The Epithelial Cell Leak Pathway.

The epithelial cell tight junction structure is the site of the transepithelial movement of solutes and water between epithelial cells (paracellular permeability). Paracellular permeability can be divided into two distinct pathways, the Pore Pathway mediating the movement of small ions and solutes and the Leak Pathway mediating the movement of large solutes. Claudin proteins form the basic paracellular permeability barrier and mediate the movement of small ions and solutes via the Pore Pathway. The Leak Pathway remains less understood. Several proteins have been implicated in mediating the Leak Pathway, including occludin, ZO proteins, tricellulin, and actin filaments, but the proteins comprising the Leak Pathway remain unresolved. Many aspects of the Leak Pathway, such as its molecular mechanism, its properties, and its regulation, remain controversial. In this review, we provide a historical background to the evolution of the Leak Pathway concept from the initial examinations of paracellular permeability. We then discuss current information about the properties of the Leak Pathway and present current theories for the Leak Pathway. Finally, we discuss some recent research suggesting a possible molecular basis for the Leak Pathway.

Modulating Cell-Surface Receptor Signaling and Ion Channel Functions by In†Situ Glycan Editing.

Glycans anchored on cell-surface receptors are active modulators of receptor signaling. A strategy is presented that enforces transient changes to cell-surface glycosylation patterns to tune receptor signaling. This approach, termed in situ glycan editing, exploits recombinant glycosyltransferases to incorporate monosaccharides with linkage specificity onto receptors in situ. α2,3-linked sialic acid or α1,3-linked fucose added in situ suppresses signaling through epidermal growth factor receptor and fibroblast growth factor receptor. We also applied the same strategy to regulate the electrical signaling of a potassium ion channel-human ether-à-go-go-related gene channel. Compared to gene editing, no long-term perturbations are introduced to the treated cells. In situ glycan editing therefore offers a promising approach for studying the dynamic role of specific glycans in membrane receptor signaling and ion channel functions.

Tracking surface glycans on live cancer cells with single-molecule sensitivity.

Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide- or alkyne-tagged glycans, a sufficiently low spatial density of dye-labeled glycans was achieved, enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked N-acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye-labeled glycans in mammary cancer cells revealed constrained diffusion of both N- and O-linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes.

Modeling membrane nanotube morphology: the role of heterogeneity in composition and material properties.

Membrane nanotubes are dynamic structures that may connect cells over long distances. Nanotubes are typically thin cylindrical tubes, but they may occasionally have a beaded architecture along the tube. In this paper, we study the role of membrane mechanics in governing the architecture of these tubes and show that the formation of bead-like structures along the nanotubes can result from local heterogeneities in the membrane either due to protein aggregation or due to membrane composition. We present numerical results that predict how membrane properties, protein density, and local tension compete to create a phase space that governs the morphology of a nanotube. We also find that there exists a discontinuity in the energy that impedes two beads from fusing. These results suggest that the membrane-protein interaction, membrane composition, and membrane tension closely govern the tube radius, number of beads, and the bead morphology.

Nucleation and growth of integrin adhesions.

We present a model that provides a mechanistic understanding of the processes that govern the formation of the earliest integrin adhesions ex novo from an approximately planar plasma membrane. Using an analytic analysis of the free energy of a dynamically deformable membrane containing freely diffusing receptors molecules and long repeller molecules that inhibit integrins from binding with ligands on the extracellular matrix, we predict that a coalescence of polymerizing actin filaments can deform the membrane toward the extracellular matrix and facilitate integrin binding. Monte Carlo simulations of this system show that thermally induced membrane fluctuations can either zip-up and increase the radius of a nucleated adhesion or unzip and shrink an adhesion, but the fluctuations cannot bend the ventral membrane to nucleate an adhesion. To distinguish this integrin adhesion from more mature adhesions, we refer to this early adhesion as a nouveau adhesion.

Visualizing glycans on single cells and tissues-Visualizing glycans on single cells and tissues.

Metabolic oligosaccharide engineering and chemoenzymatic glycan labeling have provided powerful tools to study glycans in living systems and tissue samples. In this review article, we summarize recent advances in this field with a focus on innovative approaches for glycan imaging. The presented applications demonstrate that several of the leading imaging methods, which have revolutionized quantitative cell biology, can be adapted to imaging glycans on single cells and tissues.

Tools for Studying Glycans: Recent Advances in Chemoenzymatic Glycan Labeling.

The study of cellular glycosylation presents many challenges due, in large part, to the nontemplated nature of glycan biosynthesis and glycans' structural complexity. Chemoenzymatic glycan labeling (CeGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CeGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CeGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CeGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.

Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes.

Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single ß-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, ß-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

Imaging of gene expression in living cells and tissues.

It is possible to observe gene expression within single cells using a tetracycline inducible promoter for activation. Transcription can be observed by using a fluorescent fusion protein to bind nascent RNA. Ultimately, it is desirable to activate a reporter gene within a single cell with only photons. This is achieved by preparing a chemically altered transcription factor that is functionally unable to activate a reporter gene until it is exposed to photon excitation. We apply two-photon imaging to visualize tumor cells expressing a transgene and ultimately this approach will provide the means to activate a specific gene within a single cell within any tissue to ultimately observe its functional significance in situ.

Reflectivity and topography of cells grown on glass-coverslips measured with phase-shifted laser feedback interference microscopy.

In spite of the advantages associated with the molecular specificity of fluorescence imaging, there is still a significant need to augment these approaches with label-free imaging. Therefore, we have implemented a form of interference microscopy based upon phase-shifted, laser-feedback interferometry and developed an algorithm that can be used to separate the contribution of the elastically scattered light by sub-cellular structures from the reflection at the coverslip-buffer interface. The method offers an opportunity to probe protein aggregation, index of refraction variations and structure. We measure the topography and reflection from calibration spheres and from stress fibers and adhesions in both fixed and motile cells. Unlike the data acquired with reflection interference contrast microscopy, where the reflection from adhesions can appear dark, our approach demonstrates that these regions have high reflectivity. The data acquired from fixed and live cells show the presence of a dense actin layer located ≈ 100 nm above the coverslip interface. Finally, the measured dynamics of filopodia and the lamella in a live cell supports retrograde flow as the dominate mechanism responsible for filopodia retraction.

Membrane deformation at integrin adhesions.

In order to measure the nucleation of nouveau adhesions on the ventral surface of a cell, we have combined phase shifting laser feedback interferometry with a high numerical aperture inverted fluorescence microscope. We use fluorescence to image molecules at the adhesion site and stage scanning interference microscopy in order to measure the distance between the ventral surface of a cell and the substratum with several nanometer precision. Our analytic and Monte Carlo simulations of integrin mediated adhesions predict several features of these nouveau adhesions. An analysis of the energetics of membrane bending and the effects of a composite system of freely diffusing repellers and receptors and a fixed network of ligands on the extracellular matrix predicts that a small bundle of actin filaments should be able to push the membrane down to the extracellular matrix and nucleate a nouveau adhesion with critical radius below the diffraction limit. We have obtained a map of the reflectivity of the ventral surface of fixed metastatic mammary adenocarcinoma cells and we have shown that the data are correlated with markers for a focal adhesion adaptor protein. We are modeling the interference of the incident electric field with the field reflected from the ventral surface so as to obtain the surface topography at focal adhesions from the optical phase data.

Microscopic and macroscopic structure of the precursor layer in spreading viscous drops.

We study the spreading of viscous nonvolatile liquids on smooth horizontal substrates using a phase-modulated interference microscope with sufficient dynamic range to enable the simultaneous measurement of both the inner ("microscopic") length scale and the outer ("macroscopic") flow scale in addition to the intermediate matching region. The resulting measurements of both the apparent contact angle and the lateral scale of the precursor "wetting" film agree quantitatively with theoretical predictions for a van der Waal's liquid over a wide range of capillary numbers.