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

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

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

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

Deciphering the Structure, Growth and Assembly of Amyloid-Like Fibrils Using High-Speed Atomic Force Microscopy

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins

Analyse en molécule unique de la dynamique de la tétraspanine CD9 et de sa partition à la membrane plasmique

Les tétraspanines constituent une famille de protèines transmembranaires régulant un grand nombre de fonctions cellulaires et jouant un rôle décisif dans de multiples pathologies. Elles présentent la particularité de former un réseau d'intéractions proteine/proteine à la surface des cellules, réseau qui est également dépendant de certains lipides comme le cholestérol ou les palmitates. La mise en évidence de ce réseau d'intéractions, sa composition et les bases moléculaires de sa formation se sont fait essentiellement au travers d'expériences de co-Immunoprécipitation et de microscopie de fluorescence "classique", des techniques ne permettant pas d'avoir une vue dynamique de l'organisation membranaire des tétraspanines. Dans cette optique, mon travail de thèse a porté sur la caractérisation dynamique de la tétraspanine CD9 en molécule unique, dans le contexte du réseau à tétraspanines. Pour cela, nous avons mis au point au laboratoire un montage expérimental basé sur la microscopie TIRF permettant le suivi de molécule unique en deux couleurs. Grâce à ce système, nous avons montré que la majorité des molécules CD9 sont dynamiques et diffusent dans la membrane de façon Brownienne. Certaines molécules sont également confinées, de façon transitoire ou non, au sein de régions enrichies en CD9 et en ses partenaires. Ces régions semblent jouer le rôle de plateformes, constantes en terme de forme et de localisations, qui limitent la diffusion des molécules CD9. Nous avons également montré que deux molécules présentant des trajectoires Browniennes peuvent co-diffuser dans la membrane, indiquant que des intéractions peuvent avoir lieu en dehors de ces plateformes. La partition de CD9 dans les palteformes ainsi que les colocalisations dynamiques sont toutes deux dépendantes du cholestérol membranaire et de la palmytoylation de CD9. Enfin, nous avons mis en évidence que, malgré les similitudes structurales et fonctionnelles qui existent entre les tétraspanines CD9 etCD81, leur dynamique à la surface des cellules est très différente. L'ensemble de nos résultats a ainsi mis en lumière la dynamique importante des intéractions au sein du réseau à tétraspanines et a montré que, par comparaison avec la protéine à ancrage lipidique CD55, ce réseau d'intéractions est différent des microdomaines rafts.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF