The history of cell and molecular biology displays an arms race between biological challenges and their technological solutions, regarding the detection and analysis of ever smaller structures and processes. For a long time, advancements in microscopy have kept pace with scientific motivations. But since fundamental physical laws cannot be simply overcome, appropriate instruments need to become more and more complex if modern biology does not simply want to stand still due to technical limitations. As a prominent example, the need to zoom further into biological matter in space and time recently sparked an explosion in sub-resolution techniques in microscopy. But is it actually always necessary to adapt our technology to the actual dimensions of biological matter if we want to understand fundamental processes? We know that biology works on many scales. So why not just try and upscale biological phenomena?
In 1989, the movie Honey, I Shrunk the Kids was released, in which an unfortunate scientist develops a shrinking machine and accidentally shrinks his and the neighbor's children to the size of insects. In 1992, a sequel was released under the title Honey, I Blew Up the Kid. Justifiably, it eluded the success of the previous movie. Nevertheless, the idea behind the plot shall be of interest for us. This time, the unfortunate scientist - with the help of a new invention - inflates his 2-year old son to a 34 m-tall giant that starts to terrify Los Angeles in Godzilla manner.
Why not apply this concept to cell and molecular biology? If the structure or process of interest is too small to be observed with modern microscopy, why not just magnify the very phenomenon, instead of ever increasing optical magnification? Is this possible? Yes, under certain conditions, and the key to it seems to be an approach taken from synthetic biology.
Rafts and Membrane Domains
Just to give an example: In the past ten years, membrane rafts have received much attention in the cell biology community. Until the mid-nineties, the cell membrane was generally viewed as a homo-genous fluid, in which membrane proteins are embedded.
This view was challenged in 1997 by Simons and Ikonen , and by an avalanche of consecutive research work in the following years , .
According to this "raft hypothesis", lipids organize into domains within the plasma membrane, with physical properties that distinguishes the domains from the rest of the membrane (fig. 1a). These physical properties can be, for example, lateral fluidity, surface tension, and thickness of the double layer structure. These domains, or rafts, are thought to play a major role in recruiting membrane proteins and controlling their function.
The raft hypothesis is nowadays more or less accepted, with one blemish: no one has directly observed rafts in vivo so far. Many experiments point to their existence, but they could so far not be proven microscopically. Probably this will also not be possible for a long time, since rafts are assumed to organize on the nanoscale, and may have very fast turnover. Much effort is spent to make them "visible" by other means than optical microscopy, or just to indirectly prove their existence. An alternative to that would be to "blow up" these rafts so that they can be examined in a standard fluorescence microscope. This appears to be possible by leaving the in vivo-world and entering the field of synthetic biology of minimal systems . When mimicking the cell membrane by artificial membranes containing specific raft-like lipid composition, phase separations of lipids appear as domains on the micrometer scale (fig. 1b).
These domains are supposed to be synthetic functional siblings of the assumed rafts in cells. Most importantly however, they distinguish themselves by their actual visibility in fluorescence microscopy. Such artificial membranes of appreciable size are achieved either as supported lipid bilayers by fusing small unilamellar vesicles (SUVs) with a hydrophilic support, such as glass or mica . Another possibility is to rehydrate dehydrated lipid films in the presence of an alternating current, such that so-called giant unilamellar vesicles form (GUVs), which can be seen as a very crude in vitro approximation of the biological cell membrane: a membrane bilayer separates an inner volume from the environment and therefore provide the essential boundary conditions for organelle-like function , . The ability to make domains visible in minimal cell membrane-like systems yielded deeper insight into the features and functions of the hypothetical rafts , , .
Min Proteins for the Division Site Selection
Is this application, blowing up molecular mechanisms from the nanoscopic to the microscopic scale by transformation of cellular processes into synthetic biology a singular example, or can this concept be transferred to other biological problems, e.g., fundamental ones such as structure and pattern formation?
Here, we would like to present another demonstrative example of how membrane-related processes that elude quantitative fluorescence microscopy in vivo can be easily imaged and studied by subjecting the system to a synthetic biology-like approach. It is the upscaling of oscillatory processes preceding bacterial cell division.
In Escherichia coli, cell division is facilitated by the so-called Z-ring that assembles at a midcell position between the two future daughter cells . The assembly of this Z-ring at the central cell location is again regulated by another group of proteins, the Min protein family which oscillate between the cell poles but show a concentration minimum at the cell center (fig. 2). Cell division at a midcell position is crucial for successful cell replication, and it has been shown that deletion of the corresponding Min genome in E.coli mutants results in cell septation at other potential division sites such as the polar cell caps, leading to the formation of non-replicating and DNA-deficient mini-cells .
The Min-protein family consists out of three proteins: MinC, MinD and MinE. MinC is the actual inhibitor for the assembly of the Z-Ring. Adhesion of MinC to the membrane is again regulated by the cooperative membrane adhesion behavior of MinD and MinE. Upon binding of ATP, MinD binds to the cell membrane, MinE binds to membrane-bound MinD. After hydrolysis of ATP, both proteins detach again from the membrane (fig. 3). This cooperative behavior results in an oscillatory adhesion of Min proteins to the inner membrane of E. coli, and can be observed in vivo by fluorescent labeling of the proteins , , . Unfortuntalely, due to the small size of the bacterial cells of about 1 - 2 µm, it was so far impossible to obtain a more detailed mechanistic insight into the dynamic interaction of the involved proteins in vivo.
From Small Oscillations to Big Waves
We have demonstrated that it is possible to transfer the self-organizing Min protein system into an artificial environment . Min proteins did not only exhibit dynamic binding patterns represented as planar waves on supported membranes, but the scale of dynamics is increased about 20-fold compared to the cellular environment: on the artificial membranes, protein waves have a wavelength of about 50-100 µm, compared to 1-2 µm in cells (fig. 2 and fig. 4). This increase of the scale of the dynamics allowed us to study the cooperative behavior of Min proteins in much more detail than what could be achieved by in vivo studies.
A comfortable aspect of synthetic biology of minimal systems is that one can design systems with varying combinations of components. We could prove, for example, that it needs only four components for the establishment of a dynamic protein pattern: a membrane, MinD, MinE and ATP. MinC itself is necessary for the inhibition of Z-Ring assembly at unwanted locations, but it is not necessary for the spatial positioning through the oscillation.
In summary, by combination of a synthetic "bottom up" approach of a biological phenomenon with single molecule techniques, we arrived at a better mechanistic understanding of the process. i.e., that the MinD-binding pattern is governed by a continuous migration of binding of MinD to the membrane, whereas MinE accumulates at the trailing edge by fast (quasi processive) rebinding of previously detached MinE-proteins. At the very end of the wave, the MinD-surface concentration on the membrane decreases, however, every MinD-dimer seems now to be occupied by a MinE-dimer. The addition of MinC to the system yielded further insight into the cooperativity of the system. Our minimal systems approach allowed, for the first time, the simultaneous observation of all three proteins in action. We could prove that MinE is required for the detachment of MinC. Analysis of wave profiles showed that MinC is actually not detached together with MinD and MinE, but that MinE actually displaces MinC from MinD.
Clearly, the concept of synthetic biology of minimal systems implies some negotiations with biological reality. After having resolved central molecular mechanisms in an extremely reduced system, the challenge is now to approach a more physiological situation again. In the reconstituted Min system, our main concern is now the spatial confinement of the Min protein system, resembling the situation in a single cell. Currently we investigate the effect of spatial confinement on the formation and behavior of Min protein waves in 2D and even 3D. Our goal is it not only to reproduce protein waves and determine their response to external constraints, but also to mimic true protein oscillations in closed volumes. The fact that the wavelength of Min protein waves in our in vitro assay is about 50 times larger than their cellular counterparts allows us to use closed volumes that are still well in the range of microscopic imaging.
In conclusion, such a synthetic minimal systems approach can certainly not be generally applied for magnification of processes that elude optical microscopy in vivo. But synthetic biology indeed does open up new strategies for the quantitative study of biological processes, and among many other aspects, one should at least consider the possibility of rescaling of dynamical processes and structures.
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