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Insights into the Role of Conformational Change, Membrane Interactions and ATP Hydrolysis in the Min Protein Regulators of Bacterial Cell DivisionAyed, Saud 13 August 2018 (has links)
No description available.
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Investigation of the Effect of Changes in Lipid Bilayer Properties on the Activity of the Bacterial Cell Division Regulator Protein MinDAyed, Saud 13 September 2012 (has links)
Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.
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Investigation of the Effect of Changes in Lipid Bilayer Properties on the Activity of the Bacterial Cell Division Regulator Protein MinDAyed, Saud 13 September 2012 (has links)
Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.
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Investigation of the Effect of Changes in Lipid Bilayer Properties on the Activity of the Bacterial Cell Division Regulator Protein MinDAyed, Saud January 2012 (has links)
Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.
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Min-Protein Waves on Geometrically Structured Artificial Membranes / Min-Proteinwellen auf geometrisch strukturierten künstlichen MembranenSchweizer, Jakob 04 April 2013 (has links) (PDF)
Das stäbchenförmige Bakterium Escherichia coli teilt sich in zwei gleich große Tochterzellen. Dies ist nur möglich, wenn sich die Zelle in der Mitte teilt. Bei E. coli wird die Zellteilung durch den Zusammenschluss der FtsZ-Proteine an der Membran zum Z-Ring eingeleitet. Topologische Regulierung des Z-Ringes erfolgt durch räumlich-zeitliche Oszillationen von Min-Proteinen zwischen den beiden Zellpolen. MinC, MinD und MinE binden an und lösen sich von der Membran unter Hydrolyse von ATP und in antagonistischer Art und Weise, was zu einer alternierenden Ansammlung von MinC und MinD an den Zellpolen führt. Gemittelt über die Zeit ergibt sich somit ein MinD-Verteilungsprofil, das maximale Konzentration an den Zellpolen und ein Minimum in der Zellmitte aufweist. MinC bindet an MinD und folgt somit seiner Verteilung. Der Zusammenschluss von FtsZ-Proteinen wird durch MinC unterbunden, und somit kann sich der Z-ring nur an einer Position herausbilden, die ein Minimum an MinC aufweist - der Zellmitte.
Das Min-system wurde in der Vergangenheit auch mit einem in-vitro-Ansatz untersucht, indem Min-Proteine in künstliche, aufliegende Lipiddoppelschichten (supported lipid bilayers, SLB) rekonstitutiert wurden. Dabei bildeten die Min-Proteine kein oszillierendes Muster aus, sondern organisierten sich vielmehr in parallelen und propagierenden Wellen (Loose, 2008, Science, 320). In diesen in-vitro-Experimenten war das Membransubstrat wesentlich größer als die Wellenlänge der Min-Proteinwellen. In vivo hingegen ist die Länge der Zelle in der gleichen Größenordnung wie die charakteristische Länge des Oszillationsmusters der Min-Proteine. Daher war es das Ziel dieser Arbeit, den Einfluß einer beschränkten Fläche und geometrischer Formgebung der künstlichen Lipiddoppelschichten auf die Wellenpropagation der Min-Protein zu untersuchen.
Flächige Beschränkung künstlicher Membranen erfolgte durch Mikrostrukturtechnologie. Deckglässchen wurden mit einer Goldschicht und mikroskopischen Aussparungen unterschiedlicher geometrischer Formen strukturiert. Funktionale SLBs bildeten sich nur auf Glasflächen ohne Goldbeschichtung aus. Nach der Rekonstitution der Min-Proteine, organisierten sich diese auf den Membranstücken in parallele Wellen. Dabei bestimmte die flächige Beschränkung der künstlichen Membranen die Ausbreitungsrichtung der Min-Proteinwellen. Min-Proteinwellen konnten entlang gekrümmter Membranstreifen, in Ring- und sogar in Slalomstrukturen geleitet werden. In geraden, länglichen Strukturen richteten sich die Wellen entlang der längsten Achse aus. Kopplung von Proteinwellen auf räumlich getrennten Membranstücken in Abhängigkeit des Abstandes und des sogenannten Molecular Crowdings in der wässrigen Lösung konnte ebenfalls beobachtet werden. Diese Kopplung ist ein Indiz für inhomogene Proteinverteilungen in der Lösung oberhalb der Membran. Desweiteren konnten Min-Proteinwellen auch in diversen dreidimensionalen künstlichen Membranen rekonstitituiert werden.
Im Wildtyp von E. coli ähneln die Min-Proteindynamiken der einer Oszillation mit einer charakteristischen Länge von 5 µm. Auf SLBs, bilden Min-Proteine Wellen mit einer Wellenlänge aus, die ca. zehnmal größer ist als in vivo. Dieser Unterschied zwischen der in-vivo- und der in-vitro-Welt wurde untersucht und diskutiert. In vitro konnte die Wellenlänge um 50 % durch Erhöhung des Molecular Crowding in der Lösung sowie um 33 % durch Temperaturerhöhung verkleinert werden. Das oszillierende Muster könnte dahingegen eine Folge der Kompartimentierung sein. Erste Versuche, das Min-System in geschlossene Membrankompartimente zu rekonstitutieren, wurden getestet. / Escherichia coli, a rod-like bacterium, divides by binary fission. Cell division into two daughter cells of equal size requires that fission takes place at a midcell position. In E. coli, cell division is initiated by assembly of the FtsZ-proteins at the inner membrane to the Z-ring. Topological regulation of the Z-ring is achieved by spatiotemporal pole-to-pole oscillations of Min-proteins. MinC, MinD and MinE bind to and detach from - under hydrolysis of ATP - the membrane in an antagonistic manner leading to an alternating accumulation of MinC and MinD at the cell poles. Averaged over time, the distribution profile of MinD exhibits maximal concentration at the cell poles and a minimum at the cell center. MinC binds to MinD and thus follows its distribution. FtsZ assembly is inhibited by MinC and therefore the Z-ring can only form at a cell position low in MinC - at the cell center.
In the past, the Min-system was also investigated in an in vitro approach by reconstitution of Min-proteins into a supported lipid bilayer (SLB). Here, Min-proteins did not self-organize into an oscillatory pattern but into parallel and propagating waves (Loose, 2008, Science, 320). In this in vitro assay, the membrane substrate was infinitely large compared to the wavelength. However, in vivo, the cell length is on the same order of magnitude as the respective length scale of the oscillatory pattern of Min-proteins. Therefore, we wished to investigate the effect of lateral confinement and geometric structuring of artificial lipid bilayers on the Min-protein wave propagation.
Lateral confinement of artificial membranes was achieved by microfabrication technology. Glass slides were patterned by a gold coating with microscopic windows of different geometries, and functional SLBs were only formed on uncoated areas. Upon reconstitution, Min-proteins organized into parallel waves on the geometric membrane patches. Confinement of the artificial membranes determined the direction of propagation of Min-protein waves. Min-protein waves could be guided along curved membrane stripes, in rings and even along slalom-geometries. In elongated membrane structures, the protein waves always propagate along the longest axis. Coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and level of molecular crowding of the aqueous media above the bilayer. This indicates the existence of an inhomogeneous and dynamic protein gradient in the solution above the membrane. Furthermore, reconstitution of Min-protein waves in various three-dimensional artificial membranes was achieved.
In wild-type E. coli, Min-protein dynamics resemble that of an oscillation with a characteristic length scale of 5 µm. On supported lipid bilayers, Min-proteins self-organize into waves with a wavelength approximately 10-fold larger than in vivo. These discrepancies between the in vivo and in vitro world were investigated and discussed. In vitro, the wavelength could be decreased by a factor of 50 % by increase of the molecular crowding in solution and by 33 % through temperature increase. The oscillatory pattern is thought to be a consequence of compartmentalization and first attempts to encapsulate the Min-system in closed bilayer compartments are presented.
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Min-Protein Waves on Geometrically Structured Artificial MembranesSchweizer, Jakob 06 February 2013 (has links)
Das stäbchenförmige Bakterium Escherichia coli teilt sich in zwei gleich große Tochterzellen. Dies ist nur möglich, wenn sich die Zelle in der Mitte teilt. Bei E. coli wird die Zellteilung durch den Zusammenschluss der FtsZ-Proteine an der Membran zum Z-Ring eingeleitet. Topologische Regulierung des Z-Ringes erfolgt durch räumlich-zeitliche Oszillationen von Min-Proteinen zwischen den beiden Zellpolen. MinC, MinD und MinE binden an und lösen sich von der Membran unter Hydrolyse von ATP und in antagonistischer Art und Weise, was zu einer alternierenden Ansammlung von MinC und MinD an den Zellpolen führt. Gemittelt über die Zeit ergibt sich somit ein MinD-Verteilungsprofil, das maximale Konzentration an den Zellpolen und ein Minimum in der Zellmitte aufweist. MinC bindet an MinD und folgt somit seiner Verteilung. Der Zusammenschluss von FtsZ-Proteinen wird durch MinC unterbunden, und somit kann sich der Z-ring nur an einer Position herausbilden, die ein Minimum an MinC aufweist - der Zellmitte.
Das Min-system wurde in der Vergangenheit auch mit einem in-vitro-Ansatz untersucht, indem Min-Proteine in künstliche, aufliegende Lipiddoppelschichten (supported lipid bilayers, SLB) rekonstitutiert wurden. Dabei bildeten die Min-Proteine kein oszillierendes Muster aus, sondern organisierten sich vielmehr in parallelen und propagierenden Wellen (Loose, 2008, Science, 320). In diesen in-vitro-Experimenten war das Membransubstrat wesentlich größer als die Wellenlänge der Min-Proteinwellen. In vivo hingegen ist die Länge der Zelle in der gleichen Größenordnung wie die charakteristische Länge des Oszillationsmusters der Min-Proteine. Daher war es das Ziel dieser Arbeit, den Einfluß einer beschränkten Fläche und geometrischer Formgebung der künstlichen Lipiddoppelschichten auf die Wellenpropagation der Min-Protein zu untersuchen.
Flächige Beschränkung künstlicher Membranen erfolgte durch Mikrostrukturtechnologie. Deckglässchen wurden mit einer Goldschicht und mikroskopischen Aussparungen unterschiedlicher geometrischer Formen strukturiert. Funktionale SLBs bildeten sich nur auf Glasflächen ohne Goldbeschichtung aus. Nach der Rekonstitution der Min-Proteine, organisierten sich diese auf den Membranstücken in parallele Wellen. Dabei bestimmte die flächige Beschränkung der künstlichen Membranen die Ausbreitungsrichtung der Min-Proteinwellen. Min-Proteinwellen konnten entlang gekrümmter Membranstreifen, in Ring- und sogar in Slalomstrukturen geleitet werden. In geraden, länglichen Strukturen richteten sich die Wellen entlang der längsten Achse aus. Kopplung von Proteinwellen auf räumlich getrennten Membranstücken in Abhängigkeit des Abstandes und des sogenannten Molecular Crowdings in der wässrigen Lösung konnte ebenfalls beobachtet werden. Diese Kopplung ist ein Indiz für inhomogene Proteinverteilungen in der Lösung oberhalb der Membran. Desweiteren konnten Min-Proteinwellen auch in diversen dreidimensionalen künstlichen Membranen rekonstitituiert werden.
Im Wildtyp von E. coli ähneln die Min-Proteindynamiken der einer Oszillation mit einer charakteristischen Länge von 5 µm. Auf SLBs, bilden Min-Proteine Wellen mit einer Wellenlänge aus, die ca. zehnmal größer ist als in vivo. Dieser Unterschied zwischen der in-vivo- und der in-vitro-Welt wurde untersucht und diskutiert. In vitro konnte die Wellenlänge um 50 % durch Erhöhung des Molecular Crowding in der Lösung sowie um 33 % durch Temperaturerhöhung verkleinert werden. Das oszillierende Muster könnte dahingegen eine Folge der Kompartimentierung sein. Erste Versuche, das Min-System in geschlossene Membrankompartimente zu rekonstitutieren, wurden getestet. / Escherichia coli, a rod-like bacterium, divides by binary fission. Cell division into two daughter cells of equal size requires that fission takes place at a midcell position. In E. coli, cell division is initiated by assembly of the FtsZ-proteins at the inner membrane to the Z-ring. Topological regulation of the Z-ring is achieved by spatiotemporal pole-to-pole oscillations of Min-proteins. MinC, MinD and MinE bind to and detach from - under hydrolysis of ATP - the membrane in an antagonistic manner leading to an alternating accumulation of MinC and MinD at the cell poles. Averaged over time, the distribution profile of MinD exhibits maximal concentration at the cell poles and a minimum at the cell center. MinC binds to MinD and thus follows its distribution. FtsZ assembly is inhibited by MinC and therefore the Z-ring can only form at a cell position low in MinC - at the cell center.
In the past, the Min-system was also investigated in an in vitro approach by reconstitution of Min-proteins into a supported lipid bilayer (SLB). Here, Min-proteins did not self-organize into an oscillatory pattern but into parallel and propagating waves (Loose, 2008, Science, 320). In this in vitro assay, the membrane substrate was infinitely large compared to the wavelength. However, in vivo, the cell length is on the same order of magnitude as the respective length scale of the oscillatory pattern of Min-proteins. Therefore, we wished to investigate the effect of lateral confinement and geometric structuring of artificial lipid bilayers on the Min-protein wave propagation.
Lateral confinement of artificial membranes was achieved by microfabrication technology. Glass slides were patterned by a gold coating with microscopic windows of different geometries, and functional SLBs were only formed on uncoated areas. Upon reconstitution, Min-proteins organized into parallel waves on the geometric membrane patches. Confinement of the artificial membranes determined the direction of propagation of Min-protein waves. Min-protein waves could be guided along curved membrane stripes, in rings and even along slalom-geometries. In elongated membrane structures, the protein waves always propagate along the longest axis. Coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and level of molecular crowding of the aqueous media above the bilayer. This indicates the existence of an inhomogeneous and dynamic protein gradient in the solution above the membrane. Furthermore, reconstitution of Min-protein waves in various three-dimensional artificial membranes was achieved.
In wild-type E. coli, Min-protein dynamics resemble that of an oscillation with a characteristic length scale of 5 µm. On supported lipid bilayers, Min-proteins self-organize into waves with a wavelength approximately 10-fold larger than in vivo. These discrepancies between the in vivo and in vitro world were investigated and discussed. In vitro, the wavelength could be decreased by a factor of 50 % by increase of the molecular crowding in solution and by 33 % through temperature increase. The oscillatory pattern is thought to be a consequence of compartmentalization and first attempts to encapsulate the Min-system in closed bilayer compartments are presented.
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