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1.  Deficiency of Transcription Factor Brn4 Disrupts Cochlear Gap Junction Plaques in a Model of DFN3 Non-Syndromic Deafness 
PLoS ONE  2014;9(9):e108216.
Brn4, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome–linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K+ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.
PMCID: PMC4178122  PMID: 25259580
2.  Mechanisms That Determine the Internal Environment of the Developing Brain: A Transcriptomic, Functional and Ultrastructural Approach 
PLoS ONE  2013;8(7):e65629.
We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.
PMCID: PMC3699566  PMID: 23843944
3.  The Peptidoglycan-Binding Protein SjcF1 Influences Septal Junction Function and Channel Formation in the Filamentous Cyanobacterium Anabaena 
mBio  2015;6(4):e00376-15.
Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in sjcF1 was not affected in peptidoglycan synthesis but showed an altered morphology of the septal peptidoglycan channels, which were significantly wider in the mutant than in the wild type. The mutant was impaired in intercellular exchange of a fluorescent probe to a similar extent as a sepJ deletion mutant. SjcF1 additionally bears an SH3 domain for protein-protein interactions. SH3 binding domains were identified in SepJ and FraC, and evidence for interaction of SjcF1 with both SepJ and FraC was obtained. SjcF1 represents a novel protein involved in structuring the peptidoglycan layer, which links peptidoglycan channel formation to septal junction complex function in multicellular cyanobacteria. Nonetheless, based on its subcellular distribution, this might not be the only function of SjcF1.
Cell-cell communication is central not only for eukaryotic but also for multicellular prokaryotic systems. Principles of intercellular communication are well established for eukaryotes, but the mechanisms and components involved in bacteria are just emerging. Filamentous heterocyst-forming cyanobacteria behave as multicellular organisms and represent an excellent model to study prokaryotic cell-cell communication. A path for intercellular metabolite exchange appears to involve transfer through molecular structures termed septal junctions. They are reminiscent of metazoan gap junctions that directly link adjacent cells. In cyanobacteria, such structures need to traverse the peptidoglycan layers in the intercellular septa of the filament. Here we describe a factor involved in the formation of channels across the septal peptidoglycan layers, thus contributing to the multicellular behavior of these organisms.
PMCID: PMC4488944  PMID: 26126850
4.  Transmission eigenvalue distributions in highly conductive molecular junctions 
Background: The transport through a quantum-scale device may be uniquely characterized by its transmission eigenvalues τn. Recently, highly conductive single-molecule junctions (SMJ) with multiple transport channels (i.e., several τn > 0) have been formed from benzene molecules between Pt electrodes. Transport through these multichannel SMJs is a probe of both the bonding properties at the lead–molecule interface and of the molecular symmetry.
Results: We use a many-body theory that properly describes the complementary wave–particle nature of the electron to investigate transport in an ensemble of Pt–benzene–Pt junctions. We utilize an effective-field theory of interacting π-electrons to accurately model the electrostatic influence of the leads, and we develop an ab initio tunneling model to describe the details of the lead–molecule bonding over an ensemble of junction geometries. We also develop a simple decomposition of transmission eigenchannels into molecular resonances based on the isolated resonance approximation, which helps to illustrate the workings of our many-body theory, and facilitates unambiguous interpretation of transmission spectra.
Conclusion: We confirm that Pt–benzene–Pt junctions have two dominant transmission channels, with only a small contribution from a third channel with τn << 1. In addition, we demonstrate that the isolated resonance approximation is extremely accurate and determine that transport occurs predominantly via the HOMO orbital in Pt–benzene–Pt junctions. Finally, we show that the transport occurs in a lead–molecule coupling regime where the charge carriers are both particle-like and wave-like simultaneously, requiring a many-body description.
PMCID: PMC3304317  PMID: 22428095
benzene–platinum junction; effective-field theory; isolated-resonance approximation; lead–molecule interface; many-body theory; multichannel; quantum transport; single-molecule junction; transmission eigenchannels
5.  Nonlinear Gap Junctions Enable Long-Distance Propagation of Pulsating Calcium Waves in Astrocyte Networks 
PLoS Computational Biology  2010;6(8):e1000909.
A new paradigm has recently emerged in brain science whereby communications between glial cells and neuron-glia interactions should be considered together with neurons and their networks to understand higher brain functions. In particular, astrocytes, the main type of glial cells in the cortex, have been shown to communicate with neurons and with each other. They are thought to form a gap-junction-coupled syncytium supporting cell-cell communication via propagating Ca2+ waves. An identified mode of propagation is based on cytoplasm-to-cytoplasm transport of inositol trisphosphate (IP3) through gap junctions that locally trigger Ca2+ pulses via IP3-dependent Ca2+-induced Ca2+ release. It is, however, currently unknown whether this intracellular route is able to support the propagation of long-distance regenerative Ca2+ waves or is restricted to short-distance signaling. Furthermore, the influence of the intracellular signaling dynamics on intercellular propagation remains to be understood. In this work, we propose a model of the gap-junctional route for intercellular Ca2+ wave propagation in astrocytes. Our model yields two major predictions. First, we show that long-distance regenerative signaling requires nonlinear coupling in the gap junctions. Second, we show that even with nonlinear gap junctions, long-distance regenerative signaling is favored when the internal Ca2+ dynamics implements frequency modulation-encoding oscillations with pulsating dynamics, while amplitude modulation-encoding dynamics tends to restrict the propagation range. As a result, spatially heterogeneous molecular properties and/or weak couplings are shown to give rise to rich spatiotemporal dynamics that support complex propagation behaviors. These results shed new light on the mechanisms implicated in the propagation of Ca2+ waves across astrocytes and the precise conditions under which glial cells may participate in information processing in the brain.
Author Summary
In recent years, the focus of Cellular Neuroscience has progressively stopped only being on neurons but started to include glial cells as well. Indeed, astrocytes, the main type of glial cells in the cortex, dynamically modulate neuron excitability and control the flow of information across synapses. Moreover, astrocytes have been shown to communicate with each other over long distances using calcium waves. These waves spread from cell to cell via molecular gates called gap junctions, which connect neighboring astrocytes. In this work, we used a computer model to question what biophysical mechanisms could support long-distance propagation of Ca2+ wave signaling. The model shows that the coupling function of the gap junction must be non-linear and include a threshold. This prediction is largely unexpected, as gap junctions are classically considered to implement linear functions. Recent experimental observations, however, suggest their operation could actually be more complex, in agreement with our prediction. The model also shows that the distance traveled by waves depends on characteristics of the internal astrocyte dynamics. In particular, long-distance propagation is facilitated when internal calcium oscillations are in their frequency-modulation encoding mode and are pulsating. Hence, this work provides testable experimental predictions to decipher long-distance communication between astrocytes.
PMCID: PMC2928752  PMID: 20865153
6.  A Single-Level Tunnel Model to Account for Electrical Transport through Single Molecule- and Self-Assembled Monolayer-based Junctions 
Scientific Reports  2016;6:26517.
We present a theoretical analysis aimed at understanding electrical conduction in molecular tunnel junctions. We focus on discussing the validity of coherent versus incoherent theoretical formulations for single-level tunneling to explain experimental results obtained under a wide range of experimental conditions, including measurements in individual molecules connecting the leads of electromigrated single-electron transistors and junctions of self-assembled monolayers (SAM) of molecules sandwiched between two macroscopic contacts. We show that the restriction of transport through a single level in solid state junctions (no solvent) makes coherent and incoherent tunneling formalisms indistinguishable when only one level participates in transport. Similar to Marcus relaxation processes in wet electrochemistry, the thermal broadening of the Fermi distribution describing the electronic occupation energies in the electrodes accounts for the exponential dependence of the tunneling current on temperature. We demonstrate that a single-level tunnel model satisfactorily explains experimental results obtained in three different molecular junctions (both single-molecule and SAM-based) formed by ferrocene-based molecules. Among other things, we use the model to map the electrostatic potential profile in EGaIn-based SAM junctions in which the ferrocene unit is placed at different positions within the molecule, and we find that electrical screening gives rise to a strongly non-linear profile across the junction.
PMCID: PMC4877922  PMID: 27216489
7.  A method to study electronic transport properties of molecular junction: one-dimension transmission combined with three-dimension correction approximation (OTCTCA) 
Scientific Reports  2016;6:21946.
Based on the ab initio calculation, a method of one-dimension transmission combined with three-dimension correction approximation (OTCTCA) is developed to investigate electron-transport properties of molecular junctions. The method considers that the functional molecule provides a spatial distribution of effective potential field for the electronic transport. The electrons are injected from one electrode by bias voltage, then transmit through the potential field around the functional molecule, at last are poured into the other electrode with a specific transmission probability which is calculated from one-dimension Schrödinger equation combined with three-dimension correction. The electron-transport properties of alkane diamines and 4, 4′-bipyridine molecular junctions are studied by applying OTCTCA method. The numerical results show that the conductance obviously exponentially decays with the increase of molecular length. When stretching molecular junctions, steps with a certain width are presented in conductance traces. Especially, in stretching process of 4, 4′-bipyridine molecular junction, if the terminal N atom is broken from flat part of electrode tip and exactly there is a surface Au atom on the tip nearby the N atom, the molecule generally turns to absorb on the surface Au atom, which further results in another lower conductance step in the traces as the experimental probing.
PMCID: PMC4766509  PMID: 26911451
8.  Electrostatic control over temperature-dependent tunnelling across a single-molecule junction 
Nature Communications  2016;7:11595.
Understanding how the mechanism of charge transport through molecular tunnel junctions depends on temperature is crucial to control electronic function in molecular electronic devices. With just a few systems investigated as a function of bias and temperature so far, thermal effects in molecular tunnel junctions remain poorly understood. Here we report a detailed charge transport study of an individual redox-active ferrocene-based molecule over a wide range of temperatures and applied potentials. The results show the temperature dependence of the current to vary strongly as a function of the gate voltage. Specifically, the current across the molecule exponentially increases in the Coulomb blockade regime and decreases at the charge degeneracy points, while remaining temperature-independent at resonance. Our observations can be well accounted for by a formal single-level tunnelling model where the temperature dependence relies on the thermal broadening of the Fermi distributions of the electrons in the leads.
The effect of temperature on charge transport mechanisms in molecular tunnel junctions is not fully understood. Here, charge transport studies of a redox-active molecule unveil multiple mechanistic regimes which may be explained by thermal broadening of the Fermi distributions of electrons in the leads.
PMCID: PMC4879245  PMID: 27211787
9.  High thermopower of mechanically stretched single-molecule junctions 
Scientific Reports  2015;5:11519.
Metal-molecule-metal junction is a promising candidate for thermoelectric applications that utilizes quantum confinement effects in the chemically defined zero-dimensional atomic structure to achieve enhanced dimensionless figure of merit ZT. A key issue in this new class of thermoelectric nanomaterials is to clarify the sensitivity of thermoelectricity on the molecular junction configurations. Here we report simultaneous measurements of the thermoelectric voltage and conductance on Au-1,4-benzenedithiol (BDT)-Au junctions mechanically-stretched in-situ at sub-nanoscale. We obtained the average single-molecule conductance and thermopower of 0.01 G0 and 15 μV/K, respectively, suggesting charge transport through the highest occupied molecular orbital. Meanwhile, we found the single-molecule thermoelectric transport properties extremely-sensitive to the BDT bridge configurations, whereby manifesting the importance to design the electrode-molecule contact motifs for optimizing the thermoelectric performance of molecular junctions.
PMCID: PMC4481826  PMID: 26112999
10.  Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology 
World Journal of Gastroenterology  2015;21(41):11862-11876.
AIM: To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development.
METHODS: Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1/Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function.
RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of host-microbiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that ABCB1 expression identifies a subpopulation of pro-inflammatory Th17 cells which were resistant to treatment with glucocorticoids. The evidence for the involvement of ABCC2 and ABCG2 in colonic pathophysiology was weak.
CONCLUSION: ABCB1, diet, and gut microbes mutually interact in colonic inflammation, a well-known risk factor for CRC. Further insight may be translated into preventive and treatment strategies.
PMCID: PMC4631984  PMID: 26557010
ATP-binding cassette transporters; Colorectal cancer; Intestinal; Inflammatory bowel disease; Inflammation; Adenoma-carcinoma sequence
11.  Coherent Charge Transport in Ballistic InSb Nanowire Josephson Junctions 
Scientific Reports  2016;6:24822.
Hybrid InSb nanowire-superconductor devices are promising for investigating Majorana modes and topological quantum computation in solid-state devices. An experimental realisation of ballistic, phase-coherent superconductor-nanowire hybrid devices is a necessary step towards engineering topological superconducting electronics. Here, we report on a low-temperature transport study of Josephson junction devices fabricated from InSb nanowires grown by molecular-beam epitaxy and provide a clear evidence for phase-coherent, ballistic charge transport through the nanowires in the junctions. We demonstrate that our devices show gate-tunable proximity-induced supercurrent and clear signatures of multiple Andreev reflections in the differential conductance, indicating phase-coherent transport within the junctions. We also observe periodic modulations of the critical current that can be associated with the Fabry-Pérot interference in the nanowires in the ballistic transport regime. Our work shows that the InSb nanowires grown by molecular-beam epitaxy are of excellent material quality and hybrid superconducting devices made from these nanowires are highly desirable for investigation of the novel physics in topological states of matter and for applications in topological quantum electronics.
PMCID: PMC4840339  PMID: 27102689
12.  Current–voltage characteristics of single-molecule diarylethene junctions measured with adjustable gold electrodes in solution 
We report on an experimental analysis of the charge transport through sulfur-free photochromic molecular junctions. The conductance of individual molecules contacted with gold electrodes and the current–voltage characteristics of these junctions are measured in a mechanically controlled break-junction system at room temperature and in liquid environment. We compare the transport properties of a series of molecules, labeled TSC, MN, and 4Py, with the same switching core but varying side-arms and end-groups designed for providing the mechanical and electrical contact to the gold electrodes. We perform a detailed analysis of the transport properties of TSC in its open and closed states. We find rather broad distributions of conductance values in both states. The analysis, based on the assumption that the current is carried by a single dominating molecular orbital, reveals distinct differences between both states. We discuss the appearance of diode-like behavior for the particular species 4Py that features end-groups, which preferentially couple to the metal electrode by physisorption. We show that the energetic position of the molecular orbital varies as a function of the transmission. Finally, we show for the species MN that the use of two cyano end-groups on each side considerably enhances the coupling strength compared to the typical behavior of a single cyano group.
PMCID: PMC3554105  PMID: 23365792
diarylethene; mechanically controllable break-junction; molecular electronics; photoswitching; single-molecule junctions
13.  Claudin-2-dependent paracellular channels are dynamically gated 
eLife  null;4:e09906.
Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation.
eLife digest
Epithelial cells form layers that line the inner surface of the gut, lungs and other organs. They act as barriers to control the movement of water, ions and small molecules between internal compartments within the body and the external environment. Some substances are transported across these barriers by passing through individual epithelial cells, but others pass through the spaces between adjacent cells. These spaces are sealed by tight junctions. If the tight junctions do not work properly, it can cause problems with regulating the movement of molecules across epithelial-lined surfaces. This in turn can contribute to diseases in humans, including inflammatory bowel disease and chronic kidney disease.
Proteins called claudins form channels that only allow certain molecules to pass through tight junctions. One member of this family, called claudin-2, allows sodium ions and other small positively charged ions to cross between adjacent cells. However, it is not clear how these channels work, largely due to the absence of appropriate tools to study this process. Here, Weber et al. adapted a technique called patch clamping to study the behavior of individual claudin-2 channels in the tight junctions between mammalian epithelial cells.
Weber et al. found that claudin-2 allows positively charged ions to move across a tight junction in short bursts rather than in a steady stream as had been suggested by previous work. These bursts typically begin and end in less than a millisecond. Further experiments revealed that claudin-2 channels have several states; in one state the channel is fully open, in another the channel is firmly closed, and in the third state the channel is temporarily closed but primed to open.
Further experiments show that mutations in the gene that encodes claudin-2 or drugs that inhibit claudin-2's function alter the open and closed behaviors of these trans-tight junction channels. The technique developed by Weber et al. will enable researchers to understand how channel proteins at tight junctions assemble and operate. Such studies may lead to the development of drugs that can alter the activity of these channels to treat particular diseases.
PMCID: PMC4755754  PMID: 26568313
tight junction; epithelium; ion channel; patch clamp; barrier function; intestine; Human; Other
14.  Negative differential conductance and super-Poissonian shot noise in single-molecule magnet junctions 
Scientific Reports  2015;5:8730.
Molecular spintroinic device based on a single-molecule magnet is one of the ultimate goals of semiconductor nanofabrication technologies. It is thus necessary to understand the electron transport properties of a single-molecule magnet junction. Here we study the negative differential conductance and super-Poissonian shot noise properties of electron transport through a single-molecule magnet weakly coupled to two electrodes with either one or both of them being ferromagnetic. We predict that the negative differential conductance and super-Poissonian shot noise, which can be tuned by a gate voltage, depend sensitively on the spin polarization of the source and drain electrodes. In particular, the shot noise in the negative differential conductance region can be enhanced or decreased originating from the different formation mechanisms of negative differential conductance. The effective competition between fast and slow transport channels is responsible for the observed negative differential conductance and super-Poissonian shot noise. In addition, we further discuss the skewness and kurtosis properties of transport current in the super-Poissonian shot noise regions. Our findings suggest a tunable negative differential conductance molecular device, and the predicted properties of high-order current cumulants are very interesting for a better understanding of electron transport through single-molecule magnet junctions.
PMCID: PMC4348643  PMID: 25736094
15.  Regulation of Gap Junction Dynamics by UNC-44/ankyrin and UNC-33/CRMP through VAB-8 in C. elegans Neurons 
PLoS Genetics  2016;12(3):e1005948.
Gap junctions are present in both vertebrates and invertebrates from nematodes to mammals. Although the importance of gap junctions has been documented in many biological processes, the molecular mechanisms underlying gap junction dynamics remain unclear. Here, using the C. elegans PLM neurons as a model, we show that UNC-44/ankyrin acts upstream of UNC-33/CRMP in regulation of a potential kinesin VAB-8 to control gap junction dynamics, and loss-of-function in the UNC-44/UNC-33/VAB-8 pathway suppresses the turnover of gap junction channels. Therefore, we first show a signal pathway including ankyrin, CRMP, and kinesin in regulating gap junctions.
Author Summary
The nervous system is made up of individual neurons connected by junction structures called synapses. There are two fundamentally different types of synapses: chemical synapses and electrical synapses (also called gap junctions). Through studies in different model organisms, we have gained rich knowledge about the development and dynamics of chemical synapses. However, we still know little about how gap junctions are formed during development and what regulates the dynamic of gap junctions in functional circuits. Using C. elegans PLM neurons as a model, we carried out an unbiased genetic screen and isolated mutants altering gap junctions. In this study, we focus on two of them, unc-44/ankyrin and unc-33/CRMP. Through genetic analysis in combination with live imaging, we find that UNC-44/ankyrin and UNC-33/CRMP play important roles in gap junction turnover and demonstrate that UNC-44/ankyrin acts upstream of UNC-33/CRMP and VAB-8/ kinesin to regulate the removal of gap junction channels from gap junctions.
PMCID: PMC4807823  PMID: 27015090
16.  Chemical control of electrical contact to sp2 carbon atoms 
Nature Communications  2014;5:3659.
Carbon-based nanostructures are attracting tremendous interest as components in ultrafast electronics and optoelectronics. The electrical interfaces to these structures play a crucial role for the electron transport, but the lack of control at the atomic scale can hamper device functionality and integration into operating circuitry. Here we study a prototype carbon-based molecular junction consisting of a single C60 molecule and probe how the electric current through the junction depends on the chemical nature of the foremost electrode atom in contact with the molecule. We find that the efficiency of charge injection to a C60 molecule varies substantially for the considered metallic species, and demonstrate that the relative strength of the metal-C bond can be extracted from our transport measurements. Our study further suggests that a single-C60 junction is a basic model to explore the properties of electrical contacts to meso- and macroscopic sp2 carbon structures.
Understanding metal-molecule contacts is crucial for molecular electronic devices. Here, the authors use a C60-terminated scanning tunnelling tip to probe how the chemical nature of the contacting atom on the substrate electrode determines the transport properties.
PMCID: PMC3997807  PMID: 24736561
17.  Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo  
Hepatology (Baltimore, Md.)  2016;64(4):1317-1329.
Liver kinase B1 (LKB1) and its downstream effector AMP‐activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich‐cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver‐specific (albumin‐Cre) LKB1 knockout mice (LKB1−/−) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1−/− livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6‐carboxyfluorescein diacetate (6‐CFDA), which is processed in hepatocytes into its fluorescent derivative 6‐carboxyfluorescein (6‐CF) and secreted into the canaliculi. In LKB1−/− mice, 6‐CF remained largely in hepatocytes, canalicular secretion was delayed, and 6‐CF appeared in the blood. To test whether 6‐CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6‐CFDA. In wild‐type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1−/− mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. Conclusion: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317‐1329)
PMCID: PMC5033699  PMID: 27396550
18.  Probing electron-phonon excitations in molecular junctions by quantum interference 
Scientific Reports  2016;6:20899.
Electron-phonon coupling is a fundamental inelastic interaction in condensed matter and in molecules. Here we probe phonon excitations using quantum interference in electron transport occurring in short chains of anthraquinone based molecular junctions. By studying the dependence of molecular junction’s conductance as a function of bias voltage and temperature, we show that inelastic scattering of electrons by phonons can be detected as features in conductance resulting from quenching of quantum interference. Our results are in agreement with density functional theory calculations and are well described by a generic two-site model in the framework of non-equilibrium Green’s functions formalism. The importance of the observed inelastic contribution to the current opens up new ways for exploring coherent electron transport through molecular devices.
PMCID: PMC4750039  PMID: 26864735
19.  Supramolecular Approaches to Combining Membrane Transport with Adhesion 
Accounts of Chemical Research  2013;46(12):2878-2887.
Cells carefully control the transit of compounds through their membranes using “gated” protein channels that respond to chemical stimuli. Connexin gap junctions, which are high conductance cell-to-cell channels, are a remarkable class of “gated” channel with multiple levels of assembly. A gap junction between adhering cells comprises two half-channels in each cell membrane that adhere to each other to form a continuous cell-to-cell channel. Each half-channel is a hexameric assembly of six protein transmembrane subunits. These gap junctions display both intramembrane assembly and intermembrane assembly, making them an attractive target for biomimetic studies. Although many examples of self-assembled channels have been developed, few can also mediate intermembrane adhesion. Developing systems that combine membrane adhesion with controlled transit across the membrane would not only provide a better understanding of self-assembly in and around the membrane, but would also provide a route towards smart biomaterials, targeted drug delivery and an interface with nanotechnology.
This Account describes our biomimetic approaches to combining membrane adhesion with membrane transport, using both self-assembled “sticky” pores and “sticky” nanoparticles to trigger transit across membranes. This combination links both fundamental and applied research, acting as a bridge between molecular level assembly and the formation of functional biomaterials. The ultimate goal is to create complex self-assembled systems in biological or biomimetic environments that can both interface with cells and transport compounds across bilayers in response to remote chemical or electromagnetic signals. Our research in this area started with fundamental studies of intramembrane and intermembrane self-assembly, building upon previously known channel-forming compounds to create self-assembled channels that were switchable or able to mediate vesicle–vesicle adhesion. Subsequently, nanoparticles with a “sticky” coating were used to mediate adhesion between vesicles. Combining these adhesive properties with the unique characteristics of nanosized magnetite allowed a noninvasive magnetic signal to trigger transport of compounds out of magnetic nanoparticle-vesicle assemblies. Adding an extravesicular matrix produced new responsive biomaterials for use in tissue engineering. These biomaterials can be magnetically patterned and can deliver drugs upon receipt of a magnetic signal, allowing spatiotemporal control over cellular responses.
PMCID: PMC3868445  PMID: 23682580
20.  Conformation-driven quantum interference effects mediated by through-space conjugation in self-assembled monolayers 
Nature Communications  2016;7:13904.
Tunnelling currents through tunnelling junctions comprising molecules with cross-conjugation are markedly lower than for their linearly conjugated analogues. This effect has been shown experimentally and theoretically to arise from destructive quantum interference, which is understood to be an intrinsic, electronic property of molecules. Here we show experimental evidence of conformation-driven interference effects by examining through-space conjugation in which π-conjugated fragments are arranged face-on or edge-on in sufficiently close proximity to interact through space. Observing these effects in the latter requires trapping molecules in a non-equilibrium conformation closely resembling the X-ray crystal structure, which we accomplish using self-assembled monolayers to construct bottom-up, large-area tunnelling junctions. In contrast, interference effects are completely absent in zero-bias simulations on the equilibrium, gas-phase conformation, establishing through-space conjugation as both of fundamental interest and as a potential tool for tuning tunnelling charge-transport in large-area, solid-state molecular-electronic devices.
Tunnelling currents through molecular junctions are affected by quantum interference effects, but understanding the factors leading to them remains a challenge. Here the authors show that through-space conjugation in self-assembled monolayers leads to conformation-dependent quantum interference that suppresses conductivity.
PMCID: PMC5187444  PMID: 27996036
21.  Probing the local environment of a single OPE3 molecule using inelastic tunneling electron spectroscopy 
We study single-molecule oligo(phenylene ethynylene)dithiol junctions by means of inelastic electron tunneling spectroscopy (IETS). The molecule is contacted with gold nano-electrodes formed with the mechanically controllable break junction technique. We record the IETS spectrum of the molecule from direct current measurements, both as a function of time and electrode separation. We find that for fixed electrode separation the molecule switches between various configurations, which are characterized by different IETS spectra. Similar variations in the IETS signal are observed during atomic rearrangements upon stretching of the molecular junction. Using quantum chemistry calculations, we identity some of the vibrational modes which constitute a chemical fingerprint of the molecule. In addition, changes can be attributed to rearrangements of the local molecular environment, in particular at the molecule–electrode interface. This study shows the importance of taking into account the interaction with the electrodes when describing inelastic contributions to transport through single-molecule junctions.
PMCID: PMC4734430  PMID: 26885460
current–voltage characteristics; DFT calculations; mechanically controllable break junction (MCBJ); molecule–electrode interaction; vibrational modes
22.  A Dual-Tracer Method for Differentiating Transendothelial Transport from Paracellular Leakage in Vivo and in Vitro 
Inflammation-induced impaired function of vascular endothelium may cause leakage of plasma proteins that can lead to edema. Proteins may leave the vascular lumen through two main paracellular and transcellular pathways. As the first involves endothelial cell (EC) junction proteins and the second caveolae formation, these two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during pathology that causes inflammation. Here we present a newly developed dual-tracer probing method that allows differentiation of transcellular from paracellular transport during pathology. This fluorescence-based method can be used in vitro to test changes in EC layer permeability and in vivo in various animal vascular preparations. The method is based on comparison of low molecular weight molecule (LMWM) transport to that of high molecular weight molecule (HMWM) transport through the EC layer or the vascular wall during physiological and pathological conditions. Since the LMWM will leak through mainly the paracellular and HMWM will move through paracellular (when gaps between the ECs are wide enough) and transcellular pathways, the difference in transport rate (during normal conditions and pathology) of these molecules will indicate the prevailing transport pathway involved in overall protein crossing of vascular wall. Thus, the novel approach of assessing the transport kinetics of different size tracers in vivo by intravital microscopy can clarify questions related to identification of target pathways for drug delivery during various pathologies associated with elevated microvascular permeability.
PMCID: PMC3385581  PMID: 22754530
cerebrovascular leakage; intravital microscopy; fluorescent dyes
23.  High electronic couplings of single mesitylene molecular junctions 
We report on an experimental analysis of the charge transport properties of single mesitylene (1,3,5-trimethylbenzene) molecular junctions. The electronic conductance and the current–voltage characteristics of mesitylene molecules wired into Au electrodes were measured by a scanning tunnelling microscopy-based break-junction method at room temperature in a liquid environment. We found the molecular junctions exhibited two distinct conductance states with high conductance values of ca. 10−1 G 0 and of more than 10−3 G 0 (G 0 = 2e 2/h) in the electronic conductance measurements. We further performed a statistical analysis of the current–voltage characteristics of the molecular junctions in the two states. Within a single channel resonant tunnelling model, we obtained electronic couplings in the molecular junctions by fitting the current–voltage characteristics to the single channel model. The origin of the high conductance was attributed to experimentally obtained large electronic couplings of the direct π-bonded molecular junctions (ca. 0.15 eV). Based on analysis of the stretch length of the molecular junctions and the large electronic couplings obtained from the I–V analysis, we proposed two structural models, in which (i) mesitylene binds to the Au electrode perpendicular to the charge transport direction and (ii) mesitylene has tilted from the perpendicular orientation.
PMCID: PMC4685770  PMID: 26732978
break junction; charge transport; mesitylene; single molecular junction; scanning tunnelling microscopy (STM)
24.  Limiting transport steps and novel interactions of Connexin-43 along the secretory pathway 
Histochemistry and Cell Biology  2009;132(3):263-280.
Connexins are four-transmembrane-domain proteins expressed in all vertebrates which form permeable gap junction channels that connect cells. Here, we analysed Connexin-43 (Cx43) transport to the plasma membrane and studied the effects of small GTPases acting along the secretory pathway. We show that both GTP- and GDP-restricted Sar1 prevents exit of Cx43 from the endoplasmic reticulum (ER), but only GTP-restricted Sar1 arrests Cx43 in COP II-coated ER exit sites and accumulates 14-3-3 proteins in the ER fraction. FRET-FLIM data confirm that already in ER exit sites Cx43 exists in oligomeric form, suggesting an in vivo role for 14-3-3 in Cx43 oligomerization. Exit of Cx43 from the ER can be blocked by other factors—such as expression of the β subunit of the COP I coat or p50/dynamitin that acts on the microtubule-based dynein motor complex. GTP-restricted Arf1 blocks Cx43 in the Golgi. Lastly, we show that GTP-restricted Arf6 removes Cx43 gap junction plaques from the cell–cell interface and targets them to degradation. These data provide a molecular explanation of how small GTPases act to regulate Cx43 transport through the secretory pathway, facilitating or abolishing cell–cell communication through gap junctions.
PMCID: PMC2756399  PMID: 19626334
Connexins; Gap junction; Arf; COP; FRET
25.  Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability 
Journal of Clinical Investigation  2001;107(10):1319-1327.
Tight junctions regulate paracellular conductance and ionic selectivity. These properties vary among epithelia but the molecular basis of this variation remains unknown. To test whether members of the claudin family of tight junction proteins influence paracellular ionic selectivity, we expressed human claudin-4 in cultured MDCK cells using an inducible promoter. Overexpression increased the complexity of tight junction strands visible by freeze-fracture microscopy without affecting the levels of claudin-1, -2, or -3, occludin, or ZO-1. A decrease in conductance correlated directly with the kinetics of claudin-4 induction. Dilution potentials revealed that the decrease in paracellular conductance resulted from a selective decrease in Na+ permeability without a significant effect on Cl– permeability. Flux for an uncharged solute, mannitol, and the rank order of permeabilities for the alkali metal cations were unchanged. A paracellular site for these effects was supported by the lack of apical/basal directionality of the dilution potentials, the linearity of current-voltage relationships, and the lack of influence of inhibitors of major transcellular transporters. These results provide, to our knowledge, the first direct demonstration of the ability of a claudin to influence paracellular ion selectivity and support a role for the claudins in creating selective channels through the tight-junction barrier.
PMCID: PMC209303  PMID: 11375422

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