Reconstituting geometry-modulated protein patterns in membrane compartments

Zieske, Katja; Schwille, Petra

doi:10.1016/bs.mcb.2015.02.006

Cited by 10 publications

(7 citation statements)

References 19 publications

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“…Bacteria are able to generate a wide variety of dynamic spatial patterns on the sub-micron scale even though many of their molecular parts rapidly mix due to diffusion. Within the cell, patterns range from periodic oscillations [1, 2] to the stable positioning of proteins asymmetrically at the cell poles [3, 4] (for a review see [5–7]). These spatial patterns serve many essential functions from cell division to signal reception [5, 8].…”

Section: Introductionmentioning

confidence: 99%

DNA segregation under Par protein control

Jindal

Emberly

2019

PLoS ONE

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The spatial organization of DNA is mediated by the Par protein system in some bacteria. ParB binds specifically to the parS sequence on DNA and orchestrates its motion by interacting with ParA bound to the nucleoid. In the case of plasmids, a single ParB bound plasmid is observed to execute oscillations between cell poles while multiple plasmids eventually settle at equal distances from each other along the cell’s length. While the potential mechanism underlying the ParA-ParB interaction has been discussed, it remains unclear whether ParB-complex oscillations are stable limit cycles or merely decaying transients to a fixed point. How are dynamics affected by substrate length and the number of complexes? We present a deterministic model for ParA-ParB driven DNA segregation where the transition between stable arrangements and oscillatory behaviour depends only on five parameters: ParB-complex number, substrate length, ParA concentration, ParA hydrolysis rate and the ratio of the lengthscale over which the ParB complex stimulates ParA hydrolysis to the lengthscale over which ParA interacts with the ParB complex. When the system is buffered and the ParA rebinding rate is constant we find that ParB-complex dynamics is independent of substrate length and complex number above a minimum system size. Conversely, when ParA resources are limited, we find that changing substrate length and increasing complex number leads to counteracting mechanisms that can both generate or subdue oscillatory dynamics. We argue that cells may be poised near a critical level of ParA so that they can transition from oscillatory to fixed point dynamics as the cell cycle progresses so that they can both measure their size and faithfully partition their genetic material. Lastly, we show that by modifying the availability of ParA or depletion zone size, we can capture some of the observed differences in ParB-complex positioning between replicating chromosomes in B. subtilis cells and low-copy plasmids in E. coli cells.

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Section: Introductionmentioning

confidence: 99%

DNA segregation under Par protein control

Jindal

Emberly

2019

PLoS ONE

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“…Either produce silicon wafer with microcompartments yourself using photolithography (see Zieske and Schwille for a detailed protocol 31 or Gruenberger et al for a video protocol 32 ) or order your desired silicon wafer from a foundry. For the pattern of the wafer used herein please see supplementary information.…”

Section: Protocolmentioning

confidence: 99%

<em>In Vitro</em> Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Ramm

Glock

Schwille

2018

JoVE

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Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

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“…Here, as another example of Spatiocyte application, we show that E. coli cell geometry can regulate MinD oscillation period, while the cell size controls the peak MinD concentration on the membrane. Previous works have shown that MinD dynamics can be regulated by the geometry [33][34][35][36][37] and topology [38] of the membrane. Varma and colleagues [33] 2.…”

Section: Case Studiesmentioning

confidence: 99%

Multi-Algorithm Particle Simulations with Spatiocyte

Arjunan¹,

Takahashi²

2017

Methods in Molecular Biology

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As quantitative biologists get more measurements of spatially regulated systems such as cell division and polarization, simulation of reaction and diffusion of proteins using the data is becoming increasingly relevant to uncover the mechanisms underlying the systems. Spatiocyte is a lattice-based stochastic particle simulator for biochemical reaction and diffusion processes. Simulations can be performed at single molecule and compartment spatial scales simultaneously. Molecules can diffuse and react in 1D (filament), 2D (membrane), and 3D (cytosol) compartments. The implications of crowded regions in the cell can be investigated because each diffusing molecule has spatial dimensions. Spatiocyte adopts multi-algorithm and multi-timescale frameworks to simulate models that simultaneously employ deterministic, stochastic, and particle reaction-diffusion algorithms. Comparison of light microscopy images to simulation snapshots is supported by Spatiocyte microscopy visualization and molecule tagging features. Spatiocyte is open-source software and is freely available at http://spatiocyte.org .

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Reconstituting geometry-modulated protein patterns in membrane compartments

Cited by 10 publications

References 19 publications

DNA segregation under Par protein control

DNA segregation under Par protein control

<em>In Vitro</em> Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Multi-Algorithm Particle Simulations with Spatiocyte

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