Mobility of Min-proteins in<i>Escherichia coli</i>measured by fluorescence correlation spectroscopy

Meacci, Giovanni; Ries, Jonas; Fischer‐Friedrich, Elisabeth; Kahya, Nicoletta; Schwille, Petra; Kruse, Karsten

doi:10.1088/1478-3975/3/4/003

Cited by 87 publications

(88 citation statements)

References 39 publications

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“…For a typical protein diffusion constant of D ¼ 1-10 μm 2 s −1 (42)(43)(44), the rebinding probability drops below 10% when the enzyme reactivation time becomes longer than 10 ms (Fig. 4C).…”

Section: Discussionmentioning

confidence: 96%

Spatio-temporal correlations can drastically change the response of a MAPK pathway

Takahashi

Tănase‐Nicola

Wolde

2010

Proc. Natl. Acad. Sci. U.S.A.

256

394

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Multisite covalent modification of proteins is omnipresent in eukaryotic cells. A well-known example is the mitogen-activated protein kinase (MAPK) cascade where, in each layer of the cascade, a protein is phosphorylated at two sites. It has long been known that the response of a MAPK pathway strongly depends on whether the enzymes that modify the protein act processively or distributively. A distributive mechanism, in which the enzyme molecules have to release the substrate molecules in between the modification of the two sites, can generate an ultrasensitive response and lead to hysteresis and bistability. We study by Green's Function Reaction Dynamics (GFRD), a stochastic scheme that makes it possible to simulate biochemical networks at the particle level in time and space, a dual phosphorylation cycle in which the enzymes act according to a distributive mechanism. We find that the response of this network can differ dramatically from that predicted by a mean-field analysis based on the chemical rate equations. In particular, rapid rebindings of the enzyme molecules to the substrate molecules after modification of the first site can markedly speed up the response and lead to loss of ultrasensitivity and bistability. In essence, rapid enzyme-substrate rebindings can turn a distributive mechanism into a processive mechanism. We argue that slow ADP release by the enzymes can protect the system against these rapid rebindings, thus enabling ultrasensitivity and bistability.MAP kinase | Multisite phosphorylation | Reaction diffusion | Simulation M APK cascades are ubiquitous in eukaryotic cells. They are involved in cell differentiation, cell proliferation, and apoptosis (1). MAPK pathways exhibit very rich dynamics. It has been predicted mathematically and shown experimentally that they can generate an ultrasensitive response (2-4) and exhibit bistability via positive feedback (5). It has also been predicted that they can generate oscillations (6,7,8), amplify weak but attenuate strong signals (9), and give rise to bistability due to enzyme sequestration (10, 11). MAPK pathways are, indeed, important for cell signalling, and for this reason they have been studied extensively, both theoretically (2, 3, 6-19) and experimentally (2,4,5,7,16,(20)(21)(22). However, with the exceptions of refs. 7, 9, 11, 15, and 19, the pathway is commonly modeled by using chemical rate equations (2, 3, 6, 8, 10, 12-14, 16, 17). This is a mean-field description, in which it is assumed that the system is well-stirred and that fluctuations can be neglected. Here, we perform particle-based simulations of one layer of the MAPK cascade using our recently developed GFRD algorithm (23, 24). Our simulations reveal that spatio-temporal correlations between the enzyme and substrate molecules that are ignored in the commonly employed mean-field analyses can have a dramatic effect on the nature of the response. They can not only speed up the response, but also lead to loss of ultrasensitivity and bistability.The response time, the sharpness of the ...

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Section: Discussionmentioning

confidence: 96%

Spatio-temporal correlations can drastically change the response of a MAPK pathway

Takahashi

Tănase‐Nicola

Wolde

2010

Proc. Natl. Acad. Sci. U.S.A.

256

394

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“…Although these are reasonable barebone steps to be considered, experimental observations to evaluate the quantitative details of the reaction steps have been very limited and few constraints have been imposed on these models. Only bulk diffusion coefficients of proteins have been measured directly (14,36). The surface diffusion coefficients have been estimated from FRAP experiments without analysis of exchange between membrane-bound proteins and solution (14), which turned out to be the leading cause of the fluorescence recovery according to our measurements (SI FRAP Experiments and SI Summary of the Fluorescence Recovery Time Data).…”

Section: Discussionmentioning

confidence: 99%

Multiple modes of interconverting dynamic pattern formation by bacterial cell division proteins

Ivanov

Mizuuchi

2010

Proc. Natl. Acad. Sci. U.S.A.

110

150

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Min proteins of the Escherichia coli cell division system oscillate between the cell poles in vivo. In vitro on a solid-surface supported lipid bilayer, these proteins exhibit a number of interconverting modes of collective ATP-driven dynamic pattern formation including not only the previously described propagating waves, but also near uniformity in space surface concentration oscillation, propagating filament like structures with a leading head and decaying tail and moving and dividing amoeba-like structures with sharp edges. We demonstrate that the last behavior most closely resembles in vivo system behavior. The simple reaction-diffusion models previously proposed for the Min system fail to explain the results of the in vitro self-organization experiments. We propose the hypotheses that initiation of MinD binding to the surface is controlled by counteraction of initiation and dissociation complexes; the binding of MinD/E is stimulated by MinE and involves polymerization-depolymerization dynamics; polymerization of MinE over MinD oligomers triggers dynamic instability leading to detachment from the membrane. The physical properties of the lipid bilayer are likely to be one of the critical determinants of certain aspects of the dynamic patterns observed.cell division | min system | oscillations and waves | self-organization | septum localization

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“…depend on a number of parameters, one of which is the refractive index of solution (29,30). The latter can be affected by the presence of detergents leading to the distortion of the FCS detection volume.…”

Section: Fcs Detection Volume Is Not Affected By the Presence Of Detementioning

confidence: 99%

Detergent-activated BAX Protein Is a Monomer

Ivashyna

García‐Sáez

Ries

et al. 2009

Journal of Biological Chemistry

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BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation withdetergents,suchasn-octylglucoside.DuringBAXactivationwith n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.

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Mobility of Min-proteins inEscherichia colimeasured by fluorescence correlation spectroscopy

Cited by 87 publications

References 39 publications

Spatio-temporal correlations can drastically change the response of a MAPK pathway

Spatio-temporal correlations can drastically change the response of a MAPK pathway

Multiple modes of interconverting dynamic pattern formation by bacterial cell division proteins

Detergent-activated BAX Protein Is a Monomer

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