Name: in vitro reconstitution of self-organizing protein patterns on supported lipid bilayers
Uploaded: 2018-07-28T14:45:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers Video at JoVE.com

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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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+MinDE+complex+acts+as+a+toggle+switch+that+drives+Min+oscillation+coupled+to+cytoplasmic+depletion+of+MinD.">Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD.</a> Proc Natl Acad Sci U S A. 113 (11), E1479-E1488 (2016).</li><li>Kretschmer, S., Zieske, K., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+modulation+of+reconstituted+Min+protein+patterns+and+gradients+by+defined+mutations+in+MinE's+membrane+targeting+sequence.">Large-scale modulation of reconstituted Min protein patterns and gradients by defined mutations in MinE's membrane targeting sequence.</a> PLoS One. 12 (6), e0179582 (2017).</li><li>Schweizer, J., Loose, M., Bonny, M., Kruse, K., Monch, I., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+sensing+by+self-organized+protein+patterns.">Geometry sensing by self-organized protein patterns.</a> Proc Natl Acad Sci. 109 (38), 15283-15288 (2012).</li><li>Martos, A., Raso, A., Jim&#233;nez, M., Petr&#225;&#353;ek, Z., Rivas, G., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FtsZ+polymers+tethered+to+the+membrane+by+ZipA+are+susceptible+to+spatial+regulation+by+min+waves.">FtsZ polymers tethered to the membrane by ZipA are susceptible to spatial regulation by min waves.</a> Biophys J. 108 (9), 2371-2383 (2015).</li><li>Vecchiarelli, A. G., Li, M., Mizuuchi, M., Mizuuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+affinities+of+MinD+and+MinE+to+anionic+phospholipid+influence+Min+patterning+dynamics+in+vitro.">Differential affinities of MinD and MinE to anionic phospholipid influence Min patterning dynamics in vitro.</a> Mol Microbiol. 93 (3), 453-463 (2014).</li><li>Glock, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+a+biological+reaction-diffusion+system.">Optical control of a biological reaction-diffusion system.</a> Angew Chemie Int Ed. 57 (9), 2362-2366 (2018).</li><li>Zieske, K., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+pole-to-pole+oscillations+of+min+proteins+in+microengineered+polydimethylsiloxane+compartments.">Reconstitution of pole-to-pole oscillations of min proteins in microengineered polydimethylsiloxane compartments.</a> Angew Chemie Int Ed. 52 (1), 459-462 (2013).</li><li>Miyagi, A., Ramm, B., Schwille, P., Scheuring, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Speed+Atomic+Force+Microscopy+Reveals+the+Inner+Workings+of+the+MinDE+Protein+Oscillator.">High-Speed Atomic Force Microscopy Reveals the Inner Workings of the MinDE Protein Oscillator.</a> Nano Lett. 18 (1), 288-296 (2017).</li><li>Ramirez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treadmilling+analysis+reveals+new+insights+into+dynamic+FtsZ+ring+architecture.">Treadmilling analysis reveals new insights into dynamic FtsZ ring architecture.</a> PLoS Biol. 16 (5), e2004845 (2018).</li><li>Vogel, S. K., Petrasek, Z., Heinemann, F., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+motors+fragment+and+compact+membrane-bound+actin+filaments.">Myosin motors fragment and compact membrane-bound actin filaments.</a> Elife. 2, e00116 (2013).</li><li>Zieske, K., Schweizer, J., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+topology+assisted+alignment+of+Min+protein+waves.">Surface topology assisted alignment of Min protein waves.</a> FEBS Lett. 588 (15), 2545-2549 (2014).</li><li>Loose, M., Fischer-Friedrich, E., Herold, C., Kruse, K., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Min+protein+patterns+emerge+from+rapid+rebinding+and+membrane+interaction+of+MinE.">Min protein patterns emerge from rapid rebinding and membrane interaction of MinE.</a> Nat Struct Mol Biol. 18 (5), 577-583 (2011).</li><li>Zieske, K., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+geometry-modulated+protein+patterns+in+membrane+compartments.">Reconstituting geometry-modulated protein patterns in membrane compartments.</a> Methods Cell Biol. 128, 149-163 (2015).</li><li>Gruenberger, A., Probst, C., Heyer, A., Wiechert, W., Frunzke, J., Kohlheyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+picoliter+bioreactor+for+microbial+single-cell+analysis:+fabrication,+system+setup,+and+operation.">Microfluidic picoliter bioreactor for microbial single-cell analysis: fabrication, system setup, and operation.</a> J Vis Exp. (82), e50560 (2013).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Loose, M., Kruse, K., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+self-organization:+Lessons+from+the+min+system.">Protein self-organization: Lessons from the min system.</a> Annu Rev Biophys. 40, 315-336 (2011).</li><li>Arumugam, S., Petra&#353;ek, Z., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MinCDE+exploits+the+dynamic+nature+of+FtsZ+filaments+for+its+spatial+regulation.">MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation.</a> Proc Natl Acad Sci U S A. 111 (13), E1192-E1200 (2014).</li></ol>

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 colspan="4">Reagents</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&#8242;-triphosphate disodium salt trihydrate</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-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 &#946;-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 colspan="4">Materials</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&#228;ser</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips #1 24x24 mm</td>
			<td>Menzel Gl&#228;ser</td>
			<td></td>
			<td>used only for PDMS microstructures</td>
		</tr>
		<tr>
			<td>0.5 ml reaction tube</td>
			<td>Eppendorf</td>
			<td>&#160;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 colspan="4">Instruments</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: &#196;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 colspan="4">Plasmids</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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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 Video at JoVE.com

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers Video (Video) | JoVE

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.

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