Home Page Researchers Ady
The Racah Institute of Physics
Faculty of Science
The Hebrew University, Jerusalem, 91904, Israel
Sensory processes play a fundamental roll in many aspects of living cells. Almost all living cells are equipped with molecular sensors that span the membrane bilayer and relay external signals into the cell to regulate different cellular processes. Understanding how these sensors function is a major challenge. To get unique insight into the physical functioning of such sensors we are using microscope-based fluorescence-polarization techniques that allows us to detect sub-nanometer physical responses of such molecular sensors in living cells and in real time. When complemented with other microscopy techniques these method allow systematic and quantitative analysis of such sensory systems.
The bacterial chemoreceptors: interacting sensors
As a model system, we currently study the bacterial 'chemo-receptors' that control the swimming behavior of bacterial cells, directing them along favorable gradients and away from harmful environments. Interestingly, these sensors form higher order molecular assemblies which cluster together to form two-dimensional arrays encored to the membrane bilayer (Figure 1).
Figure 1. Schematic view of the organization of bacterial chemoreceptors on the cell membrane.
In this project we are trying to understand how individual sensors physically function within clusters, and how their physical responses quantitatively relate to changes in the activity of the downstream components. We gain excess to the physical response of these sensors by labeling the receptors with fluorescent protein, YFP, and analyzing the polarization of the emitted fluorescence.
General view of the bacterial 'two component' sensory system
Microorganisms commonly use 'two-component' sensory systems to sense changes in their environment and trigger suitable physiological responses. Each system consists of a transmembrane histidine kinase receptor and a cytoplasmic phosphoryl-accepting response regulator, which in most cases acts as a transcription factor. Apart from a few well-studied cases, intracellular organization of the sensory kinases is largely unknown. Using several fluorescence techniques, we have recently provided a general picture of the spatial distribution of these sensors and of their oligomeric state in the model bacterium Escherichia coli. Out of the 25 fluorescently-tagged sensors tested, nine showed homogeneous distribution over the membrane, two showed distinct clusters, and the remaining sensors showed mild tendency for clustering. In most cases, we observed a clear density-dependent association between sensors, with only a weak tendency for self-association at low densities. Interestingly, in a few cases we detected physical associations also between sensors that belong to different signaling systems. In addition, we find examples where self-association between sensors can be modulated by changes in the environmental conditions. These data support the notion that most sensory kinases functions as dimers; however, it suggest that associations between sensors can play a role in signaling and may even lead to cross-talk between different systems.
Figure 2. TorS and EvgS sensors show distinct associations and clustering properties. (A) Fluorescence images of mYFP-tagged TorS and EvgS (B) Fluorescence anisotropy measurements of the mYFP-tagged sensors. The fluorescence anisotropy is shown as a function of the total fluorescence for the individual mYFP-tagged sensors. A total fluorescence intensity of 105 counts per second corresponds to approximately 4,000 copies of mYFP per cell.
The physical and functional thermal sensitivity of bacterial chemoreceptors
We have recently explored the temperature dependence of the receptor?s function. The bacterium Escherichia coli exhibit chemotactic behaviour at temperatures from approximately 20°C to over 42°C. We have now studied the underling molecular behaviour of these receptors in vivo and throughout their operating temperature range. We reveal a sharp modulation in the conformation of unclustered as well as clustered receptor trimers, and consequently in the kinase-activity output. These modulations occur at a characteristic temperature that depended on clustering and was lower for receptors at lower
adaptational states. However, in the presence of dynamic adaptation, the response of the kinase activity to a stimulus was sustained up to 45°C, but the sensitivity notably decreased. Thus, this molecular system exhibits clear thermal sensitivity that emerges already at the level of receptor trimers, but both receptor clustering and adaptation support the overall robust operation of the system at elevated temperatures.
Prolonged stimuli alter the bacterial chemosensory clusters
The dynamic clustering of membrane-bound receptors plays an essential role in various biological systems. A notable model system for studying this phenomenon is the bacterial chemosensory cluster that allows motile bacteria to navigate along chemical gradients in their environment. While the principle structure of these receptor arrays is becoming clear, their dynamic nature and operation are not yet understood. By measuring the fluorescence-polarization of tagged receptor-clusters in live Escherichia coli cells, we show that during a stimulus, the packing of the receptors in clusters slowly changes. Consistent with these physical changes, we find that the regulation of kinase activity by these clusters slowly evolves, altering the effective cooperativity of the response. Time-lapse fluorescence imaging indicated that despite these changes, the clusters do not generally dissociate upon ligand binding. These data suggest that prolonged stimuli induce changes in receptor packing within chemosensory clusters, which, in turn, alters the coupling between the receptors, and thus leading to non-stationary signal transduction.
List of publications in Nanoscience and Nanotechnology (2006-2010)
- Vaknin, A., and Berg, H. C., (2004). Single-cell FRET imaging of phosphatase activity in the Escherichia coli chemotaxis system. Proc. Natl. Acad. Sci. USA 101: 17072-17077.
- Vaknin, A., and Berg, H. C., (2006). Osmotic stress mechanically perturbs chemoreceptors in Escherichia coli. Proc. Natl. Acad. Sci. USA 103: 592-596.
- Vaknin, A., and Berg, H. C., (2007). Physical responses of bacterial chemoreceptors. J. Mol. Biol. 366: 1416-1423.
- Sourjik V., Vaknin A., Shimizu T.S., Berg H.C., (2007). In vivo measurement by FRET of pathway activity in bacterial chemotaxis. Methods Enzymol. 423: 365-391. Invited.
- Vaknin, A., and Berg, H. C., (2008). Direct Evidence for Coupling between Bacterial Chemoreceptors. J. Mol. Biol. 382: 573-577.
- Frank V., Koler M., Furst F. and Vaknin, A., (2011) The physical and functional thermal sensitivity of bacterial chemoreceptors. J. Mol. Biol. 411: 554-566.
- Sommer E., Koler M., Sourjik V., and Vaknin A., Cellular organization of sensory histidine kinases in Escherichia coli (in preparation)
Students, postdocs and researchers:
Research assistant: Dr. Moria Koler.
Ph.D Student: Vered Frank.
M.Sc. Student: Einat Tamar.
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