Elisabeth Fischer-Friedrich

Spatial regulators for bacterial cell division self-organize into surface waves in vitro.

In the bacterium Escherichia coli, the Min proteins oscillate between the cell poles to select the cell center as division site. This dynamic pattern has been proposed to arise by self-organization of these proteins, and several models have suggested a reaction-diffusion type mechanism. Here, we found that the Min proteins spontaneously formed planar surface waves on a flat membrane in vitro. The formation and maintenance of these patterns, which extended for hundreds of micrometers, required adenosine 5′-triphosphate (ATP), and they persisted for hours. We present a reaction-diffusion model of the MinD and MinE dynamics that accounts for our experimental observations and also captures the in vivo oscillations.

Real-time deformability cytometry: on-the-fly cell mechanical phenotyping

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Min protein patterns emerge from rapid rebinding and membrane interaction of MinE

In Escherichia coli, the pole-to-pole oscillation of the Min proteins directs septum formation to midcell, which is required for symmetric cell division. In vitro, protein waves emerge from the self-organization of MinD, a membrane-binding ATPase, and its activator MinE. For wave propagation, the proteins need to cycle through states of collective membrane binding and unbinding. Although MinD presumably undergoes cooperative membrane attachment, it is unclear how synchronous detachment is coordinated. We used confocal and single-molecule microscopy to elucidate the order of events during Min wave propagation. We propose that protein detachment at the rear of the wave, and the formation of the E-ring, are accomplished by two complementary processes: first, local accumulation of MinE due to rapid rebinding, leading to dynamic instability; and second, a structural change induced by membrane-interaction of MinE in an equimolar MinD–MinE (MinDE) complex, which supports the robustness of pattern formation.

Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy

In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct timescales are clearly visible in the correlation curves. While the faster timescale can be attributed to cytoplasmic diffusion, the slower timescale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16 microm(2) s(-1), while for MinE we find about 10 microm(2) s(-1), independently of the processes responsible for the slower time-scale. The implications of the measured values for the oscillation mechanism are discussed.

Quantification of surface tension and internal pressure generated by single mitotic cells

During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplaces law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplaces law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ≈ 40 Pa and 0.2 mNm−1 during interphase to ≈ 400 Pa and 1.6 mNm−1 during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible.

Surface Topology Engineering of Membranes for the Mechanical Investigation of the Tubulin Homologue FtsZ

In spite of their small size, bacteria display highly organized cytoskeletal structures like coils, helices, or rings. Extensive mechanical modeling has been done to explain the occurrence of such specific structures within the small volume of bacterial cells. As they are difficult to image within cells, in vitro reconstitution provides a valuable approach to quantitatively analyze their properties under defined conditions. A particularly interesting cytoskeletal feature is the Z-ring, which plays a key role in cell division for many bacteria. It is composed of FtsZ, a tubulin homologue, and other components and has been implicated in constriction force generation. Mechanisms localizing FtsZ to the center of the cell are known, but how it takes the form of a functional helical or ring-like structure remains unclear. 6] We hypothesized that intrinsically curved FtsZ filaments should initially respond to the native shape of bacteria and align using geometric cues. Thus, we devised a controlled biomimetic platform with membrane-coated glass substrates mimicking biologically relevant curvatures, to elucidate the mechanical properties of membrane-associated FtsZ. We found that E. coli FtsZ is assembled into inherently curved and twisted filaments supporting a helical geometry, which showed preferential orientations at the native bacterial cell-like curvatures. Strikingly, the FtsZ did not recognize smaller curvatures in the same way, but rather oriented themselves at an angle in higher curvatures, which does not support the idea that FtsZ alone is able to exert a constriction force. In recent studies involving high-resolution imaging and cryo-electron microscopy, the “Z-ring” has generally been described as a helical structure. Purified FtsZ has been studied extensively by electron microscopy and atomic force microscopy. Consistently, the EM and AFM images from these studies show curved filaments. Cryo-EM on reconstituted FtsZ filaments in vitro seems to contradict the presence of any local spontaneous curvature. However, in a recent study, Osawa et al. showed the ability of FtsZ filaments with an artificially introduced membrane targeting sequence (MTS) to bend membranes, with an influence of the MTS placement in FtsZ on the membrane bending direction. They used an MTS from MinD at the C-terminus of FtsZ to mimic the recruitment of FtsZ to the membrane by adaptor proteins ZipA or FtsA. Upon shifting the MTS to the Nterminus, they find that the filaments bend the membrane in opposite directions. They interpret this to be caused by a constriction force produced by partial Z-rings. A dividing bacterial cell initially has a curvature of about 2 mm , but proceeds towards much higher curvature values as the cell progresses through division. It is unknown whether a bacterial membrane, fortified with many structural proteins, osmotic pressure, and a cell wall, would be as easily deformed. The spontaneous structure of FtsZ filaments may enable them to organize into highly curved suprastructures by sensing the inner cell-membrane curvature, but they may have to recruit other mechanically active factors for cytokinesis. The distortions observed in previous studies 18] could simply be caused by a bundle of curved filaments bending the flexible membrane towards their own curvature. We first repeated the experiments with MTS-FtsZ on freestanding giant unilamellar vesicle (GUV) membranes, and quantitatively evaluated the induced radii of curvature. We found that the filaments did not bend the membranes when the unilamellar vesicles were isotonic. They aligned into filament networks similar to those on planar supported bilayers (Figure 1 b). Changing the osmotic gradient across the membranes by adding 10 mm glucose decreased intravesicular pressure and relaxed the membrane surface tension. This resulted in a curved topology of the membrane as well as the filaments (Figure 1a,b). Only when the membrane tension was low, under hypertonic conditions, could the filaments [*] Prof. Dr. P. Schwille Dept. Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry Am Klopferspitz 18, 82152 Martinsried (Germany) E-mail: schwille@biochem.mpg.de S. Arumugam, G. Chwastek, C. Ehrig Max Planck Institute for Cell Biology and Genetics Pfotenhauerstrasse 108, 01307 Dresden (Germany) and Biotechnology Center of the TU Dresden Tatzberg 47/51, 01307 Dresden (Germany)

Membrane Binding of MinE Allows for a Comprehensive Description of Min-Protein Pattern Formation

The rod-shaped bacterium Escherichia coli selects the cell center as site of division with the help of the proteins MinC, MinD, and MinE. This protein system collectively oscillates between the two cell poles by alternately binding to the membrane in one of the two cell halves. This dynamic behavior, which emerges from the interaction of the ATPase MinD and its activator MinE on the cell membrane, has become a paradigm for protein self-organization. Recently, it has been found that not only the binding of MinD to the membrane, but also interactions of MinE with the membrane contribute to Min-protein self-organization. Here, we show that by accounting for this finding in a computational model, we can comprehensively describe all observed Min-protein patterns in vivo and in vitro. Furthermore, by varying the systems geometry, our computations predict patterns that have not yet been reported. We confirm these predictions experimentally.

Intra- and intercellular fluctuations in Min-protein dynamics decrease with cell length

Self-organization of proteins in space and time is of crucial importance for the functioning of cellular processes. Often, this organization takes place in the presence of strong random fluctuations due to the small number of molecules involved. We report on stochastic switching of the Min-protein distributions between the two cell halves in short Escherichia coli cells. A computational model provides strong evidence that the macroscopic switching is rooted in microscopic noise on the molecular scale. In longer bacteria, the switching turns into regular oscillations that are required for positioning of the division plane. As the pattern becomes more regular, cell-to-cell variability also lessens, indicating cell length-dependent regulation of Min-protein activity.

Timing of Z-ring localization in Escherichia coli.

Bacterial cell division takes place in three phases: Z-ring formation at midcell, followed by divisome assembly and building of the septum per se. Using time-lapse microscopy of live bacteria and a high-precision cell edge detection method, we have previously found the true time for the onset of septation, τ(c), and the time between consecutive divisions, τ(g). Here, we combine the above method with measuring the dynamics of the FtsZ-GFP distribution in individual Escherichia coli cells to determine the Z-ring positioning time, τ(z). To analyze the FtsZ-GFP distribution along the cell, we used the integral fluorescence profile (IFP), which was obtained by integrating the fluorescence intensity across the cell width. We showed that the IFP may be approximated by an exponential peak and followed the peak evolution throughout the cell cycle, to find a quantitative criterion for the positioning of the Z-ring and hence the value of τ(z). We defined τ(z) as the transition from oscillatory to stable behavior of the mean IFP position. This criterion was corroborated by comparison of the experimental results to a theoretical model for the FtsZ dynamics, driven by Min oscillations. We found that τ(z) < τ(c) for all the cells that were analyzed. Moreover, our data suggested that τ(z) is independent of τ(c), τ(g) and the cell length at birth, L(0). These results are consistent with the current understanding of the Z-ring positioning and cell septation processes.