Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope.

An Escherichia coli mutant carrying delta malE12-18, a 21-base pair deletion confined to the coding DNA of the maltose-binding protein signal peptide, is unable to export maltose-binding protein to the periplasm efficiently. Consequently, such a strain is defective for the utilization of maltose as a sole carbon source. We obtained 16 mutants harboring extragenic delta malE12-18 suppressor mutations that exhibit partial restoration of export to the mutant maltose-binding protein. A genetic analysis of these extragenic suppressor mutations demonstrated that 15 map at prlA, at 72 min on the standard E. coli linkage map, and that 1 maps at a new locus, prlD, at 2.5 min on the linkage map. Our evidence indicates that the prlA and prlD gene products play an important role in the normal pathway for export of proteins to the cell envelope. Efficient execution of the secretory process requires that these prl gene products interact properly with each other so that a productive interaction of these gene products with the signal peptide also can occur. Our data suggest that proper assembly of a complex is required for efficient export of E. coli envelope proteins to their various extracytoplasmic compartments.     

High resolution optical imaging of infarction in intact organs

We describe a method to visualize green fluorescent protein (GFP)-labeled cells in intact organs through combined confocal and reflected laser light imaging. This method allows us a three-dimensional (3-D) view of specific cell types in situ. Imaging of tissues from transgenic mice in which the endothelial cells are labeled with GFP under the control of endothelial-specific tyrosine receptor kinase 2 (TIE2) shows the spatial distribution of the GFP-labeled endothelial cells in intact organs. We have used this method to examine the tissue necrosis in the intact heart and kidney resulting from myocardial and renal infarction. In myocardial infarction produced by surgically occluding the left anterior descending coronary artery, the border of the infarct was highly cellular and showed a disrupted endothelial network and scar tissue appearing as a dense layer of reflection. The induced renal infarction produced by ligating the renal artery in the pedicle showed a clear infarct border in the affected kidney. The 3-D reconstruction of specific cell types in the context of the surrounding tissues should be useful for studying the overall organization and the relationship between different structures in the intact organ in normal and disease states.     

Determinants of gating polarity of a connexin 32 hemichannel

There is good evidence supporting the view that the transjunctional voltage sensor (V(j)-sensor) of Cx32 and other Group 1 connexins is contained within a segment of the N-terminus that contributes to the formation of the channel pore. We have shown that the addition of negatively charged amino acid residues at several positions within the first 10 amino acid residues reverses the polarity of V(j)-gating and proposed that channel closure is initiated by the inward movement of this region. Here, we report that positive charge substitutions of the 2nd, 5th, and 8th residues maintain the negative polarity of V(j)-gating. These data are consistent with the original gating model. Surprisingly, some channels containing combinations of positive and/or negative charges at the 2nd and 5th positions display bipolar V(j)-gating. The appearance of bipolar gating does not correlate with relative orientation of charges at this position. However, the voltage sensitivity of bipolar channels correlates with the sign of the charge at the 2nd residue, suggesting that charges at this position may have a larger role in determining gating polarity. Taken together with previous findings, the results suggest that the polarity V(j)-gating is not determined by the sign of the charge lying closest to the cytoplasmic entry of the channel, nor is it likely to result from the reorientation of an electrical dipole contained in the N-terminus. We further explore the mechanism of polarity determination by utilizing the one-dimensional Poisson-Nernst-Plank model to determine the voltage profile of simple model channels containing regions of permanent charge within the channel pore. These considerations demonstrate how local variations in the electric field may influence the polarity and sensitivity of V(j)-gating but are unlikely to account for the appearance of bipolar V(j)-gating.     

Gap junction channel gating

Over the last two decades, the view of gap junction (GJ) channel gating has changed from one with GJs having a single transjunctional voltage-sensitive (V(j)-sensitive) gating mechanism to one with each hemichannel of a formed GJ channel, as well as unapposed hemichannels, containing two, molecularly distinct gating mechanisms. These mechanisms are termed fast gating and slow or 'loop' gating. It appears that the fast gating mechanism is solely sensitive to V(j) and induces fast gating transitions between the open state and a particular substate, termed the residual conductance state. The slow gating mechanism is also sensitive to V(j), but there is evidence that this gate may mediate gating by transmembrane voltage (V(m)), intracellular Ca(2+) and pH, chemical uncouplers and GJ channel opening during de novo channel formation. A distinguishing feature of the slow gate is that the gating transitions appear to be slow, consisting of a series of transient substates en route to opening and closing. Published reports suggest that both sensorial and gating elements of the fast gating mechanism are formed by transmembrane and cytoplamic components of connexins among which the N terminus is most essential and which determines gating polarity. We propose that the gating element of the slow gating mechanism is located closer to the central region of the channel pore and serves as a 'common' gate linked to several sensing elements that are responsive to different factors and located in different regions of the channel.     

A Lipid Transfer Protein Signaling Axis Exerts Dual Control of Cell-Cycle and Membrane Trafficking Systems

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Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.     

Altered Inhibition of Cx26 Hemichannels by pH and Zn2+ in the A40V Mutation Associated with Keratitis-Ichthyosis-Deafness Syndrome

Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca²⁺. This study presents two new possible contributing factors, pH and Zn²⁺. Plasma pH levels and micromolar concentrations of Zn²⁺ inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H⁺ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn²⁺. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn²⁺ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn²⁺. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism.     

Long-term monitoring of biofilm growth and disinfection using a quartz crystal microbalance and reflectance measurements

A biofilm reactor was constructed to monitor the long-term growth and removal of biofilms as monitored by the use of a quartz crystal microbalance (QCM) and a novel optical method. The optical method measures the reflectance of white light off the surface of the quartz crystal microbalance electrode (gold) for determination of the biofilm thickness. Biofilm growth of Pseudomonas aeruginosa (PA) on the surface was used as a model system. Bioreactors were monitored for over 6 days. Expressing the QCM data as the ratio of changes in resistance to changes in frequency (DeltaR/Deltaf) facilitated the comparison of individual biofilm reactor runs. The various stages of biofilm growth and adaptation to low nutrients showed consistent characteristic changes in the DeltaR/Deltaf ratio, a parameter that reflects changes in the viscoelastic properties of the biofilm. The utility of white light reflectance for thickness measurements was shown for those stages of biofilm growth when the solution was not turbid due to high numbers of unattached cells. The thickness of the biofilms after 6 days ranged from 48 mum to 68 mum. Removal of the biofilm by a disinfectant (chlorine) was also measured in real time. The combination of QCM and reflectance allowed us to monitor in real time changes in the viscoelastic properties and thickness of biofilms over long periods of time.