Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed

Tip-sample interactions in atomic force microscopy: I. Modulating adhesion between silicon nitride and glass

An adhesive interaction between a silicon nitride AFM tip and glass substrate in water is described. This adhesion is in the range 5-40 nN, of which a large component is likely to be due to hydrogen bonding between the silanol groups on both surfaces. The interaction can be modulated by a variety of buffers commonly used in biochemical and biological research, including sodium phosphate, tris(hydroxymethyl)aminomethane, glycine, and N-2-hydroxyethyl-piperazine N'-2-ethanesulfonic acid. Using these buffers it appears that there are effects of ion concentration, ion type and pH on the measured adhesion. Of the conditions examined, phosphate was most effective at reducing adhesion and could be used at concentrations as low as 10 mM at neutral pH. The results demonstrate that the chemical interactions between tip and sample can be modulated, and provide a basis for designing conditions for imaging and manipulating biological molecules and structures

Modes of remodeling in the cortical cytoskeleton of vascular endothelial cells

AbstractThe cortical cytoskeleton of vascular endothelial cells plays an important role in responding to mechanical stimuli and controlling the distribution of cell surface proteins. Here, we have used atomic force microscopy to visualize the dynamics of cortical cytoskeleton in living bovine pulmonary artery endothelial cells. We demonstrate that the cortical cytoskeleton, organized as a complex polygonal mesh, is highly dynamic and shows two modes of remodeling: intact-boundary-mode where mesh element boundaries remain intact but move at ∼0.08μm/min allowing the mesh element to change shape, and altered-boundary-mode where new mesh boundaries form and existing ones disappear

Membrane-membrane and membrane-substrate adhesion during dissection of gap junctions with the atomic-force microscope

The gap junction is a specialized region of the plasma membrane that consists of an array of cell-to-cell ion channels. These channels form where the membranes from two cells come together, and the gap junction is therefore composed of two lipid bilayers. The atomic force microscope (AFM) can be used to dissect the gap junction, removing one membrane and exposing the extracellular domains of the second. The force required to dissect the membrane, near 10^(-8) N vertical force for gap junctions adsorbed to mica, provides a measure of the strength of the interaction between the two membranes. Since a single membrane is left in contact with the mica, this interaction must be stronger than the membrane-membrane interaction. Non-junctional membrane attached to the gap junctions is easily removed with the AFM tip while the gap junction membrane remains attached to the mica, providing evidence that the interaction with the mica is mainly mediated by protein-mica interactions. Consistent with this hypothesis is the observation that material trapped under the membrane sometimes results in pieces of membrane above the material being pulled out during dissection. These results lay the foundation for examining the molecular details of the basis for membrane- membrane and membrane-substrate adhesion

Membrane-membrane and membrane-substrate adhesion during dissection of gap junctions with the atomic-force microscope

The gap junction is a specialized region of the plasma membrane that consists of an array of cell-to-cell ion channels. These channels form where the membranes from two cells come together, and the gap junction is therefore composed of two lipid bilayers. The atomic force microscope (AFM) can be used to dissect the gap junction, removing one membrane and exposing the extracellular domains of the second. The force required to dissect the membrane, near 10^(-8) N vertical force for gap junctions adsorbed to mica, provides a measure of the strength of the interaction between the two membranes. Since a single membrane is left in contact with the mica, this interaction must be stronger than the membrane-membrane interaction. Non-junctional membrane attached to the gap junctions is easily removed with the AFM tip while the gap junction membrane remains attached to the mica, providing evidence that the interaction with the mica is mainly mediated by protein-mica interactions. Consistent with this hypothesis is the observation that material trapped under the membrane sometimes results in pieces of membrane above the material being pulled out during dissection. These results lay the foundation for examining the molecular details of the basis for membrane- membrane and membrane-substrate adhesion

Adhesion force imaging in air and liquid by adhesion mode atomic force microscopy

A new imaging mode for the atomic force microscope(AFM), yielding images mapping the adhesion force between tip and sample, is introduced. The adhesion mode AFM takes a force curve at each pixel by ramping a piezoactuator, moving the silicon‐nitride tip up and down towards the sample. During the retrace the tip leaves the sample with an adhesion dip showing up in the force curve. Adhesion force images mapping parameters describing this adhesion dip, such as peak value, width, and area, are acquired on‐line together with the sample topography. Imaging in air gives information on the differences in hydrophobicity of sample features. While imaging a mercaptopentadecane‐gold layer on glass in demineralized water, the adhesion force could be modulated by adding phosphate buffered saline

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV