Confined Mobility of TonB and FepA in Escherichia coli Membranes

The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E.coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region 0.180−0.007+0.006 μm in radius in the outer membrane and TonB confined to a region 0.266−0.009+0.007 μm in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be 21−5+9 μm2/s and 5.4−0.8+1.5 μm2/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins.Yeshttp://www.plosone.org/static/editorial#pee

Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging

We report on an in vivo single-mol. study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-mol. wide-field fluorescence microscopy at 100 ms time resoln. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed. [on SciFinder (R)

Single Hepatitis-B virus capsid binding to nuclear pore complexes in HeLa cells

info:eu-repo/semantics/publishe

Craniofacial Growth at Age 6–11 Years after One-Stage Cleft Lip and Palate Repair: A Retrospective Comparative Study with Historical Controls

Background: Primary alveolar bone grafting inhibits craniofacial growth. However, its effect on craniofacial growth in one-stage cleft lip and palate protocols is unknown. This study investigated whether primary alveolar bone grafting performed during one-stage unilateral cleft lip and palate repair negatively affects growth up to 6–11 years old. Methods: The craniofacial growth, dental arch relationship and palatal morphology at 6–11 years old in children with unilateral cleft lip and palate were compared retrospectively. Two cohorts after a one-stage protocol without (Group A) and with (Group B) primary bone grafting at the same center were compared. Further, cephalometric measurements for growth were compared with an external cohort of a one-stage protocol and a heathy control. Results: Group A comprised 16 patients assessed at 6.8 years (SD 0.83), and Group B comprised 15 patients assessed at 9 years (SD 2.0). Cephalometric measurements indicated similar sagittal maxillary growth deficits and a significant deviation in maxillary inclination in both groups compared to the healthy group. Moderate to severe changes in palatal morphology were observed in 70% of the members in both groups. Conclusion: Omitting primary alveolar bone grafting under the one-stage protocol with two-flap palatoplasty studied did not improve growth at 6–11 years. The results implicate two-flap palatoplasty with secondary healing as having greater adverse effects on growth than primary alveolar bone grafting. Dental and palatal morphology was considerably compromised regardless of primary alveolar bone grafting

Parameters to fit mixed diffusion mode model

<p>Parameters to fit mixed diffusion mode model</p

Parameters to fit to variable confinement size model

<p>Parameters to fit to variable confinement size model</p

MSD of TonB and of FepA.

<p>a) MSD of TonB (solid circles) and of FepA (solid squares). b) Fit to confined diffusion model for TonB (data: solid circles, fit: open circles) which gives best fit parameters of a confinement radius of μm with short-time (microsecond timescale) diffusion coefficient in the range μm²/s. c) Fit to confined diffusion model for FepA (data: solid squares, fit: open squares) which gives best fit parameters of a confinement radius of μm with short-time (microsecond timescale) diffusion coefficient in of μm²/s.</p

Schematic of the interaction between the TonB-ExbB-ExbD complex in the inner membrane and its interaction with the Ferric enterobactin (FeEnt) TBDT FepA in the outer membrane of <i>E</i>. <i>coli</i>.

<p>In this study, either FepA was labeled extracellularly with Alexa-555 or TonB was labeled intracellularly with GFP. IM: inner membrane, PG: peptidoglycan layer, OM: outer membrane.</p

Confined Mobility of TonB and FepA in <i>Escherichia coli</i> Membranes - Fig 5

<p><b>Effect of (a) deletion of ExbB/D (triangles), (b) presence of monoclonal antibody against FepA (triangles), and (c) presence of MreB–disrupting drug A22 (triangles) on MSDs of TonB (circles).</b> Fitting suggests that the effect of deletion of ExbB/D and disruption of MreB is to reduce the size of confinement of TonB while the effect of anti-FepA is to increase the mobility of TonB which appears as a decrease in confinement size.</p