Variability and Order in Cytoskeletal Dynamics of Motile Amoeboid Cells.

The chemotactic motion of eukaryotic cells such as leukocytes or metastatic cancer cells relies on membrane protrusions driven by the polymerization and depolymerization of actin. Here we show that the response of the actin system to a receptor stimulus is subject to a threshold value that varies strongly from cell to cell. Above the threshold, we observe pronounced cell-to-cell variability in the response amplitude. The polymerization time, however, is almost constant over the entire range of response amplitudes, while the depolymerization time increases with increasing amplitude. We show that cell-to-cell variability in the response amplitude correlates with the amount of Arp2/3, a protein that enhances actin polymerization. A time-delayed feedback model for the cortical actin concentration is consistent with all our observations and confirms the role of Arp2/3 in the observed cell-to-cell variability. Taken together, our observations highlight robust regulation of the actin response that enables a reliable timing of cell movement.

Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba.

Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

Investigating Deinococcus radiodurans RecA protein filament formation on double-stranded DNA by a real-time single-molecule approach.

With the aid of an efficient, precise, and almost error-free DNA repair system, Deinococcus radiodurans can survive hundreds of double-strand breaks inflicted by high doses of irradiation or desiccation. RecA of D. radiodurans (DrRecA) plays a central role both in the early phase of repair by an extended synthesis-dependent strand annealing process and in the later more general homologous recombination phase. Both roles likely require DrRecA filament formation on duplex DNA. We have developed single-molecule tethered particle motion experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules by observing changes in DNA tether length resulting from RecA binding. We demonstrate that DrRecA nucleation on double-stranded DNA is much faster than that of Escherichia coli RecA protein (EcRecA), but the extension is slower. This combination of attributes would tend to increase the number and decrease the length of DrRecA filaments relative to those of EcRecA, a feature that may reflect the requirement to repair hundreds of genomic double-strand breaks concurrently in irradiated Deinococcus cells.

A One Donor-Two Acceptor Lipid Bilayer FRET Assay Based on Asymmetrically Labeled Liposomes.

The fusion of two opposing membranes is essential in biological functions such as fertilization, viral entry, membrane trafficking and synaptic transmission. Before the membrane bilayers are fully connected, at some stage a hemifusion intermediate-when the outer leaflets are merged but not the inner leaflets-is formed. However, the position of hemifusion in the energy landscape and the duration of it vary and have not been fully mapped out. To date, there has not been a way to differentiate lipid mixing of the two leaflets directly in a single experiment. Herein we demonstrate labeling of the outer and inner leaflets with different fluorophores, which can be distinguished by their fluorescence lifetimes. As a proof of concept, the asymmetrically labeled liposomes were used as acceptor liposomes in a novel one donor-two acceptor Förster resonance energy transfer (FRET) assay to monitor membrane fusion reactions mediated by the synaptic proteins soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) in microfluidic devices. Initial hemifusion was clearly indicated by the acceptor fluorescence lifetime originating solely from FRET acceptors on the outer leaflet (Oregon Green 488; τFl ∼ 4.8 ns). Progression to full fusion was then indicated by the significantly increasing lifetime contribution from acceptors on the inner leaflet (nitrobenzoxadiazole; τFl ∼ 6.7 ns). The new labeling strategy creates many possibilities in the design of bulk and single-molecule experiments.

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation.

Among strains of Escherichia coli that have evolved to survive extreme exposure to ionizing radiation, mutations in the recA gene are prominent and contribute substantially to the acquired phenotype. Changes at amino acid residue 276, D276A and D276N, occur repeatedly and in separate evolved populations. RecA D276A and RecA D276N exhibit unique adaptations to an environment that can require the repair of hundreds of double strand breaks. These two RecA protein variants (a) exhibit a faster rate of filament nucleation on DNA, as well as a slower extension under at least some conditions, leading potentially to a distribution of the protein among a higher number of shorter filaments, (b) promote DNA strand exchange more efficiently in the context of a shorter filament, and (c) are markedly less inhibited by ADP. These adaptations potentially allow RecA protein to address larger numbers of double strand DNA breaks in an environment where ADP concentrations are higher due to a compromised cellular metabolism.

Hypoglycemia associated with bacteremic pneumococcal infections.

OBJECTIVES: To evaluate the prevalence and associated presentations of hypoglycemia in bacteremic pneumococcal infections, and serotypes of the isolates. METHODS: This was a retrospective study of 70 episodes of pneumococcal bacteremia that occurred in 2004 and 2005. RESULTS: We found hypoglycemia (plasma glucose<3.05 mmol/l)) in six (8.6%) episodes. The patients were three children (mean age 3 years 1 month; range 1 year 5 months-4 years 5 months) and three adults (mean age 73.3 years; range 63-84 years). One child with asplenia and cyanotic heart disease had primary pneumococcal bacteremia. Of the other two children, one had meningitis and the other pneumonia. All the adults had cancer with previous chemotherapy and multilobar pneumonia, which progressed rapidly to respiratory failure. All patients developed their first hypoglycemic episode within two hours after presentation. The average plasma glucose during hypoglycemia was 1.78+/-0.78 mmol/l (range 0.33-2.94 mmol/l). One child and all of the adults died. Serotypes of isolates were those usually associated with severe pneumococcal infection: 6B and 19F in the children; 3, 14, and 23F in the adults. Only the asplenic child had received pneumococcal vaccine. CONCLUSIONS: Hypoglycemia occurred in 8.6% of bacteremic pneumococcal infections and was associated with high mortality and serotypes that cause severe invasive disease. All patients suspected of having septicemia should have their glucose checked to avoid missing hypoglycemia leading to a worsening of their already poor condition.