Lipophilic Carbocyanine Dye Research Articles

Comparative Gene Signature of Nociceptors Innervating Mouse Molar Teeth, Cranial Meninges, and Cornea.

The trigeminal ganglion (TG) collects afferent sensory information from various tissues. Recent large-scale RNA sequencing of neurons of the TG and dorsal root ganglion has revealed a variety of functionally distinct neuronal subpopulations, but organ-specific information is lacking. To link transcriptomic and tissue-specific information, we labeled small-diameter neurons of 3 specific subpopulations of the TG by local application of lipophilic carbocyanine dyes to their innervation site in the dental pulp, cornea, and meninges (dura mater). We then collected mRNA-sequencing data from fluorescent neurons. Differentially expressed genes (DEGs) were analyzed and subjected to downstream gene set enrichment analysis (GSEA), and ion channel profiling was performed. A total of 10,903 genes were mapped to the mouse genome (>500 reads). DEG analysis revealed 18 and 81 genes with differential expression (log 2 fold change > 2, Padj < .05) in primary afferent neurons innervating the dental pulp (dental primary afferent neurons [DPAN]) compared to those innervating the meninges (meningeal primary afferent neurons [MPAN]) and the cornea (corneal primary afferent neurons [CPAN]). We found 250 and 292 genes differentially expressed in MPAN as compared to DPAN and to CPAN, and 21 and 12 in CPAN as compared to DPAN and MPAN. Scn2b had the highest log 2 fold change when comparing DPAN versus MPAN and Mmp12 was the most prominent DEG when comparing DPAN versus CPAN and, CPAN versus MPAN. GSEA revealed genes of the immune and mitochondrial oxidative phosphorylation system for the DPAN versus MPAN comparison, cilium- and ribosome-related genes for the CPAN versus DPAN comparison, and respirasome, immune cell- and ribosome-related gene sets for the CPAN versus MPAN comparison. DEG analysis for ion channels revealed no significant differences between the neurons set except for the sodium voltage-gated channel beta subunit 2, Scn2b . However, in each tissue a few ion channels turned up with robust number of reads. In DPAN, these were Cacna1b , Trpv2 , Cnga4 , Hcn1 , and Hcn3 , in CPAN Trpa1 , Trpv1 , Cacna1a , and Kcnk13 and in MPAN Trpv2 and Scn11a . Our study uncovers previously unknown differences in gene expression between sensory neuron subpopulations from the dental pulp, cornea, and dura mater and provides the basis for functional studies, including the investigation of ion channel function and their suitability as targets for tissue-specific analgesia.

Testing the suitability of neuroanatomical tracing method in human fetuses with long years of postmortem delay.

Human tissues in gross anatomical archives with long years of postmortem delays are considered suboptimal relative to recently fixed materials for neuroanatomical tracing studies, yet efficacy of neuroanatomical tracing on archival fetal tissues largely unexplored. We aimed to explore the suitability of human archival tissue in neuroanatomical tracing with lipophilic carbocyanine dyes. We used crystal and paste forms 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and analogues for neuroanatomical tracing on different peripheral nerves in 15-18-year archival old formalin-fixed human fetuses. We employed bright-field, fluorescent and confocal microscopy to visualize the peripheric nerve traces, spinal cord and vibratome cut sections. Fluorescent signal of the dyes on epineurium and on axonal membranes were visualized under fluorescence and confocal microscopes and performance of the dye diffusion was assessed by semi-quantitative image analysis. We followed up seven lipophilic dye embeddings in 16-28 gestational week-old human fetuses (n = 4) with 16.75 ± 1.29-year postmortem delay. The mean distance of distally moved carbocyanine dye diffusion measured on epineurium was detected as 25.11 ± 9.1mm. Based on the results of 13 distinct studies performed neuroanatomical tracing with human tissues in the immediate postmortem hours or days, average traced distance was 16.32 ± 15.95mm, and a 95% confidence interval lower limit of 4.9mm and upper limit of 27.73mm. The tracing distances we observed in our current study fall entirely within this confidence interval. To our awareness, this is the first report to demonstrate that specific neuroanatomical tracing presented in axonal membrane level on peripheral nerves is possible on gross anatomical repositories.

Liquid-crystalline ordering of davydov-split aggregates of cyanine dyes

3,3′-ditetradecyloxacarbocyanine (DiOC14(3)) and 3,3′-Dioctadecyloxacarbocyanine (DiOC18(3)) are lipophilic carbocyanine dyes with a conjugated polymethine backbone and two long hydrocarbon tails. In this paper, we report the formation of Davydov-split aggregates of DiOC14(3) and DiOC18(3) in methanol/water mixtures with and without ammonia, which show a blue-shifted H-band and a red-shifted J-band with respect to the monomer band in their UV–vis absorption spectra. The Davydov-split aggregates of DiOC14(3) show a fiber-like morphology in the absence of ammonia and a vesicle-like morphology in the presence of ammonia. While the Davydov-split aggregates of DiOC18(3) show a vesicle-like morphology with the different lamellarities, depending on the presence of ammonia in the methanol/water mixtures. These Davydov-split aggregates with different morphologies are birefringence when being viewed through crossed polarizers, suggesting that they have liquid-crystalline phases. The Davydov-split aggregate fibers and vesicles with liquid-crystalline phases and unique excitations are great potential for photonic applications because the liquid-crystalline order and molecular exciton are synergized.

A pH-Adjustable Tissue Clearing Solution That Preserves Lipid Ultrastructures: Suitable Tissue Clearing Method for DDS Evaluation

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution. The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations.

An insight into anti-adipogenic properties of an Oroxylum indicum (L.) Kurz extract

BackgroundOroxylum indicum fruit extract (OIE) has been reported to inhibit the development of adipocytes. However, the exact mechanism of its metabolic activity is not clearly defined. This study attempted to investigate whether OIE was involved in disrupting the cell cycle, glucose metabolism, and mitochondrial function in 3 T3-L1 cells.MethodsThe effect of the OIE on cell cycle progression was measured by flow cytometry along with observing the expression of the cycle regulator by immunoblotting. The effect of the OIE on glucose metabolism was investigated. The amount of glucose uptake (2-NBDG) influenced by insulin was determined as well as the protein tyrosine phosphorylation (PY20), and glucose transporter4 (GLUT4) expression was determined by immunoblotting assay. Mitochondria are also essential to metabolic processes. This study investigated mitochondrial activity using fluorescent lipophilic carbocyanine dye (JC-1) and mitochondria mass by MitoTracker Green (MTG) staining fluorescence dyes. Finally, cellular ATP concentration was measured using an ATP chemiluminescence assay.ResultsTreatment with OIE plus adipogenic stimulators for 24 h arrested cell cycle progression in the G2/M phase. Moreover, 200 μg/mL of OIE significantly diminished the expression of the insulin receptor (IR) and GLUT4 protein compared to the untreated-adipocytes (P < 0.05). The mitochondrial membrane potential (MMP) was significantly reduced (24 h) and increased (day 12) by OIE compared to untreated-adipocytes (P < 0.05). However, OIE maintained MMP and ATP at a similar level compared to the pre-adipocytes (day 12). Transmission electron microscope (TEM) results demonstrated that OIE could protect mitochondria deformation compared to the untreated-adipocytes.ConclusionThese results suggest that the inhibitory effect of the OIE on adipogenesis may potentially inhibit the cell cycle and phosphorylation of IR, leading to a decrease in glucose uptake to the cells. The OIE also slows down the mitochondrial activity of the early phase of cell differentiation, which can also inhibit the development of fat cells.

Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO.

Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules.

Acute slice preparation for electrophysiology increases spine numbers equivalently in the male and female juvenile hippocampus: a DiI labeling study.

Hippocampal slices are widely used for in vitro electrophysiological experiments to study underlying mechanisms for synaptic transmission and plasticity, and there is a growing appreciation for sex differences in synaptic plasticity. To date, several studies have shown that the process of making slices from male animals can induce synaptogenesis in cornu ammonis area 1 (CA1) pyramidal cells, but there is a paucity of data for females and other brain regions. In the current study we use microcrystals of the lipophilic carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to stain individual neurons in the CA1 and dentate gyrus (DG) hippocampal subfields of postnatal day 21 male and female rats. We show that the preparation of sections for electrophysiology produces significant increases in spines in sections obtained from females, similar to that observed in males. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. They also show, for the first time, that in vitro electrophysiology slice preparations enhance spine numbers on hippocampal cells equivalently in both juvenile females and males.NEW & NOTEWORTHY This study introduces a new DiI technique that elucidates differences in spine numbers in juvenile female and male hippocampus, and shows that slice preparations for hippocampal electrophysiology in vitro may mask these differences.

Comparison of hippocampal neuron spine densities in acute and perfusion‐fixed slices using the lipophilic dye DiI

The hippocampus is a key brain structure involved in learning and memory. Hippocampal acute slices are widely used in ex vivo electrophysiological experiments to study cellular mechanisms of synaptic transmission and plasticity. Many approaches have been used to optimize slice viability and to maintain accurate in vivo physiological conditions during experimental procedures. Several different variables such as the age of the animal, the composition of the artificial cerebrospinal fluid (aCSF), the maintenance conditions in recording chambers, temperature and slice cutting methodology, among others, can affect hippocampal slices features. Specifically, by using electron microscopy and two‐photon microscopy it has been previously reported that dendritic spines, crucial neuronal structures that receive most of the excitatory synaptic inputs in the brain, show increased numbers in acute hippocampal slices compared to perfusion‐fixed sections. These alterations in dendritic spines could be one of the underlying mechanism explaining differences in ex vivo and in vivo electrophysiological experiments, as for instance success rates in long‐term depression (LTD) induction protocols. In our experiments, we have compared dendritic spine density in the cornu ammonis 1 (CA1) and dentate gyrus (DG) between hippocampal acute slices and aldehyde perfusion‐fixed hippocampi of Sprague‐Dawley (both males and females) using the lipophilic carbocyanine dye 1,1′‐Dioctadecyl‐3,3,3′,3′‐Tetramethylindocarbocyanine Perchlorate (DiI). We observed an increase in a range of 37–68% in dendritic spine density in the acute hippocampal slices when compared to aldehyde perfusion‐fixed sections independent of sex. These data suggest that spine plasticity in ex vivo electrophysiological experiments may not be reflective of the in vivo situation and interpretation of data should take this into account.Support or Funding InformationCanadian Institutes of Health ResearchNatural Sciences and Engineering Research CouncilThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Seebest: A pH-Adjustable Tissue Clearing Solution that Preserves Lipid Ultrastructures

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethyleneimine was required for a wide pH range adjustment of the tissue clearing solution. The combination of polyethyleneimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained by lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing while preserving lipid ultrastructures, which is suitable for multicolor deep imaging.

A novel cerebellar commissure and other myelinated axons in the Purkinje cell layer of a pond turtle (Trachemys scripta elegans).

Parallel fibers in the molecular layer of the vertebrate cerebellum mediate slow spike conduction in the transverse plane. In contrast, electrophysiological recordings have indicated that rapid spike conduction exists between the lateral regions of the cerebellar cortex of the red-ear pond turtle (Trachemys scripta). The anatomical basis for this commissure is now examined in that species using neuronal tracing techniques. Fluorescently tagged dextrans and lipophilic carbocyanine dyes placed in one lateral edge of this nonfoliated cortex are transported across the midline of living brains in vitro and along the axonal membranes of fixed tissues, respectively. Surprisingly, the labeled commissural axons traversed the cortex within the Purkinje cell layer, and not in the white matter of the molecular layer or the white matter below the granule cell layer. Unlike thin parallel fibers that exhibit characteristic varicosities, this commissure is composed of smooth axons of large diameter that also extend beyond the cerebellar cortex via the cerebellar peduncles. Double labeling with myelin basic protein antibody demonstrated that these commissural axons are ensheathed with myelin. In contrast to this transverse pathway, an orthogonal myelinated tract was observed along the cerebellar midline. The connections of this transverse commissure with the lateral cerebellum, the vestibular nuclear complex, and the cochlear vestibular ganglia indicate that this commissure plays a role in bilateral vestibular connectivity.

Nanoscale Ultrasound-Switchable FRET-Based Liposomes for Near-Infrared Fluorescence Imaging in Optically Turbid Media.

A new approach for fluorescence imaging in optically turbid media centered on the use of nanoscale ultrasound-switchable FRET-based liposome contrast agents is reported. Liposomes containing lipophilic carbocyanine dyes as FRET pairs with emission wavelengths located in the near-infrared window are prepared. The efficacy of FRET and self-quenching for liposomes with a range of fluorophore concentrations is first calculated from measurement of the liposome emission spectra. Exposure of the liposomes to ultrasound results in changes in the detected fluorescent signal, the nature of which depends on the fluorophores used, detection wavelength, and the fluorophore concentration. Line scanning of a tube containing the contrast agents with 1 mm inner diameter buried at a depth of 1 cm in a heavily scattering tissue phantom demonstrates an improvement in image spatial resolution by a factor of 6.3 as compared with images obtained in the absence of ultrasound. Improvements are also seen in image contrast with the highest obtained being 9% for a liposome system containing FRET pairs. Overall the results obtained provide evidence of the potential the nanoscale ultrasound-switchable FRET-based liposomes studied here have for in vivo fluorescence imaging.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl's blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

Labeling of neuronal morphology using custom diolistic techniques

BackgroundDiolistic labeling is increasingly utilized in neuroscience as an efficient, reproducible method for visualization of neuronal morphology. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery allows for non-toxic fluorescent labeling of multiple neurons in both living and fixed tissue. Since first described, this labeling method has been modified to fit a variety of research goals and laboratory settings. New methodDiolistic labeling has traditionally relied on commercially available devices for the propulsion of coated micro-particles into tissue sections. Recently, laboratory built biolistic devices have been developed which allow for increased availability and customization. Here, we discuss a custom biolistic device and provide a detailed protocol for its use. ResultsUsing custom diolistic labeling we have characterized alterations in neuronal morphology of the lateral/dentate nucleus of the rat cerebellum. Comparisons were made in developing rat pups exposed to abnormally high levels of 5-methyloxytryptamine (5-MT) pre-and postnatally. Using quantitative software; dendritic morphology, architecture, and synaptic connections, were analyzed. Comparison with existing method(s)The rapid nature of custom diolistics coupled with passive diffusion of dyes and compatibility with confocal microscopy, provides an unparalleled opportunity to examine features of neuronal cells at high spatial resolution in a three-dimensional tissue environment. ConclusionsWhile decreasing the associated costs, the laboratory-built device also overcomes many of the obstacles associated with traditional morphological labeling, to allow for reliable and reproducible neuronal labeling. The versatility of this method allows for its adaptation to a variety of laboratory settings and neuroscience related research goals.

Transmembrane Pathways and Mechanisms of Rod-like Paclitaxel Nanocrystals through MDCK Polarized Monolayer.

Drug nanocrystals (NCs) appear to be favorable to improving oral bioavailability of poorly water-soluble drugs as evidenced by the great success they have had in the market. However, the pathway and mechanism of drug NCs through epithelial membrane are still unclear. In an attempt to clarify their transport features, paclitaxel nanocrystals (PTX-NCs), and paclitaxel hybrid NCs with lipophilic carbocyanine dyes, were prepared and characterized as the models. The endocytosis, intracellular trafficking, paracellular transport, and transcytosis of PTX-NCs were carefully investigated with Förster resonance energy transfer (FRET) analysis, as well as a colocalization assay, flow cytometry, gene silencing, Western-blot, transepithelial electrical resistance (TEER) study and other approaches on MDCK cells. It was found that rod-like PTX-NCs could transport through the monolayer intact, and the process of endocytosis proved to be time and energy dependent. Endoplasmic reticulum (ER) and Golgi complexes were colocalized with PTX-NCs in cells, so the ER-Golgi complexes/Golgi complexes-basolateral membrane pathway may be involved in the intracellular trafficking and transcytosis of PTX-NCs. It was demonstrated here that cav-1, dynamin, and actin filament modulated the endocytosis process, and Cdc 42 regulated the transcytosis process. In addition, no paracellular transport of PTX-NCs was observed. These findings demonstrated that the rod-like nanocrystals not only enhanced the transcytosis of PTX compared with microparticles of raw drug materials but also changed the pathways of drug delivery. This study certainly provides insight for the oral absorption mechanism of nanocrystals of poorly soluble drugs.

Inter-nanocarrier and nanocarrier-to-cell transfer assays demonstrate the risk of an immediate unloading of dye from labeled lipid nanocapsules

Release studies constitute a fundamental part of the nanovector characterization. However, it can be difficult to correctly assess the release of lipophilic compounds from lipid nanocarriers using conventional assays. Previously, we proposed a method including an extraction with oil to measure the loading stability of lipophilic dyes in lipid nanocapsules (LNCs). The method indicated a rapid release of Nile Red from LNCs, while the loading of lipophilic carbocyanine dyes remained stable. This method, although interesting for a rapid screening of the fluorescence labeling stability of nanocarriers, is far from what happens in vivo, where lipid acceptor phases are nanostructured. Here, lipophilic dye loading stability has been assessed, by monitoring dye transfer from LNCs toward stable colloidal lipid nanocompartments, i.e. non-loaded LNCs, using new methodology based on size exclusion chromatography (SEC) and Förster Resonance Energy Transfer (FRET). Dye transfer between LNCs and THP-1 cells (as model for circulating cells) has also been studied by FACS. The assays reveal an almost instantaneous transfer of Nile Red between LNCs, from LNCs to THP-1 cells, between THP-1 cells, and a reversal transfer from THP-1 cells to LNCs. On the contrary, there was no detectable transfer of the lipophilic carbocyanine dyes. Dye release was also analyzed using dialyses, which only revealed a very slow release of Nile Red from LNCs, demonstrating the weakness of membrane based assays for investigations of the lipophilic compound loading stability in lipid nanocarriers. These results highlight the importance of using relevant release assays, and the potential risk of an immediate unloading of lipophilic fluorescent dyes from lipid nanocarriers, in the presence of a lipid acceptor nanocompartment. Some misinterpretations of cellular trafficking and in vivo biodistribution of fluorescent nanoparticles should be avoided.

Multispectral labeling of embryonic cells with lipophilic carbocyanine dyes.

Incubation of hatched Strongylocentrotus purpuratus sea urchin embryos or larvae with suspensions of the carbocyanine dyes DiI, DiO, and DiD resulted in the random labeling of membranes of some ectodermal epithelial cells and blastocoelar cells, producing a range of differentially colored cells that can be tracked during development. Simultaneous application of soluble Vybrant® preparations of the three dyes resulted in similar labeling of each cell. Dye labeling of the ectoderm was nearly eliminated by deciliation and some ciliated squamous epithelial cells adjacent to labelled cells were refractory to Vybrant® dye uptake irrespective of concentration or duration of treatment, together suggesting local variation in the properties of cell membranes or cilia. Furthermore, single cells possessing distinctive morphological features were detected.

Primary Acute Lymphoblastic Leukemia Cells Create a Drug-Resistant Niche By Tunneling Nanotube Signaling with Mesenchymal Stromal Cells

Background: Acute lymphoblastic leukemia (ALL) cells reside in the bone marrow microenvironment, which nurtures and protects cells from chemotherapeutic drugs. Bidirectional signaling between leukemic cells and their niche is shown to be essential for these processes. A major gap in our knowledge is the lack of insight into the functional mechanism regulating and controlling this crosstalk. Tunneling nanotubes (TNTs) have recently been reported as a novel mode of communication between eukaryotic cells, which can facilitate the transport of several types of cellular cargo.Objectives: This study aims to investigate the presence and functional importance of TNTs in the leukemic niche.Results: Here, we show for the first time that TNT signaling occurs between primary patient-derived leukemic cells and primary mesenchymal stromal cells using flow cytometry and time-lapse confocal microscopy. TNTs form within minutes after the start of co-culture and efficiently transfer lipophilic carbocyanine dye DiI from ALL cells towards MSCs. Dye transfer was significantly reduced when TNT signaling was inhibited using three independent experimental setups: actin inhibition, mechanical disruption through gentle shaking of cell cultures, and physical separation using a 3.0 µm pore-sized transwell system (4-fold, 5-fold and 35-fold reduction, respectively). In reciprocal experiments we also observed dye transfer via TNTs from MSCs to BCP-ALL cells, but the magnitude was strikingly less (175-fold, p ≤ 0.001). In addition, we show that leukemic cells use TNTs to modulate their microenvironment by directing non-malignant stromal cells to produce pro-survival cytokines. We observed patient-specific cytokine signatures after co-culture of primary ALL cells with different primary MSCs. For example, IP10/CXCL10 levels increased more than 1000-fold when patient ALL#1 cells were co-cultured with primary MSCs, but were undetectable in co-cultures from patient ALL#2 cells. Induction of all cytokines was dependent on TNT signaling, as TNT inhibition significantly lowered cytokine production (p ≤ 0.001). Importantly, we show that TNT signaling is functionally important for leukemic cell survival and stroma-driven drug resistance. Leukemic cell viability was assessed by flow cytometry after staining with Annexin V, Propidium Iodide and CD19. Primary leukemic cell survival significantly increased in 5-day co-cultures with primary MSCs compared to mono-culture (p ≤ 0.001). When TNT formation was prevented by shaking of these co-cultures or by transwell conditions, the cell viability reduced 3.5- and 3.6-fold, respectively (p ≤ 0.001). TNT inhibition similarly reduced the survival of primary ALL cells in co-culture with MSCs during prednisolone exposure (p ≤ 0.01). Co-culture with primary MSCs also induced resistance to prednisolone 2.5-fold compared to mono-culture in a proliferative setting, using the BCP-ALL cell line NALM6. Inhibition of TNT formation by shaking or transwell conditions significantly reduced this effect: cells in co-culture were only 1.4- and 1.1-fold more resistant compared to mono-culture, respectively (p ≤ 0.01).Conclusion: The presented study identifies TNT formation as a major regulator of interaction between ALL cells and their bone marrow niche, which facilitates signaling mainly from leukemic cells towards MSCs. This signaling drives the release of cytokines within the microenvironment. Disruption of tunneling nanotubes inhibits this release, diminishes the survival benefit that MSCs provide to primary ALL cells, and sensitizes ALL cells to the important anti-leukemic drug prednisolone. This observation gives insight into the pathogenesis of BCP-ALL and opens new avenues to develop more effective therapies that interfere with the leukemic niche. DisclosuresNo relevant conflicts of interest to declare.

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress. Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Imaging plasma membrane deformations with pTIRFM.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Labeling Second-Order Sensory Neurons in the Posterior Lateral-Line System of Zebrafish

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The posterior component of the system, the posterior lateral line (PLL), comprises the neuromasts on the body and tail and has its ganglion just posterior to the otic vesicle. The peripheral location of the PLL system makes it accessible and easily visualized by imaging methods. Neuromasts are innervated by a few afferent neurons (usually two, but sometimes more), which have their cell bodies clustered in cranial ganglia and project their central axons to the hindbrain, where they extend longitudinally along all rhombomeres. Positively charged lipophilic carbocyanine dyes, such as DiI, have traditionally been used to label neurons, as described here. This method is especially useful for the analysis of PLL innervation because injection of the dye into a neuromast leads to specific labeling of the afferent neurons. The method can also be used to follow the flow of PLL information to higher central nervous system levels by first labeling the central projection of chosen afferent neurons and then making a second injection of DiI within the synaptic field to label the second-order neurons that extend dendrites to this field.