > 0.209), which suggests differences in underlying etiology. However, for the individual diagnosis of the ‘classical’ ACM patient, this method might not be discriminative enough to distinguish true patients from variant carriers and controls, because of the high interindividual variability.

Knotting Two Donor−π-Acceptor AIEgens Using a Nonconjugated Linker: Tunable and Switchable Fluorescence and Fingerprinting and Live Cell Imaging Applications

Aggregation-induced emissive fluorophores (AIEgens) have drawn widespread attention over the years because of their structure-controlled tunable and external-stimuli-responsive solid-state fluorescence, which have stimulated rapid development in the optoelectronics and biomedical fields. However, precise control of organic π-conjugated molecular fluorescence remains a great challenge and depends on the molecular structure and organization. Herein, we have interlinked two triphenylamine-based donor−π acceptor (D−π-A) AIEgens using a nonconjugate spacer (xylene) and explored the effect of interlinking, positional isomerism, and acceptor structure on the tunable and switchable solid-state fluorescence. A change of the acceptor group resulted in tuning of fluorescence between 534 and 614 nm. Interestingly, interspacing modulation from o-xylene (1) to the meta (2) and para isomers (3) showed a decrease of fluorescence efficiency in the solid state. A closer spacing between the AIEgens and tighter molecular packing could restrict the intramolecular rotation in ortho isomers that resulted in enhanced fluorescence in comparison to para isomers. In contrast, more spatial freedom in para isomers allowed the molecules to respond to external pressure and produced mechanofluorochromism (MFC) in comparison to ortho isomers. Strong tunable solid-state fluorescence of ortho isomers was used to obtain clear latent fingerprinting images on three different surfaces with tunable fluorescence color. 1a,b,e (orange, green, and red, respectively) were further utilized for live cell imaging applications using E. coli cells that demonstrated clear labeling of the cell membrane and cytoplasm with strong green/red fluorescence in the cell structures. In vitro and in vivo toxicity studies of 1a,b,e confirmed the nontoxic nature of these fluorophores and indicated their potential for live cell imaging applications.

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.

Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis.

Research on extracellular vesicles (EVs) has intensified over the past decade, including fluorescent membrane labeling of EVs. An optimal fluorescent method requires the size of EVs to be preserved after labeling. Lipophilic fluorescent dyes, such as CellMask™ Green (CMG), have been widely used for this purpose. Here, we investigated conditions affecting the optimum CMG labeling of EVs derived from human choriocarcinoma cells (JAr) and different biological fluids using fluorescence NTA (fl-NTA). The effect of CMG labeling on the size, concentration and zeta potential (ZP) on JAr EVs purified with different methods were measured along with biological fluid-derived EVs. With the increase of CMG dye concentration, a significant decrease in the mean size of fluorescent nanoparticles (fl-NPs) was observed. The ZP of fl-NPs originating from JAr cells with the lowest and highest dye concentrations showed a significant shift towards more and less negative ZP values, respectively. Differences in the concentration of fl-NPs were observed for JAr EVs purified using size-exclusion chromatography (SEC) alone and SEC in combination with tangential flow filtration. The proportion of CMG labeling of NPs varied across different biological sources. CMG labeling may be a reliable technique for the detection of EVs using fl-NTA.

Spontaneous Disseminated Histiocytic Sarcoma in a Bengal Tiger (Panthera tigris tigris)

A 16-year-old, female, captive Bengal tiger (Panthera tigris tigris) had a history of a recurrent subcutaneous mass. After two attempts at surgical removal, euthanasia was elected during the third surgical attempt due to the degree of neoplastic infiltration. At necropsy, a large subcutaneous mass infiltrated the dermis, subcutis and abdominal wall with metastasis to the lungs, spleen and adrenal glands. Microscopically, the neoplasm formed nodules comprising sheets of round cells with large irregular nuclei. Toluidine blue and Giemsa stains were negative. An immunohistochemistry panel revealed membranous and cytoplasmic labelling with ionized calcium-binding adaptor molecule 1 (IBA1), cytoplasmic labelling with vimentin but no labelling with multiple myeloma oncogene 1 (MUM1), CD20 or pancytokeratin markers. Based on the gross distribution and histological and immunohistochemistry features, a diagnosis of disseminated histiocytic sarcoma was made. To the best of our knowledge, this is the first record of disseminated histiocytic sarcoma in a captive Bengal tiger.

Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line

A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50–60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.

Concentration, cellular exposure and specificity of organelle selective fluorescent dyes in fungal cell biology

This technical focus article discusses the importance of concentration, cellular exposure and specificity for the application of organelle selective fluorescent dyes in fungi using DNA, membrane and cell wall stains as examples. Nonetheless, the presented considerations are generally applicable to all fluorescent dyes applied to living cells.The association of a fluorescent dye with its target molecule generally impairs molecule and consequently organelle function. Effective dye concentration, cellular exposure time and specificity to the target molecule are key factors that influence the biocompatibility of any fluorescent dye. Prominent molecules frequently used as fluorescent staining targets in fungal cell biology are: (i) DNA for nuclear labelling, (ii) α-/β-glucans and chitin for cell wall labelling, and (iii) phospholipids for plasma membrane and endomembrane labelling. In combination with live-cell imaging settings that reduce light stress, i.e. excitation intensities and exposure times set to the minimum that still achieves good signal-to-noise ratios, is the low dosage application of fluorescent markers as so called “vital dyes” essential for visualising cellular processes in an artefact-free fashion.

Probing the Role of Chirality in Phospholipid Membranes.

Nucleotides, amino acids, sugars, and lipids are almost ubiquitously homochiral within individual cells on Earth. While oligonucleotides and proteins exist as one natural chirality throughout the tree of life, two stereoisomers of phospholipids have separately emerged in archaea and bacteria, an evolutionary divergence known as "the lipid divide". Within this review, we focus on the emergence of phospholipid homochirality and compare the stability of synthetic homochiral and heterochiral membranes in vitro. We discuss chemical probes designed to study the stereospecific interactions of lipid membranes in vitro. Overall, we aim to highlight studies that help elucidate the determinants of stereospecific interactions between lipids, peptides, and small molecule ligands. Continued work in understanding the drivers of favorable interactions between chiral molecules and biological membranes will lead to the design of increasingly selective chemical tools for bioorthogonal labeling of lipid membranes and safer membrane-associating pharmaceuticals.

A semiautomated measurement of muscle fiber size using the Imaris software.

The size and shape of skeletal muscle fibers are affected by various physiological and pathological conditions, such as muscle atrophy, hypertrophy, regeneration, and dystrophies. Hence, muscle fiber cross-sectional area (CSA) is an important determinant of muscle health and plasticity. We adapted the Imaris software to automatically segment muscle fibers based on fluorescent labeling of the plasma membrane and measure muscle fiber CSA. Analysis of muscle cross sections by the Imaris semiautomated and manual approaches demonstrated a similar decrease in CSA of atrophying muscles from fasted mice compared with fed controls. In addition, we previously demonstrated that downregulation of the Ca2+-specific protease calpain-1 attenuates muscle atrophy. Accordingly, both the Imaris semiautomated and manual approaches showed a similar increase in CSA of fibers expressing calpain-1 shRNA compared with adjacent nontransfected fibers in the same muscle cross section. Although both approaches seem valid for measurements of muscle fiber size, the manual marking method is less preferable because it is highly time-consuming, subjective, and limits the number of cells that can be analyzed. The Imaris semiautomated approach is user-friendly, requires little training or optimization, and can be used to efficiently and accurately mark thousands of fibers in a short period. As a novel addition to the commonly used statistics, we also describe statistical tests that quantify the strength of an effect on fiber size, enabling detection of significant differences between skewed distributions that would otherwise not be detected using typical methods.

Spatiotemporal Resolved Live Cell Membrane Tracking through Photo-click Reactions Enriched in Lipid Phase.

A set of photo-switchable monopeptides derived from cis-β-dibenzodiazocine-l-alanine (cis-DBDAA) have been designed and synthesized, which are capable of photo-click reacting with diaryltetrazoles or diarylsydnones in a hydrophobic phospholipid bilayer environment. The DBDAA monopeptides include both a hydrophobic tail on C-terminal, providing high affinity toward lipid membrane, and a modularized functional moiety on N-terminal, enabling rapid optimization of the self-assembly strength to form multifunctional supramolecules. With the cis-DBDAA monopeptides photo-switched into trans-configuration, we were able to disrupt the supramolecular assembly through an efficient photo-click reaction across the lipid bilayer of liposomes. We reveal that the performance of the photo-click reactions between the monopeptides and photo-generated nitrile imine intermediates is significantly enhanced by enrichment of both reactants in the hydrophobic membrane lamel of liposomes. Enrichment of the DBDAA monopeptide in lipid phase serves as a convenient method to introduce bioorthogonal chemical handles on live cell membranes, which enables fluorescence labelling of single cell's membrane with high spatiotemporal resolution to facilitate the studies on cell membrane dynamics.

Buccal mucosa cells as a diagnostic tool in patients with arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Netherlands Cardio Vascular Research Initiative (CVON): the Dutch heart foundation Background Arrhythmogenic cardiomyopathy (ACM) is predominantly caused by mutations in genes encoding desmosomal proteins (most often multiple different mutations in plakophilin-2), however also mutations in non-desmosomal proteins like phospholamban (PLN, PLNR14Del) are found. Previous investigations showed that plakoglobin protein levels and localization in cardiac tissue of ACM patients is disturbed and this could be a valuable additional tool to discriminate between non-affected family members and those being at risk during progression of the disease. Information about cardiac plakoglobin status can only be obtained via endocardial biopsies, however, this technique has major draw-backs and risks, and therefore an alternative is needed. A promising new non-invasive tool is the use of buccal mucosa cells (BMC), as it has been reported that effects of mutations in desmosomal proteins are additionally reflected in non-cardiac tissues like buccal mucosa smears that do also express those desmosomal genes. Purpose To investigate the clinical usability of BMC as tool to classify patients at risk of developing ACM by comparing healthy controls with asymptomatic and symptomatic classical ACM patients, and patients carrying a PLNR14Del mutation. Methods and results BMC of 84 human subjects, 33 ACM patients (19 males, 28 with a PKP2 mutation), 17 PLN patients (6 males) and 34 healthy controls (14 males), were collected on glass slides, fixed and labelled with anti-plakoglobin antibodies diluted 1:5.000, 1:10.000, 1:20.000 and 1:40.000, and scored for their membrane labelling. Data revealed that for each applied dilution, plakoglobin protein levels at the membrane were significantly reduced in BMC obtained from ACM patients compared to healthy controls. This effect appeared independent from age and sex of the patients. Furthermore, dilutions 1:5.000 and 1:10.000 showed a moderate correlation with the revised 2010 Task Force Criteria Score (TFC) which are commonly used for ACM diagnosis (Rs -0.67, n = 64, p &amp;lt;0.0001 and Rs -0.71, n = 64, p &amp;lt; 0.0001 resp.) In contrast, plakoglobin scores of PLN patients were similar to controls. Conclusion On the population level, there is a significant reduction of plakoglobin protein in BMC membranes of ACM patients but not for patients bearing the PLNR14Del mutation when compared to healthy controls. However, for clinical diagnosis of the individual patient, this method is most likely not discriminative enough to distinguish patients from healthy controls, because of the high interindividual variability.

Histologic, immunohistochemical, and scanning electron microscopic comparison of pre‐iridal monocellular and fibrovascular membranes in normal and glaucomatous canine globes

(i) To evaluate immunohistochemical labeling of pre-iridal monocellular and fibrovascular membranes and (ii) describe the light and scanning electron microscopic (SEM) characteristics of these membranes in glaucomatous and normal/control canine globes. All globes were evaluated with light microscopy. Immunohistochemical labeling for CD18, Smooth muscle actin (SMA), and CD117 was completed on 40 canine globes with congenital/anterior segment dysgenesis-associated glaucoma (n=10), primary/goniodysgenesis-associated glaucoma (n=10), secondary glaucoma (n=10), and normal/control globes (n=10). SEM was completed on 10 globes: 5 with monocellular membranes, 3 with fibrovascular membranes, and 2 without a histologically detectable membrane. Monocellular membranes were detected in all normal/control globes with light microscopy and appeared to be morphologically very similar to those in diseased globes. CD18 labeling was detected in 9/10 monocellular membranes in normal/control globes, 15/23 monocellular, and 7/8 fibrovascular membranes in globes with glaucoma. SMA and CD117 labeling was not detected in monocellular membranes of normal/control globes. SMA was expressed in 10/23 monocellular and 7/8 fibrovascular membranes of glaucomatous globes. CD117 was expressed in 7/23 monocellular and 5/8 fibrovascular membranes of glaucomatous globes. SEM of monocellular membranes revealed a continuous sheet of mostly spindle cells and few individual round cells that extended over the anterior iris face in normal/control and all glaucomatous globes. Pre-iridal monocellular membranes are a normal component of the anterior iris surface, and CD18 immunoreactivity suggests some cells within these are of leukocytic origin. SMA and CD117 labeling of monocellular membranes in glaucomatous, but not normal/control globes, suggest metaplastic cellular change secondary to intraocular pathology related to glaucoma.

Small Molecule Imaging Agent for Mutant KRAS G12C

Multiple potent covalent inhibitors for mutant KRAS G12C have been described and some are in clinical trials. These small molecule inhibitors potentially allow for companion imaging probe development, thereby expanding the chemical biology toolkit to investigate mutant KRAS biology. Herein, we synthesized and tested a series of fluorescent companion imaging drugs (CID) for KRAS G12C, using two scaffolds, ARS-1323 and AMG-510. We created four fluorescent derivatives of each by attaching BODIPY dyes. We found that two fluorescent derivatives (BODIPY FL and BODIPY TMR) of ARS-1323 bind mutant KRAS and can be used for biochemical binding screens. Unfortunately, these drugs could not be used as direct imaging agents in cells, likely because of non-specific membrane labeling. To circumvent this challenge, we then used a two step procedure in cancer cells where an ARS-1323 alkyne is used for target binding followed by fluorescence imaging after in situ click chemsitry with picolyl azide Alexa Fluor 647. We show that this approach can be used to image mutant KRAS G12C directly in cells. Given the current lack of mutant KRAS G12C specific antibodies, these reagents could be useful for specific fluorescence imaging.

In Situ Stable Generation of Reactive Intermediates by Open Microfluidic Probe for Subcellular Free Radical Attack and Membrane Labeling.

Subcellular stimulation by free radicals is crucial for deeper insight of cell behaviors. However, it remains a tough challenge due to the high spatial precision requirement and short life of radicals. Herein, we report a versatile open microfluidic probe for stable generation of free radical and subcellular stimulation. By optimizing parameters, the chemical reaction can be confined in a microregion with a diameter of several μm, and the real-time produced reactive radicals can attack the desired subcellular region of a single cell. In order to reveal the attacked region, fluorescent cyanine 3 labeled tyramide free radicals are synthesized, and the target microregion on a single cell is successfully stained by the covalent linking reaction between radicals and membrane proteins, which proves the feasibility of our method. We believe this method will open new avenues for short-lived reactive intermediates stimulation at the single-cell/sub-cell level and selective membrane labeling.

In Situ Stable Generation of Reactive Intermediates by Open Microfluidic Probe for Subcellular Free Radical Attack and Membrane Labeling

AbstractSubcellular stimulation by free radicals is crucial for deeper insight of cell behaviors. However, it remains a tough challenge due to the high spatial precision requirement and short life of radicals. Herein, we report a versatile open microfluidic probe for stable generation of free radical and subcellular stimulation. By optimizing parameters, the chemical reaction can be confined in a microregion with a diameter of several μm, and the real‐time produced reactive radicals can attack the desired subcellular region of a single cell. In order to reveal the attacked region, fluorescent cyanine 3 labeled tyramide free radicals are synthesized, and the target microregion on a single cell is successfully stained by the covalent linking reaction between radicals and membrane proteins, which proves the feasibility of our method. We believe this method will open new avenues for short‐lived reactive intermediates stimulation at the single‐cell/sub‐cell level and selective membrane labeling.

Abnormal N-Glycosylation of Human Lens Epithelial Cells in Type-2 Diabetes May Contribute to Cataract Progression.

PurposeIn order to better understand cataract development, we analyzed the glycosylation profile of human lens epithelial cells (HLECs) from anterior lens capsules of type 2 diabetes mellitus (T2DM) and non-diabetic (ND) patients undergoing routine cataract surgery.SettingResearch Department of the Asociación para Evitar la Ceguera, Hospital “Dr. Luis Sánchez Bulnes”, Mexico.DesignExperimental study.MethodsEvaluation of anterior lens capsules from T2DM and ND patients undergoing phacoemulsification and free from other ocular diseases.ResultsHematoxylin-eosin staining revealed HLECs alterations in T2DM samples. From lectins with different sugar specificities used, concanavalin A showed significant differences, labeling homogeneously both in the cytoplasm and in cell membranes in ND capsules, while in T2DM capsules, in addition to membrane and cytoplasm labeling, there were perinuclear vesicles with high concanavalin A labeling. Two-dimensional gel electrophoresis showed that T2DM patients have a ~65-kDa spot with an isoelectric point of 5.5 with a higher density compared to ND capsules, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed 62% homology with type-1 cytokeratin. Immunohistochemistry using anti-pan cytokeratin antibody revealed co-localization with concanavalin A, and a lectin blot revealed with concanavalin A showed a band of ~65 kDa, a molecular weight that corresponds to human type 1 cytokeratin.ConclusionThese results suggest that over-expression of N-glycosidically linked human type 1 cytokeratin may induce capsule disruption and affect selective permeability, allowing the entry of different molecules to the lens that facilitate cataract progression.

Abstract IA015: Niche-dependent control of tumor cell dormancy

Abstract Dormancy is an elusive and deadly component of cancers. Rare, therapy resistant cells lay dormant for decades and when reactivated cause disease progression and relapse. Eradicating dormant cancer cells is key to curing cancers yet is an unrealized goal. The skeleton remains a common location for dissemination and dormancy, yet our understanding of the cellular and molecular pathways that control dormant cancer cells in the the skeleton is limited. We hypothesized that dormant cancer cells occupy a common niche in the skeleton and this supports long-term dormancy. To test this we developed technology to identify and analyse dormant cancers cells from different cancer types and the compartment in the skeleton in which they reside. Membrane label retention was able to distinguish dormant cancer cells from reactivated cancer cells. Intravital imaging showed that dormant cancer cells were found associated with endosteal bone surface suggesting that different cancers may occupy a common niche. Single cell RNA sequencing of dormant cancer cells showed they expressed a distinct gene signature that was enriched for myeloid genes. Single cell RNA sequencing of &amp;gt;130,000 cells isolated from the endosteal bone compartment and the bone marrow identified 32 distinct cell clusters. Detailed transcriptional analysis facilitated construction of a map of all of the cell types/states present in the endosteal bone compartment. In silico ligand/receptor interaction mapping enabled identification of the cell types and the molecular pathways that may mediate dormant cell niche formation in vivo. Non-haemopoietic cells, particularly cells of the osteoblast lineage and endothelia cells were the most enriched for dormant cell binding partners. This was common across three different dormant tumor types. Detailed analysis of cells of the osteoblast lineage showed greatest enrichment for binding partners in LeprHigh/Cxcl12High mesenchymal cells. Further analysis of the molecular pathways that can interact with binding partners identified a number of potential molecular regulators of dormancy. For example, Gas6, which is expressed by LeprHigh/Cxcl12High mesenchymal cells, has the binding partners Axl expressed by dormant myeloma cells, Mertk in dormant breast cancer cells and Mertk and Tyro3 in dormant prostate cancer. Treatment of mice bearing myeloma cells with small molecule inhibitors of Axl reduced dormant cells and increased tumor burden suggesting the Axl/Gas6 interaction is functional important in controlling dormancy. Together these data show that single cell sequencing can be used to define the cells and molecular pathways that facilitate dormant cancer cell niche formation in the skeleton. This approach suggests that that cancer cell specific molecules interact with common molecules in the endosteal niche, including LeprHigh/Cxcl12High mesenchymal cells, to switch on common molecular pathways to control dormancy. Citation Format: Peter Croucher, Weng Hua Khoo, Ryan Chai, Alex Corr, James Smith, Qihao Ren, Paul Baldock, Michelle McDonald, Sheila Stewart, Tri G. Phan. Niche-dependent control of tumor cell dormancy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA015.

Solvatochromic and pH-Sensitive Fluorescent Membrane Probes for Imaging of Live Cells.

Membrane trafficking is essential for all cells, and visualizing it is particularly useful for studying neuronal functions. Here we report the synthesis, characterization, and application of several membrane- and pH-sensitive probes suitable for live-cell fluorescence imaging. These probes are based on a 1,8-naphthalimide fluorophore scaffold. They exhibit a solvatochromic effect, and one of them, ND6, shows a substantial fluorescence difference between pH 6 and 7. The solvatochromic effect and pH-sensitivity of those probes are explained using quantum chemical calculations, and molecular dynamics simulation confirms their integration and interaction with membrane lipids. For live-cell fluorescence imaging, we tested those probes in a cancer cell line (MCF7), cancer spheroids (MDA-MB-468), and cultured hippocampal neurons. Confocal imaging showed an excellent signal-to-noise ratio from 400:1 to about 1300:1 for cell membrane labeling. We applied ND6 during stimulation to label nerve terminals via dye uptake during evoked synaptic vesicle turnover. By ND6 imaging, we revealed cholesterol's multifaced role in replenishing synaptic vesicle pools. Our results demonstrate these fluorescent probes' great potential in studying membrane dynamic and synaptic functions in neurons and other secretory cells and tissues.

Solid-Phase Synthesis of Fluorescent Probes for Plasma Membrane Labelling.

The cellular plasma membrane plays a fundamental role in biological processes, including cell growth, signaling and transport. The labelling of the plasma membrane with targeted fluorescent probes offers a convenient and non-invasive way to image the morphological changes and dynamics of a membrane in real-time and, despite many examples of fluorescent plasma membrane probes, a “universal targeting/anchoring moiety” is still required. In this study, a small number of stearic acid-based probes labelled with 6-carboxyfluorescein was designed and fabricated via solid-phase synthesis in which variations in both charge and hydrophobicity were explored. To ease the synthesis process, a gram-scale synthesis of the Fmoc-Lys(6-carboxyfluoresein diacetate)-OH building block was developed, allowing the discovery of optimal probes that carried a positively charged amino group and a stearic acid tail that exhibited intense plasma membrane brightness and robust retention.