Single molecule imaging reveals a slice of life

JGP study describes method to trace the real-time movements of individual membrane proteins in live tissue slices.

Directly observing the movements of single, fluorescently labeled molecules can provide crucial information about a molecule’s interactions in living cells. Plasma membrane proteins, for example, may freely diffuse around the lipid bilayer, pausing only when they collide and interact with other proteins. These movements can be followed relatively easily in single-cell organisms or cultured mammalian cells but are much more challenging to observe in multicellular organisms, where cell–cell interactions can dramatically alter the properties of the plasma membrane. In this issue of JGP, Mashanov et al. describe a new method to image and track individual plasma membrane proteins in living tissue slices (1).Justin Molloy (left), Gregory Mashanov (right), and colleagues describe a method to image single plasma membrane proteins in live tissue slices. By tracking individual M₂ muscarinic acetylcholine receptors in cardiac tissue over time, the researchers can construct a super-resolution map of the tissue, encompassing both the round cardiomyocytes and the ultrathin nerve fibers that innervate them.Justin Molloy’s group at The Francis Crick Institute in London are interested in how the M₂ muscarinic acetylcholine receptor regulates the heartbeat. This G protein–coupled receptor diffuses through the plasma membrane and, in response to acetylcholine, alters the resting potential of cardiomyocytes via a G_βγ-mediated interaction with inwardly rectifying potassium GIRK channels (2, 3).“It’s a diffusion-limited signaling cascade, so it’s important to look at the movement of the molecules within the membrane,” Molloy explains. “We’ve tracked the movements of single M₂ receptors in cultured cardiomyocytes, but we wanted to do it in tissues where the cells are in their native environment.”Molloy and colleagues, led by Gregory Mashanov, developed a technique to image single M₂ receptors in cardiac tissue slices (1). Freshly extracted mouse hearts are quickly placed in a custom-made, 3-D–printed cutting block, then sectioned by a multi-blade assembly into 1-mm-thick slices. These slices are treated with a fluorescently labeled ligand that tightly binds to M₂ receptors, before being transferred to coverslips for TIRF video microscopy.Mashanov immediately noticed that cardiomyocytes in living tissue are much more rounded than they are in cell culture. More remarkable still, however, were the differences Mashanov observed when he compared the movements of single M₂ receptors in cells and tissues. “The M₂ receptors move around the membrane around four times faster in tissue than they do in cultured cells,” Mashanov says.The reason for this increased mobility in tissues remains unclear, but Mashanov et al. saw a similarly rapid movement of M₂ receptors in zebrafish hearts, which the researchers were also able to dissect and prepare for TIRF microscopy with their new technique, even though these organs measure just ∼0.5 mm in length.In addition, the researchers discovered that they could use their single-molecule tracking data to create super-resolution images of the cardiac tissue slices. “When we average our tracking data over time, the paths of individual M₂ receptors combine to delineate the cellular structure of the tissue,” Molloy explains.Because neurons also express M₂ receptors, these super-resolution tissue maps include not only the cardiomyocytes but also the nerve fibers that innervate them. “These nerve fibers are only ∼0.2 μM in diameter and they aren’t really visible by light microscopy,” Mashanov says. “But we could see hundreds of them. Every cardiomyocyte has a nerve fiber associated with it.”Mashanov et al.’s technique should be easily adapted for other tissues and membrane proteins and may even facilitate single-molecule imaging in entire model organisms like zebrafish or fruit flies. For Molloy’s laboratory, though, the next step is to develop dual-color labeling of M₂ receptors and the downstream proteins in the pathway, G_βγ and GIRK, so that the kinetics of the molecules’ interactions can be studied in living tissues.     

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing.     

Selective induction of targeted cell death and elimination by near-infrared femtosecond laser ablation

The techniques for inducing the death of specific cells in tissue has attracted attention as new methodologies for studying cell function and tissue regeneration. In this study, we show that a sequential process of targeted cell death and removal can be triggered by short-term exposure of near-infrared femtosecond laser pulses. Kinetic analysis of the intracellular accumulation of trypan blue and the assay of caspase activity revealed that femtosecond laser pulses induced immediate disturbance of plasma membrane integrity followed by apoptosis-like cell death. Yet, adjacent cells showed no sign of membrane damage and no increased caspase activity. The laser-exposed cells eventually detached from the substrate after a delay of >54 min while adjacent cells remained intact. On the base of in vitro experiments, we applied the same approach to ablate targeted single cardiac cells of a live zebrafish heart. The ability of inducing targeted cell death with femtosecond laser pulses should find broad applications that benefit from precise cellular manipulation at the level of single cells in vivo and in vitro.     

DNA modification of live cell surface

We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell–cell interactions.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Revealing Compartmentalized Diffusion in Living Cells with Interferometric Scattering Microscopy

The spatiotemporal organization and dynamics of the plasma membrane and its constituents are central to cellular function. Fluorescence-based single-particle tracking has emerged as a powerful approach for studying the single molecule behavior of plasma-membrane-associated events because of its excellent background suppression, at the expense of imaging speed and observation time. Here, we show that interferometric scattering microscopy combined with 40 nm gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons with up to 3 nm precision and 25 μs temporal resolution. The achievable spatiotemporal precision enabled us to reveal signatures of compartmentalization in neurons likely caused by the actin cytoskeleton.     

Downscaling the Analysis of Complex Transmembrane Signaling Cascades to Closed Attoliter Volumes

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell’s plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.     

Adaptive Geometric Tessellation for 3D Reconstruction of Anisotropically Developing Cells in Multilayer Tissues from Sparse Volumetric Microscopy Images

The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM) image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2–4 slices/cell) and wide range of cell shapes and sizes. The proposed method, named as the ‘Adaptive Quadratic Voronoi Tessellation’ (AQVT), is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM) and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells.     

Trafficking of astrocytic vesicles in hippocampal slices

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.     

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.     

Hyperpolarization of the Membrane Potential in Cardiomyocyte Tissue Slices by the Synchronization Modulation Electric Field

Our previous studies have shown that a specially designed, so-called synchronization modulation electric field can entrain active transporter Na/K pumps in the cell membrane. This approach was previously developed in a study of single cells using a voltage clamp to monitor the pump currents. We are now expanding our study from isolated single cells to aggregated cells in a 3-dimensional cell matrix, through the use of a tissue slice from the rat heart. The slice is about 150 μm in thickness, meaning the slices contain many cell layers, resulting in a simplified 3-dimensional system. A fluorescent probe was used to identify the membrane potential and the ionic concentration gradients across the cell membrane. In spite of intrinsic cell-to-cell interactions and the difficulty in stimulating cell aggregation in the tissue slice, the oscillating electric field increased the intracellular fluorescent intensity, indicating elevation of the cell ionic concentration and hyperpolarization of the cell membrane. Blockage of these changes by ouabain confirmed that the results are directly related to Na/K pumps. These results along with the backward modulation indicate that the synchronization modulation electric field can influence the Na/K pumps in tissue cells of a 3-dimensional matrix and therefore hyperpolarize the cell membrane.     

Argosomes: membrane fragments on the run

Any biology student will know that the plasma membrane separates the inside of a cell from the outside world. Indeed, the plasma membrane is the guardian of a cell's physiology. Fewer students will be aware that cells can export bits of plasma membrane to distant sites in a tissue. Recent work demonstrates the transfer of membrane fragments in a live epithelium and, importantly, suggests that these fragments might be used as a vehicle to transport morphogens in a developing tissue.     

Imaging Membrane Lipid Order in Whole, Living Vertebrate Organisms

We report the first imaging of membrane lipid order in a whole, living vertebrate organism. This was achieved with the phase-sensitive, membrane-partitioning probe Laurdan in conjunction with multiphoton microscopy to image cell membranes in various tissues of live zebrafish embryos in three dimensions, including hindbrain, retina, muscle, gut, and kidney. The data also allowed quantitative analysis of membrane order, which showed high lipid order in the apical surfaces of polarized epithelial cells. The transition of membrane order imaging from cultured cell lines to living organisms is an important step forward in understanding the physiological relevance of membrane microdomains including lipid rafts.     

Mycoplasma Suppression of THP-1 Cell TLR Responses Is Corrected with Antibiotics

Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 β was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1β and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.     

High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells

Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.     

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.     

Small molecule screening by imaging

Useful small molecule tools can be discovered in imaging screens that measure phenotype in single cells or small organisms. Recent examples include identification of small molecule inhibitors of processes such as cell migration, cytokinesis, mitotic spindle length determination, melanogenesis, aggresome formation, membrane transport and nuclear export. Imaging screens are currently limited by challenges in the areas of image analysis and target identification. We discuss the use of model organisms such as zebrafish in screens and review different methods of target identification. The emerging field of automated image analysis is also introduced.