Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Distribution and dynamics of gap junction channels revealed in living cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Bayesian inference for improved single molecule fluorescence tracking

Single molecule tracking is widely used to monitor the change in position of lipids and proteins in living cells. In many experiments in which molecules are tagged with a single or small number of fluorophores, the signal/noise ratio may be limiting, the number of molecules is not known, and fluorophore blinking and photobleaching can occur. All these factors make accurate tracking over long trajectories difficult and hence there is still a pressing need to develop better algorithms to extract the maximum information from a sequence of fluorescence images. We describe here a Bayesian-based inference approach, based on a trans-dimensional sequential Monte Carlo method that utilizes both the spatial and temporal information present in the image sequences. We show, using model data, where the real trajectory of the molecule is known, that our method allows accurate tracking of molecules over long trajectories even with low signal/noise ratio and in the presence of fluorescence blinking and photobleaching. The method is then applied to real experimental data.     

Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation

The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic β-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically only to those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.     

A mathematical description of metabolizing systems: II

Some of the laboratory procedures available for determining the functions in the integral equation established in part I are discussed. The tracer or tagged molecule technique is shown to be especially promising including the use of “double tracer” molecules. Conversely, the integral equation may be a convenient device for correlating and integrating some of the work now being done with tracer molecules in biological systems.     

Testing independence between two random sets for the analysis of colocalization in bioimaging

Colocalization aims at characterizing spatial associations between two fluorescently tagged biomolecules by quantifying the co-occurrence and correlation between the two channels acquired in fluorescence microscopy. Colocalization is presented either as the degree of overlap between the two channels or the overlays of the red and green images, with areas of yellow indicating colocalization of the molecules. This problem remains an open issue in diffraction-limited microscopy and raises new challenges with the emergence of superresolution imaging, a microscopic technique awarded by the 2014 Nobel prize in chemistry. We propose GcoPS, for Geo-coPositioning System, an original method that exploits the random sets structure of the tagged molecules to provide an explicit testing procedure. Our simulation study shows that GcoPS unequivocally outperforms the best competitive methods in adverse situations (noise, irregularly shaped fluorescent patterns, and different optical resolutions). GcoPS is also much faster, a decisive advantage to face the huge amount of data in superresolution imaging. We demonstrate the performances of GcoPS on two biological real data sets, obtained by conventional diffraction-limited microscopy technique and by superresolution technique, respectively.     

Expression and function of potassium channels in the human placental vasculature

In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.     

Relationship between placental vascular endothelial growth factor expression and placental/endometrial vascularity in the pig

We investigated the temporal association between placental vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis and vascular permeability, and changes in placental/endometrial vascularity on selected days throughout gestation in the pig. Placental and endometrial tissues were collected from sows on Days 25 (n = 4), 36 (n =6), 44 (n = 6), 70 (n =5), 90 (n =5 ), and 112 (n = 7) of gestation. Cross sections of the placental/endometrial interface of each conceptus were used to estimate the number of blood vessels per unit area via image analysis and the intensity of VEGF staining via immunohistochemistry. Placental tissues were also collected on these days to evaluate VEGF mRNA expression. Placental VEGF mRNA expression and the numbers of blood vessels per unit area of placental and adjacent endometrial tissue were low and decreasing from Day 25 to Day 44, before increasing (P < 0.05) markedly and progressively through Day 112. These data are consistent with the marked increase in VEGF immunostaining in the chorionic and uterine luminal epithelium from early to late gestation. Further, these increases in placental VEGF mRNA were positively correlated with fetal weight (r = 0.73; P < 0.0001) and placental efficiency (fetal weight/placental weight ratio; r = 0.66, P < 0.0001). These data are consistent with a role for VEGF in increasing the number of blood vessels at the placental endometrial interface, resulting in an increased capacity for nutrient transfer from the maternal to the fetal compartment.     

Noninvasive Cardiac Flow Assessment Using High Speed Magnetic Resonance Fluid Motion Tracking

Cardiovascular diseases can be diagnosed by assessing abnormal flow behavior in the heart. We introduce, for the first time, a magnetic resonance imaging-based diagnostic that produces sectional flow maps of cardiac chambers, and presents cardiac analysis based on the flow information. Using steady-state free precession magnetic resonance images of blood, we demonstrate intensity contrast between asynchronous and synchronous proton spins. Turbulent blood flow in cardiac chambers contains asynchronous blood proton spins whose concentration affects the signal intensities that are registered onto the magnetic resonance images. Application of intensity flow tracking based on their non-uniform signal concentrations provides a flow field map of the blood motion. We verify this theory in a patient with an atrial septal defect whose chamber blood flow vortices vary in speed of rotation before and after septal occlusion. Based on the measurement of cardiac flow vorticity in our implementation, we establish a relationship between atrial vorticity and septal defect. The developed system has the potential to be used as a prognostic and investigative tool for assessment of cardiac abnormalities, and can be exploited in parallel to examining myocardial defects using steady-state free precession magnetic resonance images of the heart.     

Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo

We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models.     

Noninvasive cineangiography by magnetic resonance global coherent free precession

Cardiovascular disease is primarily diagnosed using invasive X-ray cineangiography. Here we introduce a new concept in magnetic resonance imaging (MRI) that, for the first time, produces similar images noninvasively and without a contrast agent. Protons in moving blood are 'tagged' every few milliseconds as they travel through an arbitrary region in space. Simultaneous with ongoing tagging of new blood, previously tagged blood is maintained in a state of global coherent free precession (GCFP), which allows acquisition of consecutive movie frames as the heart pushes blood through the vascular bed. Body tissue surrounding the moving blood is never excited and therefore remains invisible. In 18 subjects, pulsating blood could be seen flowing through three-dimensional (3D) space for distances of up to 16 cm outside the stationary excitation region. These data underscore that our approach noninvasively characterizes both anatomy and blood flow in a manner directly analogous to invasive procedures.     

Probing functionalized gold colloids for single particle tracking experiments

Functionalized submicroscopic particles are currently used to label proteins or lipids at the surface of living cells for single particle tracking experiments. In many cases, it can be of crucial importance for the particle to be anchored to a single molecule. We have addressed this question for the labeling at the plasma membrane of NRK cells of the mu-opioid receptor bearing a T7 epitope at the N-terminus. Using biophysical methods we were able to prepare quasi-monovalent anti-T7 antibody conjugated gold colloids (40 nm diameter) leading to stable and specific binding to the receptor. The rational method, we report here, can be extended to design customized probes for the labeling of various tagged molecules.     

The differential equations for specific activities in a distributed tracer system

Under the assumption of simple Fick-law diffusion with convection and/or imposed force fields, the flux law for tagged molecules in a tracer system is derived, and the Sheppard-Householder interfusion coefficient identified. The partial differential equations for concentrations of tagged species and for specific activities in an open distributed reaction system are derived and compared.     

A quantitative histochemical study of the microvasculature of irradiated skin

Short-term X-ray damage to the microvasculature of the skin of newborn rats has been quantitated using Horseradish Peroxidase as a tracer. Image analysis of thick sections on which peroxidase was demonstrated histochemically revealed a radioinduced increase in vascular volume coupled with a decrease in vascular length and an altered frequency distribution of blood vessel calibers which resulted in early telangiectasia. The results afforded by direct counting of peroxidase positive macrophagic cells and microphotometric evaluation of peroxidase present in the connective tissue indicate a progressive increase in capillary permeability as a function of dose and time post-irradiation. The accuracy with which the affected region of blood vessels coincided with the area exposed to the beam favours the hypothesis of direct damage to the vessel wall as a major cause of radioinduced lesion.     

Imaging and tracking of single GFP molecules in solution

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.     

Segmentation-Less,Automated, Vascular Vectorization

Recent advances in two-photon fluorescence microscopy (2PM) have allowed large scale imaging and analysis of blood vessel networks in living mice. However, extracting network graphs and vector representations for the dense capillary bed remains a bottleneck in many applications. Vascular vectorization is algorithmically difficult because blood vessels have many shapes and sizes, the samples are often unevenly illuminated, and large image volumes are required to achieve good statistical power. State-of-the-art, three-dimensional, vascular vectorization approaches often require a segmented (binary) image, relying on manual or supervised-machine annotation. Therefore, voxel-by-voxel image segmentation is biased by the human annotator or trainer. Furthermore, segmented images oftentimes require remedial morphological filtering before skeletonization or vectorization. To address these limitations, we present a vectorization method to extract vascular objects directly from unsegmented images without the need for machine learning or training. The Segmentation-Less, Automated, Vascular Vectorization (SLAVV) source code in MATLAB is openly available on GitHub. This novel method uses simple models of vascular anatomy, efficient linear filtering, and vector extraction algorithms to remove the image segmentation requirement, replacing it with manual or automated vector classification. Semi-automated SLAVV is demonstrated on three in vivo 2PM image volumes of microvascular networks (capillaries, arterioles and venules) in the mouse cortex. Vectorization performance is proven robust to the choice of plasma- or endothelial-labeled contrast, and processing costs are shown to scale with input image volume. Fully-automated SLAVV performance is evaluated on simulated 2PM images of varying quality all based on the large (1.4×0.9×0.6 mm³ and 1.6×10⁸ voxel) input image. Vascular statistics of interest (e.g. volume fraction, surface area density) calculated from automatically vectorized images show greater robustness to image quality than those calculated from intensity-thresholded images.     

The permeability of nonhuman primate vaginal epithelium: a freeze-fracture and tracer-perfusion study

The lining of the vaginal mucosa in primates is a keratinized stratified squamous epithelium. As in other structurally similar epithelia, one function of the vaginal epithelium is to provide a barrier between the external environment and the underlying tissues. The vaginal lining is aglandular and the source of true vaginal fluid has been suggested to be the intercellular channels of the epithelium. On the other hand, other structurally similar epithelia have been shown to have a barrier to the movement of water-soluble molecules through these channels. In the present study, we have examined the permeability of rhesus monkey vaginal epithelium to lanthanum and horseradish peroxidase. Both tracer molecules penetrated the intercellular channels in the lower layers of the epithelium, but were excluded from the channels at and above the granular layer. Neither tracer penetrated significantly between cells at the free surface of the epithelium and usually did not penetrate between cells in the upper layers to any degree from the cut edges of the biopsy. These results are consistent with tracer studies in other structurally similar epithelia and strongly suggest that the upper layers of vaginal epithelium present a barrier to the movement of water-soluble molecules through the intercellular channel system. Freeze-fracture analysis of the epithelium revealed gap junctions and desmosomes between cells in the lower layers, but the former disappear in the upper layers. Unlike other keratinizing epithelia that have been described, random intramembranous particles do not disappear from the plasma membranes of the fully differentiated cells. Fracture planes through the upper layers reveal particle-free lamellae in the intercellular spaces, supporting the idea that intercellular lipids may be one of the components that limits the permeability of the intercellular spaces in this epithelium.     

Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.     

A public software for energy filtering transmission electron tomography (EFTET-J): application to the study of granular inclusions in bacteria from Riftia pachyptila

Energy-filtering transmission electron microscopy (EFTEM) allows the determination of elemental distributions out of a sequence of energy filtered images. Combined with electron tomography, EFTEM is a powerful tool to obtain three-dimensional chemical maps from sub-cellular structures. However, there is no existing software in the public-domain for the computation and analysis of 3D-chemical maps. Here, we present a Java-based program to compute 3D-elemental distribution. This program is available as a set of plug-ins for the public-domain Java image processing program Image J inspired by NIH Image. Its implemented algorithms have been successfully applied to the three-dimensional localization of iron granules in semi thin (200 nm) epon sections from the vent worm Riftia pachyptalia.