We thank Michael Heymann and Frank Siedler for production of silicon wafers, Core Facility MPI-B for assistance in protein purification and Simon Kretschmer and Leon Harrington for comments on the manuscript.

<ol>
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The authors have nothing to disclose.

We have described a protocol for the in vitro reconstitution of MinCDE self-organization on planar supported lipid bilayers and in lipid bilayer covered 3D structures, using the example of rod-shaped PDMS microstructures. In order to obtain valuable data from these assays, the most important factors to control are protein and membrane quality.
To ensure protein quality, protein mass should be confirmed using SDS-PAGE and mass spectrometry. Furthermore, it should be verified that proteins are soluble and not aggregated, by using analytical gel filtration or dynamic light scattering. Gel filtration can be used to remove any aggregated fraction of proteins. Careful pH adjustment and quality of added nucleotides is critical, as the addition of non-adjusted or partially degraded nucleotide to protein stocks or self-organizing assays is sufficient to eliminate protein activity, therefore abolishing self-organization.
Next to protein quality, membrane quality is most critical, and improper membrane formation is most often the cause for defective self-organization and the origin of artefactual surface structures.
When performing the protocol for the first time, it is helpful to label the supported lipid bilayers by including labeled lipids such as Atto-655-DOPE or DiI at low molar percentages (0.05%). Thereby the properties and quality of the membrane can be judged directly. Using FRAP, the fluidity of the membrane can be assessed. Furthermore, one can directly assess the quality of washing of the SLB, as there will either be too many vesicles, no fluid membrane, or no membrane at all, if it has been washed off. The open chamber approach allows to rigorously wash the membrane, and hence also to remove vesicles that are sticking on the surface of the SLB. The most crucial factors for obtaining fluid and homogenous supported lipid bilayers are the cleaning and hydrophilicity of the support surface and the correct size and homogeneity of the SUVs. It can be helpful to check SUV size and size distribution using dynamic light scattering. For narrow size distributions, we recommend extruding the vesicles rather than sonicating them. Other methods of cleaning coverslips, e.g., treatments with strong bases, basic detergents, or using coverslips directly after rinsing with water, may yield good results, depending on the application and lipid mixture.
The first half of the protocol presented here, in vitro reconstitution on planar supported lipid bilayers in open chambers, has the advantage of rendering the surface accessible for optical microscopies, such as TIRF microscopy30, FRAP analysis6, single-particle tracking34, as well as surface probe techniques such as atomic force microscopy26. The large homogeneous area allows for better statistics at defined concentrations. Furthermore the open chamber approach allows to precisely control protein concentration and a rapid and simple addition of further components, hence permitting to titrate protein concentration in a single chamber20. The assay can also be expanded by addition of other bacterial divisome components such as FtsZ22,35, ZipA22 or the chimeric protein FtsZ-YFP-MTS10,35.
Other groups have taken a similar approach to reconstituting the Min system in vitro, but use a flow-cell instead of an open chamber17,18,19. Flow-cells have certain advantages, in particular when a fully enclosed 3D environment is needed18, the influence of flow17,19,23 or membrane composition23 on MinCDE patterns is investigated, or if protein patterns are to be observed under protein limiting conditions19. Nonetheless, local control of molecular concentrations is more difficult. Protein components, especially MinD, strongly bind to the membrane they first encounter18,19. In our experience, the proteins frequently exhibit non-specific binding to tubing, inlets, syringes and other microfluidic parts. Hence, local protein concentrations differ from input concentrations18 and also vary over the length of the flow-cell, resulting in a variety of different protein patterns on the membrane between inlet and outlet, as observed by others19.
The second half of the protocol presented here, the in vitro reconstitution in rod-shaped microstructures re-using the open chamber approach on a patterned support covered by lipid bilayers allows for a simple mimic of in vivo protein behavior even though precise control over protein concentrations is lost due to buffer removal. Note that because the wavelength of MinDE is about one order of magnitude larger in vitro than in vivo the rod-shaped microcompartments are also about one order of magnitude larger (10 x 30 &#181;m) than a rod-shaped E. coli cell.
Overall, this protocol allows for the precise control of all conditions including protein concentration, buffer composition and membrane properties. The use of 3D structured supports enables the reaction to be studied under spatial confinement, mimicking in vivo behavior without the need for complex microfluidics equipment.

Spatiotemporal patterns are essential in nature, regulating complex tasks both on the multicellular and cellular level, from morphogenesis to regulated cell division1,2. Reaction-diffusion systems play an important role in establishing these patterns, but are still not well understood. A prime example of a reaction-diffusion system and the best characterized biological system so far is the Escherichia coli MinCDE system3,4,5,6,7. The MinCDE system oscillates from cell pole to cell pole in E. coli to determine the middle of the cell as the future division site. This system is based on the ATPase MinD, the&#160;ATPase activating protein MinE, and the membrane as a spatial reaction matrix8. MinC is not part of the pattern formation mechanism, but is the actual functional agent: an inhibitor of the main divisome protein FtsZ5,6. MinC binds to MinD and therefore follows the oscillations, resulting in a time-averaged protein concentration gradient that is maximal at the cell poles and minimal at the cell middle, only allowing FtsZ to polymerize at midcell9,10. The MinCDE system is part of the larger family of Walker A ATPases that are key to the spatiotemporal organization in bacteria2, for positioning and transporting protein complexes11 and plasmids12 and for regulating cell division13 and chromosome segregation14. Hence, the MinCDE reaction-diffusion system not only represents an archetypal reaction-diffusion system, but has also attracted attention because of its relevance for the spatiotemporal organization in bacteria.
Detailed functional studies of the MinCDE system in vivo are complicated, as manipulation of proteins and gene deletion typically result in cell division defects. Furthermore, changing the membrane composition or the properties of the cytosol in vivo is very challenging15,16. Changes to the system and influencing factors are hard to interpret in the complex environment of the cell, even more so if it is disturbed in such an essential function as cell division. We and others have therefore turned to an in vitro reconstitution approach, reducing the system to its core components: MinD, MinE, ATP as an energy source, and the supported lipid bilayer as a reaction matrix6,17,18. This bottom-up approach allows to probe the mechanism of self-organization in detail without the complexity of a living cell. The proteins form traveling surface waves6 and other kinds of patterns17,19 under these conditions, albeit with a wavelength that is usually about a magnitude larger than in vivo. The use of an open chamber facilitates precise control over all aspects influencing pattern formation: protein concentrations6, protein properties20, membrane composition10, buffer composition, and ATP concentration6, as well as addition of other factors such as crowding agents21 and other divisome proteins22. In comparison, the in vitro reconstitution of the MinCDE system in a flow-cell18,19,23 can be used to probe the influence of flow17,23, protein limiting conditions19, membrane composition19 and full 3D confinement18 on protein patterns, but renders an exact control of protein/component concentration and sequential component addition much more complicated.
Using this open chamber, we also patterned the support of the planar lipid bilayers by which one can probe how geometrical boundaries influence pattern formation21, a phenomenon that has recently also been investigated in vivo using bacteria molded into microstructures7. We also employed this assay to investigate how defined mutations in MinE affect pattern formation of the system20. Furthermore, the same basic assay format has been employed to investigate how pattern formation can be controlled by light, introducing an azobenzene-crosslinked MinE peptide into the assay, and imaging with TIRF microscopy24.
We found that, in order to replicate the MinDE pattern formation observed in vivo in an in vitro system, confinement was key. Using rod-shaped microcompartments, with dimensions adjusted to the larger wavelength of MinDE in vitro (10 x 30 &#181;m), clad with a supported lipid bilayer allowed the reconstitution of MinDE pole-to-pole oscillations and protein gradient formation10,25. In this assay, the supported lipid bilayers are deposited on a patterned PDMS substrate that contains several hundred replicas of rod-shaped microcompartments that remain open on the top. By this, the reaction can be set up in an open chamber, and subsequently the buffer is lowered to the rim of the microcompartments, thereby confining the reaction&#160;to a small volume. Even though these compartments have an air-buffer interface on one side and hence do not represent a full 3D confinement by membrane, the protein dynamics mimicked in vivo oscillations10,25. Compared to full 3D confinement, which shows very similar results18, the open microstructures assay is relatively simple and easy to handle and can also be performed by laboratories that are not equipped with specialized microfluidics equipment and clean-room facilities.
Here, we present an experimental protocol for reconstituting MinCDE pattern formation on supported lipid bilayers in vitro using an open chamber that allows for control of all components and easy access by optical microscopy and, with minor modifications, is also adaptable for surface-probe techniques26. Next to planar supported lipid bilayers, we also show how protein confinement can be obtained using simple patterned supported lipid bilayers on rod-shaped PDMS microstructures. These assays, although optimized for the MinCDE system, can also be transferred to other protein systems that interact in a similar way with the membrane, such as FtsZ27 or a minimal actin cortex28.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850375</td><td></td></tr><tr><td>DOPG</td><td>Avanti Polar Lipids</td><td>840475</td><td></td></tr><tr><td>E.coli polar lipid extract</td><td>Avanti Polar Lipids</td><td>100600</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt trihydrate</td><td>Roche</td><td></td><td></td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dihydrate</td><td>Sigma</td><td>A5285-1G</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>27810.295</td><td></td></tr><tr><td>Potassium chloride</td><td>Roth</td><td>6781.1</td><td></td></tr><tr><td>Tris-base</td><td>Sigma Aldrich</td><td>T1503-1kg</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Roth</td><td>9277.1</td><td></td></tr><tr><td>TCEP-HCl</td><td>Termo Fisher Scientific</td><td>20491</td><td></td></tr><tr><td>Ethylene Diamine Tetraacetate</td><td>Merck Millipore</td><td>1.08418.1000</td><td></td></tr><tr><td>Sulfuric Acid 98%</td><td>Applichem</td><td>173163.1611</td><td></td></tr><tr><td>Hydrogen Peroxide 50%</td><td>Applichem</td><td>147064.1211</td><td></td></tr><tr><td>HEPES</td><td>Biomol</td><td>05288.1</td><td></td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>Merck</td><td>102950</td><td>Uvasol</td></tr><tr><td>Glycerol 86%</td><td>Roth</td><td>4043.1</td><td></td></tr><tr><td>TB medium</td><td></td><td></td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Roth</td><td>2316.x</td><td></td></tr><tr><td>Atto-655-DOPE</td><td>Atto Tec</td><td>AD 655-161</td><td></td></tr><tr><td>Ni-NTA agarose</td><td>Qiagen</td><td>30210</td><td></td></tr><tr><td>PDMS base</td><td>Dow Corning Corporation</td><td></td><td>SYLGARD 184</td></tr><tr><td>PDMS crosslinker</td><td>Dow Corning Corporation</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>UV Glue</td><td>Norland Products</td><td>6801</td><td>#68 and #63 both work well</td></tr><tr><td>Coverslips #1.5 24x24 mm</td><td>Menzel Gl&amp;auml;ser</td><td></td><td></td></tr><tr><td>Coverslips #1 24x24 mm</td><td>Menzel Gl&amp;auml;ser</td><td></td><td>used only for PDMS microstructures</td></tr><tr><td>0.5 ml reaction tube</td><td>Eppendorf</td><td>&amp;nbsp;0030123301</td><td></td></tr><tr><td>culture flask 2L</td><td>Corning</td><td>e.g. 734-1905</td><td></td></tr><tr><td>His-Trap HP</td><td>GE Healthcare Life Sciences</td><td></td><td></td></tr><tr><td>Gelfiltration column: HiLoad Superdex 75 PG or 200 PG</td><td>GE Healthcare Life Sciences</td><td></td><td></td></tr><tr><td>Econo-Pac 10DG desalting column prepacked column</td><td>Biorad</td><td>7322010</td><td></td></tr><tr><td>dialysis device: Slide-A-Lyzer Dialysis Cassettes, 3.5K MWCO, 0.1 - 0.5 mL or 0.5-3 mL</td><td>Termo Fisher Scientific</td><td>66333 or 66330</td><td></td></tr><tr><td>razor blade</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ultrapure water: Milli-Q Type 1 Ultrapure Water Systems</td><td>Merck</td><td></td><td></td></tr><tr><td>automated protein purification system: &amp;Auml;kta Pure</td><td>GE Healthcare Life Sciences</td><td></td><td></td></tr><tr><td>bath sonicator</td><td>Branson</td><td></td><td>e.g. Model 1510</td></tr><tr><td>ARE-250 mixer</td><td>Thinky Corporation</td><td></td><td></td></tr><tr><td>Plasma cleaner Zepto</td><td>Diener electronic</td><td></td><td>use oxygen as process gas</td></tr><tr><td>positive displacement pipettes</td><td>Brand</td><td></td><td>Transferpettor models with glass tips</td></tr><tr><td>LSM780 confocal laser scanning microscope</td><td>Zeiss</td><td></td><td>Fitted with Zeiss C-Apochromat 40X/1.20 water-immersion objective</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET28a-His-MinD_MinE</td><td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td><td></td><td>plasmid encoding His-MinD and non-tagged MinE to improve yield</td></tr><tr><td>pET28a-His-MinE</td><td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td><td></td><td>plasmid encoding His-MinE</td></tr><tr><td>pET28a-His-EGFP-MinD</td><td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td><td></td><td>plasmid encoding His-EGFP-MinD</td></tr><tr><td>pET28a-His-mRuby3-MinD</td><td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td><td></td><td>plasmid encoding His-mRuby3-MinD</td></tr><tr><td>pET28a-His- MinC</td><td>Department of Cellular and Molecular Biophysics, MPI of Biochemistry, Prof. Schwille</td><td></td><td>plasmid encoding His-MinC</td></tr></tbody></table>

in vitro reconstitution of self-organizing protein patterns on supported lipid bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

Protein purification following our protocol should yield Min proteins of adequate purity. As a reference, Figure 1 provides an SDS-PAGE image of MinD, fluorescently labelled MinD, MinE, and MinC. The individual steps of the procedure to perform a MinDE self-organization assay on non-patterned supported lipid bilayers are described in Figure 2. Using this protocol, regular MinDE traveling surface waves can be observed throughout the chamber (Figure 3). The wavelength can vary slightly within the chamber, but in general patterns look similar. The edges of the chamber should not be used for quantitative comparisons, as membranes that form on the UV glue seem to have different properties than on the glass surface (see Figure 3C). The traveling surface waves can be analyzed by plotting the intensity along the propagation direction (Figure 3B). While MinD fluorescence plateaus rather fast from the leading edge of the wave and then sharply decreases at the trailing edge, MinE fluorescence increases almost linearly from the start of the MinD wave and reaches its maximum after MinD at the trailing edge, where it falls off markedly6.
Next to protein quality, the quality of the supported lipid bilayers is most critical for a regular self-organization of MinCDE. On the one hand if the membrane is washed too excessively or the underlying surface has been cleaned and thus charged too strongly, holes can form in the membrane (Figure 6A, top). On the other hand if the membrane is not washed properly or the underlying surface is not cleaned/hydrophilized, vesicles will stick to the membrane or the membrane fluidity will be compromised (Figure 6A, bottom). Even though not as apparent as when observing the membrane directly via labeled lipids, these problems can also be detected from the MinD fluorescence signal, as patterns are not regular and the fluorescence in the maxima is not homogenous but contains &#34;holes&#34; or bright spots as shown in the middle panel of Figure 6A.
For the MinDE self-organization in rod-shaped PDMS microstructures the procedure is summarized in Figure 4. Several protocol steps do not need to be repeated, as proteins and lipids can be reused. Like on non-patterned substrates, the substrate is cleaned and hydrophilized (by plasma-cleaning), a supported lipid bilayer is formed on the PDMS and the self-organization assay is set up in a volume of 200 &#181;L. To check that a proper membrane has formed and MinDE self-organizes on the membrane, the chambers are imaged. When a proper membrane has been formed, MinDE forms regular traveling surface waves on the surface of the PDMS between the individual microstructures and also self-organizes at the bottom of the microstructures as the waves can freely move over the entire membrane-covered surface (Figure 5A). After buffer removal, the surface between the compartments should not show any propagating MinDE patterns (Figure 5B), as it should be entirely dry. If MinDE patterns are still moving, more buffer needs to be removed. The proteins are now confined in the rod-shaped microcompartments by the membrane-clad PDMS and by air on the upper interface (Figure 5C), in which they will self-organize. Under these conditions the two proteins can perform pole-to-pole oscillations as shown in Figure 5D. As a fraction of MinD and MinE is always membrane-bound, also during buffer removal, the concentrations after buffer removal are not comparable to input concentrations. Due to this effect the concentrations also vary between individual microstructures on the same coverslip as they depend on the position of the patterns before buffer removal. Silicon wafer production or PDMS molding from the silicon wafer can result in incomplete microstructures that cannot be used for analysis (Figure 6B). Furthermore, due to the buffer removal microstructures might dry out during the process, and hence, should be excluded from further analysis&#160;(Figure 6B). As a result only a fraction of the microstructures in one chamber shows the desired pole-to-pole oscillations. To analyze protein dynamics in the microstructures, a kymograph can be obtained by drawing a selection over the entire structure (Figure 5E). When MinCDE oscillate from pole-to-pole, MinC and MinD will show a time-averaged concentration gradient with maximum&#160;concentration at the compartment poles and minimal concentration in the middle of the compartment&#160; (Figure 5F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58139/58139fig1.jpg" /> 
Figure 1: SDS-PAGE showing the final products of protein purifications. His-MinD (33.3 kDa), His-eGFP-MinD (60.1 kDa), His-mRuby3-MinD (59.9 kDa), His-MinE (13.9 kDa) and His-MinC (28.3 kDa) are shown in order. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/58139fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58139/58139fig2.jpg" /> 
Figure 2: Process flow diagram showing the individual steps and timing of the protocol for a self-organization on non-patterned supported lipid bilayers (Steps 1-6). Dashed boxes indicate that one of these two options can be used for cleaning. Arrows marked by circles indicate where the protocol can be paused and resumed later. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/58139fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58139/58139fig3.jpg" /> 
Figure 3: Imaging of MinDE assay by confocal microscopy. A) Regular Min spiral, from which wave propagation speed, intensity plot and speed measurements can be obtained. Concentrations used: 0.6 &#181;M MinD (30% eGFP-MinD), 1.8 &#181;M His-MinE (30% His-MinE-Alexa647) B) Example normalized intensity plot for the region marked in A. C) Overview of entire assay chamber (scale bar: 1 mm, same protein concentrations as above). Spirals turning either direction as well as target patterns can be observed. The magnified region shows how wave patterns differ on the UV-glue. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/58139fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58139/58139fig4.jpg" /> 
Figure 4: Process flow diagram showing the individual steps and timing of the protocol for&#160;self-organization in rod-shaped microstructures (Steps 1-5, 7, 8). Grey boxes indicate steps where products can be reused from the protocol on non-patterned supported lipid bilayers. Arrows marked by circles indicate where the protocol can be paused and resumed later. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/58139fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58139/58139fig5.jpg" /> 
Figure 5: Representative results for MinDE pattern formation in rod-shaped PDMS microcompartments. A) MinDE self-organize on the surface of the PDMS forming traveling surface waves (1 &#181;M MinD (30% EGFP-MinD), 2 &#181;M MinE and 2.5 mM ATP). B) After the buffer is lowered to the height of the microstructures, the protein self-organization stops on the planar surface between the microcompartments. C) Schematic of one rod-shaped microcompartment. D) Representative images of MinDE pole-to-pole oscillations after buffer removal. E) Kymograph of the oscillations along the highlighted line shown in D). F) Image and profile of the average fluorescence intensity of the time-series shown in D) clearly showing the protein gradient that is maximal at microcompartment poles and minimal at compartment middle. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/58139fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58139/58139fig6.jpg" /> 
Figure 6: Examples of negative experimental outcomes. A) Over-washed membranes accumulate holes, while suboptimal vesicle preparations and lipid compositions lead to sticking vesicles. The two center panels show a combination of both problems and how they become visible when observing Min oscillations. Membranes were labelled with 0.05% Atto655-DOPE. (scale bars: 50 &#181;m) B) Top panel: Dried out microcompartments can be caused by too much buffer removal or when the buffer evaporates over time. Bottom panel: Incomplete compartments can be formed during wafer production or PDMS molding. (scale bars: 30 &#181;m) <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/58139fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1:&#160;Plasmid map for His-MinD.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/01_pET28a-MinD-MinE.gb " target="_blank">Please click here to download this file.</a>
Supplementary File 2:&#160;Plasmid map for His-EGFP-MinD.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/02_pET28a-EGFP-MinD.gb" target="_blank">Please click here to download this file.</a>
Supplementary File 3:&#160;Plasmid map for His-mRuby3-MinD.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/03_pET28a-mRuby3-MinD.gb" target="_blank">Please click here to download this file.</a>
Supplementary File 4:&#160;Plasmid map for His-MinE.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/04_pET28a-MinE.gb" target="_blank">Please click here to download this file.</a>
Supplementary File 5:&#160;Plasmid map for His-MinC.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/05_pET28a-MinC.gb" target="_blank">Please click here to download this file.</a>
Supplementary File 6:&#160;CAD file for silicon wafer of rod-shaped microcompartments.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58139/JoVe_wafer_rod-like-structures_3x3.dwg" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers at JoVE.com

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

in-vitro-reconstitution-self-organizing-protein-patterns-on-supported

Universidad de Cartagena UDEC

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JoVE Journal

Biochemistry

12.1K Views.  Max Planck Institute of Biochemistry. We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Video: In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioengineering

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Light-Controlled Reversible Protein Patterning on Lipid Membranes

Dynamic Light-Induced Protein Patterns at Model Membranes

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

Author Spotlight: Photo Switchable Protein Recruitment for Reversible Patterning in Artificial Cellular Systems

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

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Chapters in this video

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Dynamic Light-Induced Protein Patterns at Model Membranes

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers