Membrane Dye Research Articles

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer.

Extracellular vesicles (EVs) are membranous particles released by most cells in our body, which are involved in many cell-to-cell signaling processes. Given the nanometer sizes and heterogeneity of EVs, highly sensitive methods with single-molecule resolution are fundamental to investigating their biophysical properties. Here, we demonstrate the sizing of EVs using a fluorescence-based flow analyzer with single-molecule sensitivity. Using a dye that selectively partitions into the vesicle's membrane, we show that the fluorescence intensity of a vesicle is proportional to its diameter. We discuss the constraints in sample preparation which are inherent to sizing nanoscale vesicles with a fluorescent membrane dye and propose several guidelines to improve data consistency. After optimizing staining conditions, we were able to measure the size of vesicles in the range ∼35-300 nm, covering the spectrum of EV sizes. Lastly, we developed a method to correct the signal intensity from each vesicle based on its traveling speed inside the microfluidic channel, by operating at a high sampling rate (10 kHz) and measuring the time required for the particle to cross the laser beam. Using this correction, we obtained a threefold greater accuracy in EV sizing, with a precision of ±15-25%.

Intercalation of Dyes in Graphene Oxide Thin Films and Membranes.

Intercalation of dyes into thin multilayered graphene oxide (GO) films was studied by neutron reflectivity and X-ray diffraction. Methylene blue (MB) penetrates the interlayer space of GO in ethanol solution and remains intercalated after the solvent evaporation, as revealed by the expansion of the interlayer lattice and change in chemical composition. The sorption of MB by thin GO films is found to be significantly stronger compared to the sorption of Crystal violet (CV) and Rose bengal (RB). This effect is attributed to the difference in the geometrical shape of planar MB and essentially nonflat CV and RB molecules. Graphite oxides and restacked GO films are found to exhibit different methylene blue (MB) sorptions. MB sorption by precursor graphite oxide and thin spin-coated films of GO is significantly stronger compared to freestanding micrometer-thick membranes prepared by vacuum filtration. Nevertheless, the sorption capacity of GO membranes is sufficient to remove a significant part of the MB from diluted solutions tested for permeation in several earlier studies. High sorption capacity results in strong modification of the GO structure, which is likely to affect permeation properties of GO membranes. Therefore, MB is not suitable for testing size exclusion effects in the permeation of GO membranes. It is not only hydration or solvation diameter but also the exact geometrical shape of molecules that needs to be taken into account considering size effects for penetration of molecules between GO layers in membrane applications.

Fluid-phase and membrane markers reveal spatio-temporal dynamics of membrane traffic and repair in the green alga Chara australis

We investigated the mechanisms and the spatio-temporal dynamics of fluid-phase and membrane internalization in the green alga Chara australis using fluorescent hydrazides markers alone, or in conjunction with styryl dyes. Using live-cell imaging, immunofluorescence and inhibitor studies we revealed that both fluid-phase and membrane dyes were actively taken up into the cytoplasm by clathrin-mediated endocytosis and stained various classes of endosomes including brefeldin A- and wortmannin-sensitive organelles (trans-Golgi network and multivesicular bodies). Uptake of fluorescent hydrazides was poorly sensitive to cytochalasin D, suggesting that actin plays a minor role in constitutive endocytosis in Chara internodal cells. Sequential pulse-labelling experiments revealed novel aspects of the temporal progression of endosomes in Chara internodal cells. The internalized fluid-phase marker distributed to early compartments within 10 min from dye exposure and after about 30 min, it was found almost exclusively in late endocytic compartments. Notably, fluid cargo consecutively internalized at time intervals of more than 1h, was not targeted to the same vesicular structures, but was sorted into distinct late compartments. We further found that fluorescent hydrazide dyes distributed not only to rapidly recycling endosomes but also to long-lived compartments that participated in plasma membrane repair after local laser injury. Our approach highlights the benefits of combining different fluid-phase markers in conjunction with membrane dyes in simultaneous and sequential application modus for investigating vesicle traffic, especially in organisms, which are still refractory to genetic transformation like characean algae.

Some Special Aspects of Liver Repair after Resection and Administration of Multipotent Stromal Cells in Experiment

Changes in rat liver after resection and injection of autologous multipotent mesenchymal stromal cells of bone marrow origin (MSCs) transfected with the GFP gene and cell membranes stained with red-fluorescent lipophilic membrane dye were studied by light microscopy. It was found that after the introduction of MSCs into the damaged liver, their differentiation into any cells was not found. However, under the conditions of MSCs use, the number of neutrophils in the parenchyma normalizes earlier, and necrosis and hemorrhages disappear more quickly. It was concluded that the use of MSCs at liver resection for the rapid restoration of an organ is inappropriate, since the injected cells in vivo do not differentiate either into hepatocytes, into epithelial cells of bile capillaries, into endotheliocytes and pericytes of the vascular membranes, into fibroblasts of the scar or other connective tissue structures, or into any other cells present in the liver.

Specific Labelling of Phagosome-Derived Vesicles in Macrophages with a Membrane Dye Delivered with Microfabricated Microparticles

Phagocytosis performed by a macrophage is a cellular process characterized by complex membrane trafficking and reorganization involving phagosomes and various other cellular membranous structures. The present work reports on development of a novel technique for characterizing this process. The technique is based on the use of microfabricated poly(N-isopropylacrylamide) microparticles to deliver a membrane dye into a macrophage phagosome and consequently label membranous vesicles derived from the phagosome. By using this technique, it has been found that the phagosome-derived vesicles can be formed from a phagosome without undergoing resolution and possess properties of the lysosomes. This technique is applicable to another common membrane dye and promises to have wide applications.

Abstract 2714: The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models

Abstract Introduction: Progression into metastatic disease is a critical step in most malignancies. Cancer cells may enter circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. CSCs are known to form cell clusters in vitro (spheroids/tumorspheres). We reported that tumorspheres also circulate in blood in vivo. We found that they carry cancer stem cells and strongly correlate with metastatic disease. We collected tumorspheres from blood by filtration. This project aims to test the metastatic potential of tumorspheres by developing an assay using in vivo circulation models. The goal is to establish a novel prognostic test for metastatic predisposition in cancer patients. Methods: To develop an in vivo assay for metastasis we use the chorioallantoic circulation in the chicken embryo, with several advantages: easy access to circulation, lack of immune response, rapid turnaround time (5 to 10 days) and inexpensive and simple technique. For clinical applications we introduced three technical innovations: (i) to make fertilized chicken embryos easily available we worked out a method to store viable embryos beyond 30 days; (ii) we developed a new coverglass chamber technique for micromanipulations; and (iii) for microscopic analysis new over-drained chorioallantois preparations can accommodate high resolution objectives. To test tumorspheres in embryonic circulation we used both highly metastatic (e.g. MDA-MB-436) and non-metastatic (e.g. MCF7) cell lines with GFP label, DiI membrane dye and CFDA-SA dye. For molecular studies DNA was extracted 5 days after cancer spheroid implantation and used qPCR to detect human sequences. Results: In traditional storage few embryos survive beyond 6 days. As fertilized eggs carry pluripotent stem cells, we optimized temperature, gas and humidity combinations to match known stem cell optimums. By suppressing metabolism, blocking embryo adhesion and controlling gas and humidity exchange we extended viable embryo storage beyond 30 days. We modified the traditional window method by forming visually clear microscopic coverglass chambers, and the new over-draining approach lowered the chorioallantois membranes and allowed high resolution imaging. The results showed invasive and hematogenic dissemination of the cancer spheroids. Molecular studies of human markers detected metastatic progression and generated quantitative data. Conclusions: We optimized the in vivo embryonic metastasis assay. We show that it has the potential for a sensitive and quantitative method to diagnose metastatic predisposition in cancer patients. The molecular approach (DNA extraction, PCR markers) is easily up-scalable, the instrumentation is available in most hospitals and offers cost effective and critical early diagnosis of cancer progression. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Kathleen Marinelli, Christine Kyritsis, Viktoria Denes. The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2714.

Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) is frequently used to study diffusion in cell membranes, primarily the plasma membrane. The diffusion coefficients reported in the plasma membrane of the same cell type and even within single cells typically display a large spread. We have investigated whether this spread can be explained by variations in membrane topography throughout the cell surface, that changes the amount of membrane in the FCS focal volume at different locations. Using FCS, we found that diffusion of the membrane dye DiI in the apical plasma membrane was consistently faster above the nucleus than above the cytoplasm. Using live cell scanning ion conductance microscopy (SICM) to obtain a topography map of the cell surface, we demonstrate that cell surface roughness is unevenly distributed with the plasma membrane above the nucleus being the smoothest, suggesting that the difference in diffusion observed in FCS is related to membrane topography. FCS modeled on simulated diffusion in cell surfaces obtained by SICM was consistent with the FCS data from live cells and demonstrated that topography variations can cause the appearance of anomalous diffusion in FCS measurements. Furthermore, we found that variations in the amount of the membrane marker DiD, a proxy for the membrane, but not the transmembrane protein TCRζ or the lipid-anchored protein Lck, in the FCS focal volume were related to variations in diffusion times at different positions in the plasma membrane. This relationship was seen at different positions both at the apical cell and basal cell sides. We conclude that it is crucial to consider variations in topography in the interpretation of FCS results from membranes.

Over-expression of Caj1, a plasma membrane associated J-domain protein in Saccharomyces cerevisiae, stabilizes amino acid permeases

Hsp70: J-domain protein (JDP) machines, along with the cellular protein degradation systems play a central role in regulating cellular proteostasis. An equally robust surveillance system operates at the plasma membrane too that affects proper sorting, stability as well as the turnover of membrane proteins. Although plausible, a definitive role of the Hsp70: JDP machine in regulating the stability of plasma membrane proteins is not well understood in Saccharomyces cerevisiae. Here we show that a moderate over-expression of Caj1, one of the thirteen JDPs residing in the nucleo-cytosolic compartment of S. cerevisiae reduced the cold sensitivity of tryptophan auxotrophic yeast cells by stabilizing tryptophan permeases, Tat1 and Tat2 in a J-domain dependent manner. Concomitantly, higher Caj1 levels also caused slow growth and increased plasma membrane damage at elevated temperatures possibly due to the stabilization of thermolabile plasma membrane proteins. Finally, we show that although majorly cytosolic, Caj1 also co-localizes with the membrane dye FM4-64 at the cellular periphery suggesting that Caj1 might interact with the plasma membrane. Based on the results presented in this study, we implicate the Hsp70: Caj1 chaperone machine in regulating the stability or turnover of plasma membrane proteins in budding yeast.

Abstract 376: Functional Significance of Anatomically Heterogeneous Distribution of Transversal-axial Tubule System in Mouse and Human Atrial Myocardium

Background: In cardiac myocytes, efficient and synchronous excitation-contraction coupling (ECC) occurs through a specialized network of membrane invaginations called transverse-axial tubule system (TATS). In contrast to an extensive TATS in ventricular myocytes, atrial cells show a significantly heterogeneous organization of TATS, varying from a ventricular-like organized TATS to absent/irregular TATS. However, anatomical distribution of atrial TATS and its functional significance remains unknown. We hypothesized that atrial myocytes with a different TATS organization have a specific anatomical location within the atria that determines their impact on atrial ECC and contraction. Methods and Results: In order to avoid the potential bias associated with cell isolation, we applied a mosaic imaging protocol at the whole-mount isolated mouse atrial preparation stained with a membrane dye RH-237. We developed an algorithm that can automatically remove the outer membrane and analyze TATS organization. This analysis showed that myocytes located within the inter-caval region (ICR, i.e., between the superior and inferior vena cava and between the crista terminalis in atrioventricular junction) have a lower TATS organization than cells located within the right atrial appendage (RAA): 0.21±0.01 A.U. vs. 0.50±0.02 A.U. in ICR vs. RAA, respectively, P<0.01. Fluorescent optical mapping of Ca 2+ transients from isolated mouse atrial preparations revealed that ICR region has a longer Ca 2+ transient rise up time compared to RAA (7.8±0.3 ms vs. 6.3±0.3 ms, P<0.01). Moreover, a similar region-specific TATS organization was found in human non-failing (donor) atrial tissue samples. The analysis of TATS density in specimens selected from ICR and RAA regions of the human hearts (n=4) showed a significantly lower density of TATS in ICR compared to RAA region (5.8±0.2% vs. 10.6±0.4%, P<0.01). Conclusion: Our findings show a region-specific structural and functional differences of myocytes from RAA and ICR regions of mouse and human heart that could represent a general organization of the mammalian atria. It may have important implications into establishment of different functional roles of cells with a specific TATS structure in atrial physiology and pathology.

Factors affecting the degradation of remazol turquoise blue (RTB) dye by titanium dioxide (TiO2) entrapped photocatalytic membrane

This study investigated the photocatalytic degradation of remazol turquoise blue (RTB) dye using titanium dioxide (TiO2) entrapped polyvinylidene fluoride (PVDF) membrane synthesized by non-solvent induced phase separation method. Numerous experiments were conducted to investigate the effects of catalyst loading in membranes, the concentration of dye, feed temperature, pH of the solution, and the addition of H2O2 to the dye solution on the removal of dye. Results were compared with the performance of virgin PVDF and modified PVDF/TiO2 membranes. The experimental results indicated that the optimum TiO2 loading in the membrane was 2 wt%, which enhanced the membrane morphology, permeability, and dye removal performance. The rate of photocatalytic degradation dropped with the increase in dye concentration. The photocatalytic efficiency of the membrane depends on the pH and the temperature of the solution.

Erbium (III) molybdate as a new nanofiller for fabrication of antifouling polyethersulfone membranes

The effect of erbium (III) molybdate (Er2MoO6) nanomaterial as a new nanofiller on fabrication of polyethersulfone (PES) mixed matrix membranes was examined in terms of membrane morphology, hydrophilicity, permeability, mechanical strength, dye and protein separation and antifouling property. The synthesized Er2MoO6 nanoparticles were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The morphology and surface properties of the prepared nanocomposite membranes were evaluated by SEM images, surface roughness parameters using atomic force microscopy (AFM) and contact angle measurement. The pure water flux of the nanocomposite membranes improved significantly (195.3 L/m2 h at 3 bar for 1 wt.%) after adding of Er2MoO6 due to the higher hydrophilicity in comparison with bare one (82.5 L/m2 h). The fouling resistance results exhibited that 1 wt% Er2MoO6 blended membrane had the best antifouling characteristic (flux recover ratio of 87.6 %). Evaluation of membrane separation performance was performed by examining the Reactive Red 195 rejection. The dye rejection data showed that the Er2MoO6/PES membranes have better dye removal capacity rather than the unfilled PES. It could be say that the Er2MoO6 nanocomposite membranes showed the suitable antibiofouling properties tested by BSA filtration due to low surface roughness and high hydrophilicity prompted by the blended nanomaterial.

A loose polyamide nanofiltration membrane prepared by polyether amine interfacial polymerization for dye desalination

It is crucial for fractionating dye/salt mixture in dyeing wastewater treatment. Herein, a polyamide nanofiltration (NF) membrane (PEA-TMC) for high efficient dye desalination was prepared through polyether amine (PEA) and trimesoyl chloride interfacial polymerization. Due to the large molecular size and hydrophilicity of PEA, the resulting NF membrane possessed a relatively loose membrane structure and good hydrophilicity, which improved membrane water flux and dye desalination performance. The influences of PEA concentration and reaction time on the NF performance were systematically investigated. Under the optimal conditions, the membrane exhibited a high water flux of 66.4 L m-2h−1 at low operating pressure (0.4 MPa), and showed a high methyl blue rejection (95.4%) and low NaCl rejection (16.4%) when nanofiltrated methyl blue/ NaCl mixture solution. Moreover, PEA-TMC membrane exhibited good stability in continuous operation and antifouling performance. These results disclosed the promising applications of PEA-TMC membrane in dye/salt separation process.

β-cyclodextrin functionalized MWCNTs as a promising antifouling agent in fabrication of composite nanofiltration membranes

In this research, the manufacture and properties of polyethersulfone (PES)-based mixed matrix nanofiltration (MM-NF) membranes modified with β-cyclodextrin functionalized multiwalled carbon nanotubes (β-CD/MWCNTs) were studied. The β-CD/MWCNTs nanocomposite was synthesized using the soft cutting method. Stable dispersivity of the resulting nanocomposite was checked for 12 h. Then, the membranes containing up to 1 wt% β-CD/MWCNTs were prepared using conventional phase inversion method. The impact of varied mass loadings of the β-CD/MWCNTs on the morphological features and performance of the amended membranes was evaluated. It was deduced that membrane hydrophilicity, porosity, pore size, water contents, dye rejection and antifouling capacity were noticeably improved after incorporating the β-CD/MWCNTs nanomaterials. In comparison with the bare membrane, the β-CD/MWCNTs amended membranes displayed higher hydrophilicity, and consequently pure water flux (PWF). Since β-CD can exploit the structure advantages of MWCNTs via enhancement of the dispersivity, devoting unique features to the β-CD/MWCNTs nanomaterial. The 0.1 wt% nanocomposite membrane sample revealed the highest pure water flux of 21.5 Kg/m2.h due to increase in pore size (5.0 nm). While, the membrane contributed with the β-CD/MWCNTs-0.5 revealed better performance in terms of dye rejection and antifouling capability than the bare and modified membranes. It indicated a dye rejection of above 92%, flux recovery ratio (FRR) of 89% and an irreversible resistance of 11.1%. The β-CD/MWCNTs-0.5 acted as a potential nanofiller in nanofiltration (NF) membranes improving their antifouling performance at different operational conditions.

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

Differentiating between Membrane Topography and Molecular Clustering

According to single-molecule localisation microscopy (SMLM) almost every plasma membrane protein is organised into clusters. However, an appreciable fraction of this clustering could be due to variations in membrane topography rather than genuine clustering since an increase in the local membrane density can make molecules that are randomly distributed appear to be clustered. Cells are neither flat nor smooth and membrane topography variations can be caused by membrane folding, undulations, protrusions like microvilli and lamellipodia as well as invaginations like caveolae and clathrin coated pits. To differentiate between topography-induced and genuine clustering variations in the membrane density needs to be factored out. Live cell dual colour SMLM, with the fluorophore DiI showing the membrane density together with the proteins the transferrin receptor (TfR) or the GPI-anchored CD59 was performed to demonstrate that apparent clustering due to topography variations can be identified in SMLM datasets. Pair correlation analysis reported both the two proteins and the membrane dye as being clustered. Converting the localisations to images and performing signal distribution analysis, on the other hand, revealed that the DiI and CD59 clusters are simply caused by variations in topography, i.e. the CD59 clusters are not genuine clusters whereas the TfR clusters are legitimate. Performing simulations on localisation distributions varying the partition ratio to clusters, blurring, cluster number, cluster size and image size we demonstrate that signal distribution analysis is a powerful tool to differentiate clustered from random distributions and to detect very low levels of clustering. We conclude that topography must be considered when deciding whether clusters are genuine or artefactual. Our findings imply that clustering may be less ubiquitous than the current literature suggests.

Characterization of Small Objects in Homogenates of the Squid Optic Lobe

The squid (Loligo pealei) optic lobe is unique among sources of CNS synaptosomes via differential centrifugation, both in its ease and speed of dissection and preparation, and in the purity of the final result (Pekkurnaz et al. Biol. Bull. 2011). Despite the high purity of ∼1 µm-diameter synaptosomes in the synaptosome fraction, another class of 2-10 μm diameter objects were detected by one us (GP) in DIC microscopy of thin layers of this fraction. Recently, a publication assumed that these inclusions were large synaptosomes (Cefaliello et al. Molec. Neurobio. 2019). To test that assumption, and to further characterize these inclusions, we carried out a series of fluorescent labeling and dye exclusion experiments by direct imaging for size classification. Two major classes of objects were found, a larger class (L) 5-10 μm in diameter and a smaller class (S) that appeared more refractile, perfectly spherical, and averaged 2 μm in diameter. To identify some of the macromolecular content of these objects, we labeled them with fluorescent dyes for DNA, lipid, and protein aggregates. ‘L’ stained positive for the membrane dye BODIPY-sphingomyelin and the DNA marker Hoechst. The exclusion of rhodamine-dextran (10kDa) further corroborated the membrane-enclosed nature of the ‘L’ object. Electron microscopy showed a large ∼10 μm spheroid with a double membrane and dark material draping the internal membrane, suggesting that the ‘L’ inclusions were nuclei. The majority of the inclusions in the ‘S’ class did not exclude dyes, did not stain with the DNA dye Hoechst, and were positive for a rotamer fluorescent marker for intracellular aggregated misfolded proteins. Currently, efforts are underway to identify the protein composition of the ‘S’ droplets.

Fructose-1,6-bisphosphatase degradation in the methylotrophic yeast Komagataella phaffii occurs in autophagy pathway.

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. After shift of methanol-grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase, formate dehydrogenase, and fructose-1,6-bisphosphatase [FBP]) along with peroxisomal enzymes of methanol metabolism is observed. Mechanisms of inactivation of cytosolic enzymes remain unknown. To study the mechanism of FBP inactivation, the changes in its specific activity of the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of Komagataella phaffii with or without the addition of the MG132 (proteasome degradation inhibitor) were investigated after shift of methanol-grown cells in glucose medium. Western blot analysis showed that inactivation of FBP in GS200 occurred due to protein degradation whereas inactivation in the strains SMD1163 and gss1Δ was negligible in such conditions. The effect of the proteasome inhibitor MG132 on FBP inactivation was insignificant. To confirm FBP degradation pathway, the recombinant strains with GFP-labeled Fbp1 of K. phaffii and red fluorescent protein-labeled peroxisomes were constructed on the background of GS200 and SMD1163. The fluorescent microscopy analysis of the constructed strains was performed using the vacuolar membrane dye FM4-64. Microscopic data confirmed that Fbp1 degrades by autophagy pathway in K. phaffii. K. phaffii transformants, which express heterologous β-galactosidase under FLD promoter, have been constructed.

Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Dibenzothiophene Sulfone Derivatives as Plasma Membrane Dyes.

Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO-based dyes to include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent microscopy. The photophysical properties of the two new DBTOO derivatives were determined and both have good fluorescent quantum yields and a visible blue emission. Due to agreeable wavelengths of excitation and emission, a standard 4',6-diamindino-2-phenylindole (DAPI) filter set worked well with these dyes. After co-staining, it was confirmed that both DBTOO dyes localized in the plasma membrane. The quality of the overlap was quantified using Pearson correlation coefficient, which indicated a strong overlap between the DBTOO dyes and the standard plasma membrane dye. The novel dyes also displayed relatively low toxicity to the HeLa cells with IC50 between 10 and 100µm. Thus, this work reports a new use of DBTOO derivatives as fluorescent microscopy stains.

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.