Effect of Energy Metabolism on Protein Motility in the Bacterial Outer Membrane

Winther, Tabita; Xu, Lei; Berg-Sørensen, Kirstine; Brown, Stanley; Oddershede, Lene B.

doi:10.1016/j.bpj.2009.06.027

Cited by 20 publications

(33 citation statements)

References 27 publications

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“…Plasmids lacking a partition system are highly mobile in E. coli cells growing on agarose pads, but slow down or stop in nongrowing cells imaged on glass slides, perhaps because of nutrient and energy depletion (45). Furthermore, Winther et al (46) used optical tweezers to track the motion of single molecules of LamB, an E. coli outer membrane protein that serves as the receptor for bacteriophage λ. LamB motility decreased an order of magnitude when cells were depleted of energy by treatment with azide and arsenate. A similar decrease in motion was seen upon treatment with ampicillin, an antibiotic that inhibits cross-linking of the peptidoglycan, indicating that PBP activity is the dominant source of fluctuations driving LamB motion.…”

Section: Discussionmentioning

confidence: 99%

Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci

Weber

Spakowitz

Theriot

2012

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

314

313

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Chromosomal loci jiggle in place between segregation events in prokaryotic cells and during interphase in eukaryotic nuclei. This motion seems random and is often attributed to Brownian motion. However, we show here that locus dynamics in live bacteria and yeast are sensitive to metabolic activity. When ATP synthesis is inhibited, the apparent diffusion coefficient decreases, whereas the subdiffusive scaling exponent remains constant. Furthermore, the magnitude of locus motion increases more steeply with temperature in untreated cells than in ATP-depleted cells. This "superthermal" response suggests that untreated cells have an additional source of molecular agitation, beyond thermal motion, that increases sharply with temperature. Such ATP-dependent fluctuations are likely mechanical, because the heat dissipated from metabolic processes is insufficient to account for the difference in locus motion between untreated and ATP-depleted cells. Our data indicate that ATP-dependent enzymatic activity, in addition to thermal fluctuations, contributes to the molecular agitation driving random (sub)diffusive motion in the living cell.T he cytoplasm is a crowded and dynamic medium, with molecules constantly jostling around and colliding with each other. This molecular motion is often attributed to Brownian motion, the random movement of suspended particles driven by thermal fluctuations of the solvent (1, 2). Classic Brownian motion theory assumes a system at thermal equilibrium. However, cells are far from equilibrium. They use the chemical energy of ATP (and GTP) to drive active biological processes, such as transport and metabolism.Recent work in eukaryotic cells demonstrates that biological activity generates nonthermal fluctuations of greater magnitude than thermal fluctuations (3-8). These active fluctuations can drive diffusive-like motion of molecules inside the cell, a phenomenon known as "active" diffusion (9, 10). In vitro experiments and analytical theory suggest that these active fluctuations are generated by the cytoskeletal molecular motor myosin (11-13). Thus, random molecular motion in vivo, at least in eukaryotic cytoplasm, may be due to active motor-driven forces in addition to passive thermal forces.Here we present evidence suggesting that ATP-dependent fluctuations contribute to the motion of chromosomal loci in bacterial and yeast cells. By modulating the temperature at which cells are observed, we were able to identify nonthermal forces that contribute to intracellular motion. Unlike active microrheology (7,8,11), temperature modulation presents a simple perturbation that can be applied to any experimental system to explore the physical processes underlying molecular motion in vivo. Our results suggest that "active" diffusion is not unique to systems containing eukaryotic cytoskeletal motors. This phenomenon may in fact be a general property of macromolecular motion in all living cells. Resultsis calculated to determine the subdiffusive scaling exponent α and the apparent diffusion coefficient D ap...

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Section: Discussionmentioning

confidence: 99%

Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci

Weber

Spakowitz

Theriot

2012

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

314

313

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“…This could be investigated using single-molecule techniques. Finally, we believe that our experimental design forms a valuable addition to existing techniques to study OM protein mobility, such as FRAP after chemical labeling treatments [8], tracking of single molecule fluorescence [35,36] as well as single particle tracking [4,5]. …”

Section: Discussionmentioning

confidence: 99%

“…For example, recent work on the mobility of integral OMP LamB suggests that it is confined to a region of size ~50 nm [4,5]. This was based on the motion of a marker bead or quantum dot attached to a surface-exposed biotinylated loop of LamB.…”

Section: Introductionmentioning

confidence: 99%

Absence of long-range diffusion of OmpA in E. coliis not caused by its peptidoglycan binding domain

2013

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BackgroundIt is widely believed that integral outer membrane (OM) proteins in bacteria are able to diffuse laterally in the OM. However, stable, immobile proteins have been identified in the OM of Escherichia coli. In explaining the observations, a hypothesized interaction of the immobilized OM proteins with the underlying peptidoglycan (PG) cell wall played a prominent role.ResultsOmpA is an abundant outer membrane protein in E. coli containing a PG-binding domain. We use FRAP to investigate whether OmpA is able to diffuse laterally over long-range (> ~100 nm) distances in the OM. First, we show that OmpA, containing a PG binding domain, does not exhibit long-range lateral diffusion in the OM. Then, to test whether PG interaction was required for this immobilization, we genetically removed the PG binding domain and repeated the FRAP experiment. To our surprise, this did not increase the mobility of the protein in the OM.ConclusionsOmpA exhibits an absence of long-range (> ~100 nm) diffusion in the OM that is not caused by its PG binding domain. Therefore, other mechanisms are needed to explain this observation, such as the presence of physical barriers in the OM, or strong interactions with other elements in the cell envelope.

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“…4b), was shown to be connected more tightly to the membrane than typical eukaryotic membrane proteins and to have a higher diffusion constant 37,48 . Interestingly, the diffusion of the l-receptor is dependent on the bacterial metabolism of the host cell: if the cell is depleted of energy, the proteins move significantly less 14,49 . Figure 4c shows a time series of the positions visited by a particular l-receptor both before and after poisoning the cell with azide and arsenate to effectively stop electron transport and ATP synthesis.…”

Section: Getting a Handle On The Biological Systemmentioning

confidence: 99%

“…However, the results obtained in vitro may not faithfully reflect properties of the same molecule in vivo. For instance, the diffusion of membrane proteins in vivo has been shown to be rather different from that in vitro in artificial membranes, as the protein motility depends on cellular metabolism 14 . Also, molecular motors seem to have somewhat different characteristics in vivo than in vitro, dyneins have shorter runs inside a living cell 15 than in a test tube 16 , and ribosomes seem to translate over ten times slower along mRNAs in vitro 6 than they do in ensemble in vivo measurements 17 .…”

mentioning

confidence: 99%

Force probing of individual molecules inside the living cell is now a reality

Oddershede

2012

Nat Chem Biol

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Biological systems can be quantitatively explored using single-molecule manipulation techniques such as optical or magnetic tweezers or atomic force microscopy. Though a plethora of discoveries have been accomplished using single-molecule manipulation techniques in vitro, such investigations constantly face the criticism that conditions are too far from being physiologically relevant. Technical achievements now allow scientists to take the next step: to use single-molecule manipulation techniques quantitatively in vivo. Considerable progress has been accomplished in this realm; for example, the interaction between a protein and the membrane of a living cell has been probed, the mechanical properties of individual proteins central for cellular adhesion have been measured and even the action of molecular motors in living cells has been quantified. Here, we review the progress of in vivo single-molecule manipulation with a focus on the special challenges posed by in vivo conditions and how these can be overcome.

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Effect of Energy Metabolism on Protein Motility in the Bacterial Outer Membrane

Cited by 20 publications

References 27 publications

Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci

Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci

Absence of long-range diffusion of OmpA in E. coliis not caused by its peptidoglycan binding domain

Force probing of individual molecules inside the living cell is now a reality

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