Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - ABCvolume (The ABC of Cell Volume Regulation)

Teaser

“What is life” is one of the most intriguing and difficult questions to answer. In 1932, the physicist Niels Bohr even wrote: “ The existence of life must be considered as an elementary fact that cannot be explained”. Another physicist, Erwin Schrödinger, argued in...

Summary

“What is life” is one of the most intriguing and difficult questions to answer. In 1932, the physicist Niels Bohr even wrote: “ The existence of life must be considered as an elementary fact that cannot be explained”. Another physicist, Erwin Schrödinger, argued in 1945 that biological problems could be thought of in physical terms, as a limited number of atoms in a large molecule that could produce an unlimited number of arrangements. Actually, Schrödinger postulated the concept of “genes”, although at that time he thought that genes were made up of proteins rather than DNA. In the 1960s, Richard Feynman argued that “Everything that living systems do is done by atoms that act according to the laws of physics”, and this is the basis we (all) build on. Today, it is still difficult to define life, even at the cellular scale. At the molecular level, however, it is well established that life is a system of self-sustained chemical processes. Complex networks of proteins, nucleic acids and small molecules sustain the essential processes of energy provision, gene expression and cell reproduction that characterize living matter. Biochemical networks direct cell growth and division, and through the uptake of nutrients, conservation of metabolic energy and the excretion of waste, they maintain a dynamic state far from thermodynamic equilibrium. In fact, a living system that reaches equilibrium is dead.

One of the grand challenges in chemistry is the bottom-up construction of functional far-from-equilibrium systems, which are prominent in living systems but difficult to realize in test tubes. The challenge in multidisciplinary research, such as synthetic biology, is to construct such living systems from molecular building blocks, that is, to assemble and engineer the components that allow a synthetic cell to grow and divide.

The prospect of creating synthetic life has inspired people for many years. The Venter Institute, for instance, has recently demonstrated that a de novo synthesized genome containing less than 500 genes can lead to viable cells. While creating a reduced cell by selectively removing components from a wild-type genome is an impressive achievement, this top-down approach does neither reveal how the remaining gene products act together to create life nor capture the links between metabolism, compartmentalization and the information contained in DNA. As a result, it has not yet been possible to rationally design and construct, using a bottom-up constructive approach, a simple form of life based on a limited number of molecular building blocks. While our fundamental understanding of the individual building blocks of life is rapidly growing, putting a minimal set of components together such that life-like properties emerge remains a formidable, yet exciting challenge.

The overall aim of ABCVolume is to understand cell volume regulation at the molecular level, including the gating mechanisms of osmoregulatory transporters and the interplay of the network with the physicochemistry of the cell. The specific aims are:
(i) To develop a network for cell volume regulation by synthesizing a vesicle system with sustained production of metabolic energy and capacity to build up sufficient osmotic pressure to expand, yet prevent it from lysing;
(ii) To elucidate the molecular mechanism of gating and translocation of an osmoregulatory ABC transporter, a key component of the volume regulatory network, using state-of-the-art reconstitution technology and single-molecule optical microscopy.

Work performed

One of the grand challenges in building synthetic forms of life is the construction of functional out-of-equilibrium systems, which are essential in living cells. One requires control over the formation and degradation of interacting chemicals in membrane-bounded open systems. We report the in vitro construction of a pathway for sustained ATP production that performs at least an order of magnitude better than any system described so far. We maintain energy homeostasis while the load on the system is varied. One of the crucial ATP-requiring networks in biology controls the volume of the cell and hence influences the osmotic pressure, ionic strength, internal pH, membrane tension, and molecular crowding. We use the ATP provision to control the transmembrane fluxes of osmolytes and demonstrate control of the internal pH and ionic strength, which stabilizes the enzymes that we have reconstituted in our synthetic cells. Our work allows a further understanding of metabolic energy conservation and cell volume regulatory mechanisms in a context and at a level of complexity minimally needed for life.

Final results

Cells contain many proteins that constantly move about within the cell to carry out tasks that keep the cell running. A protein of average size makes contact with all the other proteins in a typical bacterial cell in a few seconds. This moving about mixes the cell’s contents, which is crucial for its survival and reproduction.

Proteins can group together, or with other molecules, to form bigger units. This grouping together depends on the properties of protein surfaces; for example, opposite electrical charges on the surfaces of proteins can cause them to group together. Grouping together proteins causes them to move around cells more slowly. Indeed the ribosome, a protein unit that constructs new proteins and is found in every known species, moves over a hundred times more slowly than the average protein.

Finding out how the total electric charge inside the cells of different species affects the mobility of proteins would give us a better idea of how fast tasks can be carried out in cells. We have recently shown that protein surface charge matters a great deal in the cytoplasm of the cell. Negatively charged and neutral proteins move about rapidly whereas positively charged proteins move up to 100 times more slowly. We also find that positively charged proteins slow down because they bind to negatively charged ribosomes. Because ribosomes are found in all living cells, understanding how they affect how other proteins move around the cell has a wide range of possible applications. For example, biologists and biotechnologists often produce proteins in E. coli for convenient study. Yet very positively charged proteins may bind to ribosomes in E. coli, causing experiments to fail. Using cells that shield charges better, such as H. volcanii or Lactococcus lactis or engineered synthetic cells could solve this issue.

Website & more info

More info: http://www.membraneenzymology.com.