Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.Instituto de Física de Líquidos y Sistemas Biológico

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.Instituto de Física de Líquidos y Sistemas Biológico

Rat endopeptidase-24.18 α subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells

AbstractEndopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoenzyme. The α and β submits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E-24.18 α subunit and to test the functionality of the astacin-like domain in the α subunit when expressed alone, COS-1 cells were transfected with a cloned cDNA for rat α subunit. Despite the presence of its putative transmembrane domain, the α subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the α subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Taken together our results suggest that the α subunit of Endopeptidase-24.18 contains a latent astacin-like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine

AFM Imaging of Lipid Domains in Model Membranes

Characterization of the two-dimensional organization of biological membranes is one of the most important issues that remains to be achieved in order to understand their structure-function relationships. According to the current view, biological membranes would be organized in in-plane functional microdomains. At least for one category of them, called rafts, the lateral segregation would be driven by lipid-lipid interactions. Basic questions like the size, the kinetics of formation, or the transbilayer organization of lipid microdomains are still a matter of debate, even in model membranes. Because of its capacity to image structures with a resolution that extends from the molecular to the microscopic level, atomic force microscopy (AFM) is a useful tool for probing the mesoscopic lateral organization of lipid mixtures. This paper reviews AFM studies on lateral lipid domains induced by lipid-lipid interactions in model membranes

Modification of the expression of the aquaporin ZmPIP2;5 affects water relations and plant growth

The maize plasma membrane PIP2;5 aquaporin plays a role in controlling root radial water movement, leaf hydraulic conductivity, and plant growth. The plasma membrane intrinsic protein PIP2;5 is the most highly expressed aquaporin in maize (Zea mays) roots. Here, we investigated how deregulation of PIP2;5 expression affects water relations and growth using maize overexpression (OE; B104 inbred) or knockout (KO; W22 inbred) lines. The hydraulic conductivity of the cortex cells of roots grown hydroponically was higher in PIP2;5 OE and lower in pip2;5 KO lines compared with the corresponding wild-type plants. While whole-root conductivity decreased in the KO lines compared to the wild type, no difference was observed in OE plants. This paradox was interpreted using the MECHA hydraulic model, which computes the radial flow of water within root sections. The model hints that the plasma membrane permeability of the cells is not radially uniform but that PIP2;5 may be saturated in cell layers with apoplastic barriers, i.e. the endodermis and exodermis, suggesting the presence of posttranslational mechanisms controlling the abundance of PIP in the plasma membrane in these cells. At the leaf level, where the PIP2;5 gene is weakly expressed in wild-type plants, the hydraulic conductance was higher in the PIP2;5 OE lines compared with the wild-type plants, whereas no difference was observed in the pip2;5 KO lines. The temporal trend of leaf elongation rate, used as a proxy for that of xylem water potential, was faster in PIP2;5 OE plants upon mild stress, but not in well-watered conditions, demonstrating that PIP2;5 may play a beneficial role in plant growth under specific conditions

Surface topography of membrane domains

金沢大学理工研究域数物科学系Elucidating origin, composition, size, and lifetime of microdomains in biological membranes remains a major issue for the understanding of cell biology. For lipid domains, the lack of a direct access to the behaviour of samples at the mesoscopic scale has constituted for long a major obstacle to their characterization, even in simple model systems made of immiscible binary mixtures. By its capacity to image soft surfaces with a resolution that extends from the molecular to the microscopic level, in air as well as under liquid, atomic force microscopy (AFM) has filled this gap and has become an inescapable tool in the study of the surface topography of model membrane domains, the first essential step for the understanding of biomembranes organization. In this review we mainly focus on the type of information on lipid microdomains in model systems that only AFM can provide. We will also examine how AFM can contribute to understand data acquired by a variety of other techniques and present recent developments which might open new avenues in model and biomembrane AFM applications. © 2009 Elsevier B.V. All rights reserved

Treatment use in a prospective naturalistic cohort of children and adolescents with catatonia

Abstract We aimed to (1) describe the treatment used in a large sample of young inpatients with catatonia, (2) determine which factors were associated with improvement and (3) benzodiazepine (BZD) efficacy. From 1993From to 2011 patients between the ages of 9 and 19 years were consecutively hospitalized for a catatonic syndrome. We prospectively collected sociodemographic, clinical and treatment data. In total, 51 (77 %) patients underwent a BZD trial. BZDs were effective in 33 (65 %) patients, who were associated with significantly fewer severe adverse events (p = 0.013) and resulted in fewer referrals for electroconvulsive therapy (ECT) (p = 0.037). Other treatments included ECT (N = 12, 18 %); antipsychotic medications, mostly in combination; and treatment of an underlying medical condition, when possible. For 10 patients, four different trials were needed to achieve clinical improvement. When all treatments were combined, there was a better clinical response in acute-onset catatonia (p = 0.032). In contrast, the response was lower in boys (p = 0.044) and when posturing (p = 0.04) and mannerisms (p = 0.008) were present as catatonic symptoms. The treatment response was independent of the underlying psychiatric or systemic medical condition. As in adults, BZDs should be the first-line symptomatic treatment for catatonia in young patients, and ECT should be a second option. Additionally, the absence of an association between the response to treatment and the underlying psychiatric condition suggests that catatonia should be considered as a syndrome

Mechanical manipulation of magnetic nanoparticles by magnetic force microscopy

A method has been developed in this work for the mechanical manipulation of magnetic nanoparticles (MNPs). A helical curve was designed as the capture path to pick up and remove the target nanoparticle on a mica surface by a magnetic probe based on the magnetic force microscope (MFM). There were magnetic, tangential and pushing forces acting on the target particle during the approaching process when the tip followed the helical curve as the capture path. The magnetic force was significant when the tip was closer to the particle. The target particle can be attached on the surface of the magnetic probe tip and then be picked up after the tip retracted from the mica surface. Theoretical analysis and experimental results were presented for the pick-up and removal of MNPs. With this method, the precision and flexibility of manipulation of MNPs were improved significantly compared to the pushing or sliding of the target object away from the corresponding original location following a planned path