Pilizota, T. | Search bioRxiv

bioRxiv2019

Comparison of Escherichia coli surface attachment methods for single-cell, in vivo microscopy

For in vivo, single-cell imaging bacterial cells are commonly immobilised via physical confinement or surface attachment. Different surface attachment methods have been used both for atomic force and optical microscopy (including super resolution), and some have been reported to affect bacterial physiology. However, a systematic comparison of the effects these attachment methods have on the bacterial physiology is lacking. Here we present such a comparison for bacterium Escherichia coli, and assess the growth rate, size and intracellular pH of cells growing attached to different, commonly used, surfaces. We demonstrate that E. coli grow at the same rate, length and internal pH on all the tested surfaces when in the same growth medium. The result suggests that tested attachment methods can be used interchangeably when studying E. coli physiology.

systems biology↗

bioRxiv2019

Escherichia coli’s physiology can turn membrane voltage dyes into actuators

The electrical membrane potential (Vm) is one of the components of the electrochemical potential of protons across the biological membrane (proton motive force), which powers many vital cellular processes, and Vm also plays a role in signal transduction. Therefore, measuring it is of great interest, and over the years a variety of techniques has been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favourite strategy, and their cytoplasmic accumulation depends on Vm according to the Nernst equation. However, a careful calibration of Nernstian probes that takes into account the trade-offs between the ease with which the signal from the dye is observed, and the dyes interactions with cellular physiology, is rarely performed. Here we use a mathematical model to understand such trade-offs and, based on the knowledge gained, propose a general work-flow for the characterization of Nernstian dye candidates. We demonstrate the work-flow on the Thioflavin T dye in Escherichia coli, and identify conditions in which the dye turns from a Vm probe into an actuator.\n\nSIGNIFICANCE STATEMENT\n\nThe phospholipid bilayer of a biological membrane is virtually impermeable to charged molecules. Much like in a rechargeable battery, cells harness this property to store an electrical potential that fuels life reactions but also transduces signals. Measuring this electrical potential, also referred to as membrane voltage, is therefore of great interest and a variety of techniques have been employed for the purpose, starting as early as the 1930s. For the case of bacteria, which are smaller in size and possess a stiffer cell wall, arguably the most popular approach to measuring membrane voltage are Nernstian probes that accumulate across the bacterial membrane according to the Nernst potential. The present study characterizes the undesired effects Nernstian probes can have on cell physiology, which can be crucial for the accurate interpretation of experimental results. Using mathematical modelling and experiments, the study provides a general, simple workflow to characterise and minimise these effects.

biophysics↗

bioRxiv2019

Dynamic clustering regulates activity of mechanosensitive membrane channels

Mechanosensitive channels regulate the flux of solutes between the cell and its environment in response to mechanical stimuli. Recent in vitro experiments have suggested that bacterial mechanosensitive channels separate into 2D liquid-like clusters, exhibiting cooperative activity. To understand the functional role of the process and its physical mechanism, we develop a coarse-grained model of mechanosensitive channels embedded in a membrane, exposed to an osmotic shock. We find that the channel aggregation into liquid-like clusters promotes the closure of individual channels. The extent of clustering is highly dependent on the channel concentration and membrane stress. We show that this behaviour yields a tightly regulated gating system, whereby at high tensions channels gate individually, and at lower tensions the channels spontaneously aggregate and inactivate. We suggest that this positive feedback protects the cell against excessive loss of cytoplasmic content. Our results point to a novel role of functional micro-phase separation in the cell membrane.

biophysics↗