This work was funded by the European Research Council ERC Starting Grant ARTIST (# 757593 S.V.W.). DDI thanks the Alexander von Humboldt Foundation for a postdoctoral fellowship.

1. Experimental preparation
<ol>
	<li>Express and purify biotinylated-disiLID (b-disiLID) and mOrange-Nano (see Table of Materials) by following previously reported procedures7,8.</li>
	<li>Prepare lipid mixtures in glass vials with the selected lipid composition and concentrations. First, dissolve lipids in chloroform to obtain a final lipid solution with 1 mg/mL concentration.
	<ol>
		<li>Mix lipids in order to obtain a composition of 94.9 mol% 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 5 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineN-(cap biotinyl) sodium salt (DOPE-biotin) and 0.1 mol% 1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-Tetramethylindodicarbocyanine (DiD) (see Table of Materials). 
		NOTE: The lipid mixture can be adjusted with varying ratios and DOPE-biotin concentration and/or different membrane dyes. The recommended DOPE-biotin concentration (5 mol%) allows the formation of a high-density Streptavidin (SAv) layer in the following steps.</li>
	</ol>
	</li>
	<li>Prepare small unilamellar vesicles (SUVs) following previously reported methods7,8,12. For this step, it is recommended to prepare SUVs with a diameter &#8804;100 nm.</li>
	<li>For this study, sonication method is employed. First, evaporate the chloroform solution in the glass vial with a nitrogen stream while rotating the vials in order to form a thin lipid film. Next, remove the residual chloroform for at least 1 h under vacuum.
	<ol>
		<li>Rehydrate the dried film in ultrapure water with a final concentration of 1 mg/mL lipids by vortexing. Finally, sonicate the obtained solution for 10 min until the opaque solution turns clear. 
		NOTE: Store the lipid mixture in a microcentrifuge tube in the refrigerator for a maximum of 2 weeks. Different SUV preparation methods (e.g., extrusion method) can also be used as long as the size of the final SUVs is &#8804; 100 nm.</li>
	</ol>
	</li>
</ol>
2. mOrange-Nano recruitment to disiLID functionalized SLBs
<ol>
	<li>Add 150 &#181;L of 2 M NaOH in each well of the &#181;-slide 18 well glass-bottom chamber (see Table of Materials) and incubate for 1 h at room temperature. Subsequently, remove the NaOH and wash the wells 3-5 times first with 150 &#181;L ultrapure&#160;water, and then 3 times with 150 &#181;L buffer (10 mM Tris pH 7.4, 100 mM NaCl) containing 10 mM CaCl2.</li>
	<li>Add 15 &#181;L of freshly prepared SUVs (stock concentration 1 mg/mL in water) into the wells containing 150 &#181;L buffer with 10 mM CaCl2 in order to have approximately a factor 10 dilution of SUVs in the buffer. Let the SUVs incubate for 30 min at room temperature. After incubation time, biotinylated-SLBs will be formed.</li>
	<li>Wash the SLBs at least 7 times with buffer (10 mM Tris pH 7.4, 100 mM NaCl) without CaCl2 by first removing the solution and subsequently adding fresh buffer in each step. It is recommended to use 80 &#181;L of buffer for each washing step. 
	NOTE: An optimal washing of the SLBs is obtained by pipetting fresh solution up and down several times without touching the surface. Pipetting of the solution in the wells containing the newly formed SLBs should be gentle in order to reduce the formation of small air bubbles that would damage the formed SLBs. From this moment onwards, wells should contain a sufficient volume of buffer in order to prevent the SLBs from drying out.</li>
	<li>For the further functionalization of the biotinylated SLBs with SAv, add a solution of SAv to a final concentration of 250 nM and incubate for 30 min at room temperature. Subsequently, remove the excess of SAv by washing it with a buffer (10 mM Tris pH 7.4, 100 mM NaCl) at least 5 times.</li>
	<li>From this moment onwards, keep the samples under a protective red light in order to avoid unwanted photoactivation of photoswitchable proteins. Add b-disiILD (see Table of Materials) to a final concentration of 1 &#181;M in the well. After 30 min incubation at room temperature, remove the excess protein by washing with buffer at least 5 times.</li>
	<li>Add mOrange-Nano (see Table of Materials) to a final concentration of 200 nM and keep the sample in the dark by covering it with aluminum foil.</li>
	<li>Place the &#181;-slide under the fluorescence microscope and adjust the imaging settings. Set the 552 nm laser for excitation of mOrange-Nano. Adjust the emission range to optimize the mOrange signal. The photoactivation of disiLID is achieved with a 488 nm laser, using light pulses with 2.58 s intervals.</li>
</ol>
3. Preparation of GUVs
<ol>
	<li>Prepare a 5% (w/v) solution of polyvinyl alcohol (PVA, see Table of Materials) (MW: 145 000 g/ mol) with 100 mM sucrose in ultrapure water, mixing overnight at 80 &#176;C at 400 rpm.</li>
	<li>Prepare a lipid solution in chloroform with a desired composition (final concentration 10 mg/mL). For this method, it is recommended a composition consisting of 10 mg/mL POPC, 10 mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (POPG), 2 mol% DOPE-biotin and 1 mol% DiD (see Table of Materials).</li>
	<li>Prepare GUVs with the hydration technique7,8. First, spread 40 &#181;L of the prepared PVA solution as a homogeneous thin layer on top of a 60 mm x 24 mm glass slide, preferably with a pipette tip. Next, dry the thin layer at 50 &#176;C for 30 min.</li>
	<li>Spread 5 &#181;L of the lipid solution with a needle on the PVA layer and let dry at 30 &#176;C for 1 h.</li>
	<li>Assemble a chamber on the functionalized glass slide using a spacer (~40 mm &#215; 24 mm &#215; 2 mm, see Table of Materials) and a second glass slide.</li>
	<li>Add 1 mL of rehydration buffer (10 mM Tris pH 7.4, 100 mM NaCl) into the chamber for 1 h at room temperature to form GUVs. After 1 h, invert the chamber and gently tap on the glass surfaces with a pipette tip.</li>
	<li>Carefully remove the glass slide on one side to open the built chamber and harvest the GUVs with a pipette.</li>
	<li>Place the solution in a plastic tube and let the GUVs settle for 2 h.</li>
</ol>
4. mOrange-Nano Recruitment to disiLID functionalized GUVs
<ol>
	<li>Add a solution of SAv to the freshly harvested GUVs and let sit for 30 min at room temperature. 
	NOTE: The following steps should be performed with protective red light in order to avoid photoactivation of disiLID.</li>
	<li>Add 1 &#181;M of b-disiLID to the GUVs solution and place the sample for 30 min in the dark, covering it with aluminum foil.</li>
	<li>Pretreat a &#181;-slide 18-well glass bottom chamber with 150 &#181;L BSA solution (3% w/v in water) for 10 min. Next, remove the BSA solution and wash the wells with 150 &#181;L ultrapure water 3 times.</li>
	<li>Add 145 &#181;L of 200 nM mOrange-Nano in buffer (10 mM Tris pH 7.4, 100 mM NaCl) to the well.</li>
	<li>Next, add 5 &#181;L of GUVs decorated with b-disiLID to the solution and wait ~15 min for the GUVs to settle.</li>
	<li>Place the &#181;-slide under the confocal microscope. Excite the sample at 552 nm to visualize mOrange (&#955;ex = 557 nm; &#955;em = 576 nm) fluorescence and at 638 nm to visualize DiD (&#955;ex = 644 nm; &#955;em = 665 nm) in the GUV membranes. The mOrange-Nano recruitment is triggered with blue light pulses (488 nm, intensity 1%) every 5.3 s in order to minimize undesired photobleaching effects. 
	NOTE: The excitation wavelengths can be adapted based on the type of microscope in use. Other common excitation wavelengths available for microscopes are also 532 nm or 561 nm and 633 nm, 647 nm, 639 nm, or 640 nm lasers.</li>
</ol>

Light-Controlled Reversible Protein Patterning on Lipid Membranes

<ol>
	<li>Kretschmer, S., 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=Pattern+formation+on+membranes+and+its+role+in+bacterial+cell+division.">Pattern formation on membranes and its role in bacterial cell division.</a> Curr Opin Cell Biol. 38, 52-59 (2016).</li><li>Yang, H. W., Collins, S. R., Meyer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+excitable+Cdc42+signals+steer+cells+during+chemotaxis.">Locally excitable Cdc42 signals steer cells during chemotaxis.</a> Nat Cell Biol. 18 (2), 191-201 (2016).</li><li>Caldwell, R. M., 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=Optochemical+control+of+protein+localization+and+activity+within+cell-like+compartments.">Optochemical control of protein localization and activity within cell-like compartments.</a> Biochem. 57 (18), 2590-2596 (2018).</li><li>Kennedy, M. 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=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nat Methods. 7 (12), 973-975 (2010).</li><li>Guntas, 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=Engineering+an+improved+light-induced+dimer+(iLID)+for+controlling+the+localization+and+activity+of+signaling+proteins.">Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.</a> Proc Natl Acad Sci. 112 (1), 112-117 (2015).</li><li>Levskaya, A., Weiner, O. D., Lim, W. A., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+cell+signalling+using+a+light-switchable+protein+interaction.">Spatiotemporal control of cell signalling using a light-switchable protein interaction.</a> Nature. 461 (7266), 997-1001 (2009).</li><li>Bartelt, S. M., 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=Dynamic+blue+light-switchable+protein+patterns+on+giant+unilamellar+vesicles.">Dynamic blue light-switchable protein patterns on giant unilamellar vesicles.</a> Chem Commun. 54 (8), 948-951 (2018).</li><li>Di Iorio, D., Bergmann, J., Higashi, S. L., Hoffmann, A., Wegner, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+disordered+tether+to+iLID+improves+photoswitchable+protein+patterning+on+model+membranes.">A disordered tether to iLID improves photoswitchable protein patterning on model membranes.</a> Chem. Commun. 59 (29), 4380-4383 (2023).</li><li>Hartzell, E. J., Terr, J., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+blue+light+inducible+Spytag+system+(BLISS).">Engineering a blue light inducible Spytag system (BLISS).</a> J Am Chem Soc. 143 (23), 8572-8577 (2021).</li><li>Xu, D., Bartelt, S. M., Rasoulinejad, S., Chen, F., Wegner, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+light+lithography:+a+general+strategy+to+create+active+protein+and+cell+micropatterns.">Green light lithography: a general strategy to create active protein and cell micropatterns.</a> Mater Horiz. 6 (6), 1222-1229 (2019).</li><li>Jia, H., 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=Light-induced+printing+of+protein+structures+on+membranes+in+vitro.">Light-induced printing of protein structures on membranes in vitro.</a> Nano Lett. 18 (11), 7133-7140 (2018).</li><li>Di Iorio, D., Verheijden, M. L., vander Vries, E., Jonkheijm, P., Huskens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weak+Multivalent+Binding+of+influenza+hemagglutinin+nanoparticles+at+a+sialoglycan-functionalized+supported+lipid+bilayer.">Weak Multivalent Binding of influenza hemagglutinin nanoparticles at a sialoglycan-functionalized supported lipid bilayer.</a> ACS Nano. 13 (3), 3413-3423 (2019).</li><li>Kasahara, M., Torii, M., Fujita, A., Tainaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FMN+binding+and+photochemical+properties+of+plant+putative+photoreceptors+containing+two+LOV+domains,+LOV/LOV+proteins.">FMN binding and photochemical properties of plant putative photoreceptors containing two LOV domains, LOV/LOV proteins.</a> J Biol Chem. 285 (45), 34765-34772 (2010).</li></ol>

The authors have nothing to disclose.

We have described a method for the localized recruitment of mOrange-Nano proteins on model membranes, such as supported lipid bilayer and giant unilamellar vesicles by using the photoswitchable protein disiLID8. Aspects that contribute to the quality of the pattern include the quality of the proteins as well as the good quality of SLBs and GUVs.
To ensure a good protein quality after expression and purification, it is important to first evaluate the photoswitchable properties of disiLID. To this purpose, the absorption of the FMN cofactor has to be measured in the dark and after blue light illumination. UV-Vis spectra of disiLID are expected to show the characteristic triple peak of the cofactor FMN in the dark, which significantly decreases upon blue light illumination and recovers in the dark13. This photoswitchable behavior is crucial to obtaining reproducible and reversible recruitment in the following steps. Working with protective red light and exposing disiLID to minimum external illumination during the preparation of the samples increases the performance of the experiments.
Another critical step, and perhaps the most crucial, is the formation of proper SLBs. Defects in the membranes and/or the formation of inhomogeneous SLBs (i.e., the presence of multilayers or patched SLBs) will affect the quality of the protein patterning. Therefore, for inexperienced users, it is recommended to reproduce the protocol by labeling the SUVs with some membrane dyes, such as DiD and DiO, in order to form fluorescently labeled SLBs. In this way, the properties and quality of SLBs can be well characterized with fluorescence microscopy. FRAP measurements represent a typical approach to assessing the quality of an SLB by evaluating the fluidity of the membranes. Alternatively, in the case of biotinylated SLBs such as the ones described in this protocol, fluorescently labeled SAv (e.g., Atto 488-SAv) can be used to visualize and assess the quality of SLBs.
The first part of the protocol describes the formation of patterns on SLBs. To ensure an optimal result, it is important to add mOrange-Nano onto the SLBs and let the sample incubate in the dark for 15 min. During the photoactivation, the selection of the ROI is not restricted to a specific size. However, laser intensity and exposure time need to be regulated in order to reduce undesired photobleaching of the fluorescent proteins.
This method is not restricted to biotinylated proteins, and other approaches can be used to anchor disiLID to SLBs. For example, His-tagged disiLID can be expressed and anchored onto Ni- NTA-containing SLBs. However, it is crucial to express Nano and disiLID with different tags in order to avoid replacement of the proteins on the SLBs. This method also allows the possibility to invert the order of the proteins, thus functionalizing SLBs with Nano and recruiting disiLID (or disiLID-fused proteins) upon blue light illumination.
For dynamic control of protein localization, the reversible localization of the protein to the selected region should be possible repeatedly. To achieve this, the concentration of Nano (200 nM) in the solution is a critical parameter to obtain high reversibility.
Another concern is the recruitment of Nano to the disiLID-functionalized GUV surface. As in the case of protein patterning on SLBs, this method can be extended to different membrane functionalization strategies. In this protocol, the entire GUV was illuminated with blue light to recruit mOrange-Nano on the entire GUV surface. However, the selection of small ROIs localized on the GUV membrane should lead to the precise localization of proteins in a more restricted area.
This method presents only a limitation related to the choice of fluorophore employed for imaging of the Nano recruitment at the SUVs&#39; or GUVs&#39; membrane. In particular, fluorophores with an excitation spectrum in the blue-light range must be avoided, as their usage will interfere with the photoactivation of (dis)iLID. Therefore, the choice of fluorophores in the green or red-light range (e.g., mOrange or Cy5) is recommended for this type of experiment.
The disiLID design offers a simple and adaptable way to improve local protein recruitment to membranes and broadens the dynamic range of iLID and Nano from optogenetics4. These methods focus on the recruitment of Nano onto mimic membranes such as lipid bilayers and GUVs. Nevertheless, this approach is extendable to the numerous optogenetic tools in cells where (dis)iLID or Nano are linked to a membrane.

The formation of protein patterns on cell membranes within subcellular regions gives rise to numerous biological processes, including migration, division, and localized cell-to-cell communication1,2. These protein patterns are regulated in space and time, and are highly dynamic. Replicating such protein patterns in synthetic cells is essential for mimicking cellular processes that arise from them and for gaining a better understanding of how such regulation works at a molecular level. Analogous to what is observed for membranes in living cells, methods for generating protein patterns on artificial membranes must capture their dynamics and provide precise spatiotemporal control.
Among various stimuli, light stands out for providing the highest spatiotemporal control and several additional advantages3. Through regulation with light, it is straightforward to illuminate a desired area at any desired time with unmatched precision. In addition, light provides high tunability as both the light intensity and the pulse durations can be adjusted. Furthermore, visible light is harmless to biomolecules, including proteins, and it is possible to even address multiple functionalities with different wavelengths. Hence, light-responsive approaches based on visible light emerge as promising avenues for the controlled and biorthogonal regulation of protein patterns in space and time4,5,6. Utilizing photoswitchable protein pairs from optogenetics, acting as light-inducible dimerizers, provides a straightforward method for recruiting&#160;specific proteins to membranes. In particular, protein patterns have successfully been formed on artificial membranes using the blue light-triggered interaction between iLID (improved light-inducible dimer, based on the photoswitchable LOV2 domain from&#160;Avena sativa) and Nano (wild-type SspB)7,8, the blue light-inducible SpyTag system (BLISS)9, the green light-responsive&#160;protein tetramer CarH10 and the red light-inducible interaction between PhyB and PIF611.
It has been demonstrated that the photoswitchable interaction between iLID&#160;and Nano5 can be used to photo-pattern proteins on model membranes using blue light7. The iLID/Nano interaction is reversible in the dark, highly specific, and operates under physiological conditions. Anchoring iLID onto lipid membrane models, such as giant unilamellar vesicles (GUVs) or supported lipid bilayers (SLBs), enables light-regulated recruitment of Nano to these membranes, which is reversible in the dark. Notably, we observed that introducing a disordered domain to the N-terminus of iLID (resulting in a protein named disiLID) as a tether to a model lipid membrane enhances Nano recruitment efficiency and reversion dynamics8.
By employing the disiLID/Nano interaction, we have developed a method for generating high contrast patterns of Nano-fused proteins of interest (POI) on SLBs and the external membranes of GUVs. This method allows for the creation of protein patterns with remarkable spatial and temporal resolution and high reversibility within minutes. The detailed protocol outlines the process for locally recruiting proteins onto artificial membranes. Specifically, this is achieved by immobilizing a biotinylated version of disiLID on SLBs and GUVs through the biotin-streptavidin (SAv) interaction. Subsequently, fluorescently labeled Nano (mOrange-Nano) is recruited to these disiLID functionalized membranes under blue light illumination. Our experimental protocol offers a straightforward and adaptable approach for achieving localized protein recruitment to membranes. Importantly, this methodology is not confined to the reported SLB and GUV interfaces or mOrange-Nano; it can be extended to other disiLID-functionalized materials and proteins fused to Nano.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Slide 18 Well</td>
			<td>Ibidi</td>
			<td>81817</td>
			<td>For SLB preparation</td>
		</tr>
		<tr>
			<td>25 &#181;L Microliter Syringe&#160;</td>
			<td>Hamilton</td>
			<td>Model 702 N</td>
			<td>For the preparation of lipid mixture and spreading the lipid solution on the PVA layer</td>
		</tr>
		<tr>
			<td>Biotinyl Cap PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)) (sodium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>870273C</td>
			<td>For SUVs preparation</td>
		</tr>
		<tr>
			<td>CaCl2 (Calcium chloride)</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>For SLB formation</td>
		</tr>
		<tr>
			<td>Cover Slips 24 mm x 60 mm</td>
			<td>Engelbrecht</td>
			<td>K12460</td>
			<td>For GUVs formation</td>
		</tr>
		<tr>
			<td>DiD (1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;- 
			Tetramethylindodicarbocyanine)</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>D7757</td>
			<td>Membrane dye</td>
		</tr>
		<tr>
			<td>disiLID</td>
			<td></td>
			<td></td>
			<td>Sequence: MGGSGLNDIFEAQKIEWHEGGSH 
			HHHHHGSMAATELRGVVGPGPAA 
			IAALGGGGAGPPVGGGGGRGDA 
			GPGSGAASGTVVAAAAGGPGPG 
			AGGVAAAGPPAPPTGGSGGSGA 
			GGSGSAGEFLATTLERIEKNFVIT 
			DPRLPDNPIIFASDSFLQLTEYSR 
			EEILGRNCRFLQGPETDRATVRK 
			IRDAIDNQTEVTVQLINYTKSGKK 
			FWNVFHLQPMRDYKGDVQYFIG 
			VQLDGTERLHGAAEREAVMLIKK 
			TAFQIAEAANDENYF</td>
		</tr>
		<tr>
			<td>DOPC (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine)</td>
			<td>Avanti Polar Lipids</td>
			<td>850375P</td>
			<td>For SUVs lipid composition</td>
		</tr>
		<tr>
			<td>Eppendorf Protein LoBind 
			microcentrifuge tubes</td>
			<td>Merk</td>
			<td>EP0030108116-100EA</td>
			<td>For collecting freshly made GUVs</td>
		</tr>
		<tr>
			<td>mOrange-Nano</td>
			<td></td>
			<td></td>
			<td>Sequence: MRGSHHHHHHGSKIEEGKLVI 
			WINGDKGYNGLAEVGKKFEKDT 
			GIKVTVEHPDKLEEKFPQVAATG 
			DGPDIIFWAHDRFGGYAQSGLLA 
			EITPDKAFQDKLYPFTWDAVRYN 
			GKLIAYPIAVEALSLIYNKDLLPNP 
			PKTWEEIPALDKELKAKGKSALM 
			FNLQEPYFTWPLIAADGGYAFKY 
			ENGKYDIKDVGVDNAGAKAGLTF 
			LVDLIKNKHMNADTDYSIAEAAFN 
			KGETAMTINGPWAWSNIDTSKVN 
			YGVTVLPTFKGQPSKPFVGVLSA 
			GINAASPNKELAKEFLENYLLTDE 
			GLEAVNKDKPLGAVALKSYEEELA 
			KDPRIAATMENAQKGEIMPNIPQM 
			SAFWYAVRTAVINAASGRQTVDEA 
			LKDAQTNSSSNNNNNNNNNNLGI 
			EGTTENLYFQGSVSKGEENNMAI 
			IKEFMRFKVRMEGSVNGHEFEIE 
			GEGEGRPYEGFQTAKLKVTKGG 
			PLPFAWDILSPQFTYGSKAYVKH 
			PADIPDYFKLSFPEGFKWERVMN 
			FEDGGVVTVTQDSSLQDGEFIYK 
			VKLRGTNFPSDGPVMQKKTMG 
			WEASSERMYPEDGALKGEIKMR 
			LKLKDGGHYTSEVKTTYKAKKPV 
			QLPGAYIVGIKLDITSHNEDYTIVE 
			QYERAEGRHSTGGMDELYKGG 
			SGTSSPKRPKLLREYYDWLVDN 
			SFTPYLVVDATYLGVNVPVEYVK 
			DGQIVLNLSASATGNLQLTNDFIQ 
			FNARFKGVSRELYIPMGAALAIYA 
			RENGDGVMFEPEEIYDELNIG</td>
		</tr>
		<tr>
			<td>NaCl (Sodium chloride)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>For buffer</td>
		</tr>
		<tr>
			<td>NaOH (Sodium hydroxide)</td>
			<td>Sigma-Aldrich</td>
			<td>1064980500</td>
			<td>For surface activation in SLB formation</td>
		</tr>
		<tr>
			<td>POPC (1-Palmitoyl-2- 
			oleoylphosphatidylcholine)</td>
			<td>Avanti Polar Lipids</td>
			<td>850457C</td>
			<td>For GUVs lipid composition</td>
		</tr>
		<tr>
			<td>POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-racglycerol))</td>
			<td>Avanti Polar Lipids</td>
			<td>840457C</td>
			<td>For GUVs lipid composition</td>
		</tr>
		<tr>
			<td>PVA (Polyvinyl alcohol) fully hydrolyzed</td>
			<td>Sigma-Aldrich</td>
			<td>8148940101</td>
			<td>For GUVs formation</td>
		</tr>
		<tr>
			<td>SP8 confocal laser scanning microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>TermoFisher</td>
			<td>434301</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>84097</td>
			<td>For GUVs formation</td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>10812846001</td>
			<td>For buffer</td>
		</tr>
	</tbody>
</table>

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.

The described procedures allow the formation of SLBs to recruit mOrange-Nano on the synthetic membranes. The formation of defined mOrange-Nano patterned on the SLBs functionalized with b-disiLID is shown in Figure 1A. As a squared (24 &#181;m &#215; 24 &#181;m) region of interest (ROI) on the SLB is illuminated with 488 nm blue light, a rapid increase of fluorescence signal is observed in the mOrange channel (shown in red) in the ROI within 200 s. The pattern shows very defined and sharp edges (Figure 1B), indicating high spatial control over the photoactivated area. The interaction is fast and fully reversible as the illumination with blue light is interrupted. This method also allows the formation of patterns over several illumination cycles (Figure 2). Alternate cycles of ~200 s of blue light and 200 s of dark lead to reversible recruitment of mOrange-Nano in the selected area for multiple times with comparable values of &#916; intensity of fluorescence in the patterns.
Figure 3 shows the schematic representation of the GUVs preparation. The recruitment of mOrange-Nano is also observed on GUVs. It is shown that GUVs placed in the dark do not exhibit mOrange fluorescence (Figure 4A). As the GUVs are globally illuminated with blue light, mOrange fluorescence is observed, colocalizing with the GUV membrane dye (DiD). The interaction is highly reversible as the illumination is terminated. The quantification of the mOrange intensity at the GUV membrane over time shows the fast and effective recruitment of proteins as well as the full reversibility (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66531/66531fig01.jpg" /> 
Figure 1: Fluorescence microscopy images of SLBs functionalized with b-disiLID. The fluorescence images in the presence of mOrange-Nano before (A) and during (B) local blue light (488 nm) illumination in the ROI. Scale bar = 20 &#181;m. (C) Fluorescence intensity of mOrange measured in the ROI for SLBs functionalized with b-disiLID (at = 200 s). The figure is adapted from Di Iorio et al.8. <a href="https://www.jove.com/files/ftp_upload/66531/66531fig01large.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/66531/66531fig02v3.jpg" /> 
Figure 2: Fluorescence intensity of mOrange recruited in the ROI on SLBs decorated with b-disiLID during three recruitment cycles. After each photoactivation step, the mOrange-Nano fluorescence increased within the ROI. The pattern reaches saturation within 120 s, and the fluorescence decreases within 120 s, almost to background levels. No loss of pattern quality is observed over different blue light/dark cycles. The figure is adapted from Di Iorio et al.8. <a href="https://www.jove.com/files/ftp_upload/66531/66531fig02large.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/66531/66531fig03v2.jpg" /> 
Figure 3: Schematic representation of the GUVs preparation using the gentle hydration method. The scheme offers a visual representation of the several steps and of the chamber built using two glass slides and a spacer. <a href="https://www.jove.com/files/ftp_upload/66531/66531fig03large.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/66531/66531fig04.jpg" /> 
Figure 4: Fluorescence microscopy measurements of the light-dependent recruitment of mOrange-Nano on GUVs membranes. (A) Fluorescence images of a disiLID-functionalized GUV in the presence of mOrange-Nano. In green is the membrane dye of the GUVs, and in red is the mOrange fluorescence before, during, and after blue light illumination. Scale bars = 10 &#181;m. (B) Fluorescence intensity of mOrange localized on GUV over time. Upon illumination, the mOrange fluorescence (shown in red) on the lipid membrane reaches maximum intensity within 60 s, with a 5.9-fold fluorescence intensity increase. As the illumination is stopped, the mOrange fluorescence decreases to almost pre-illumination values within 60 s (with a 90% recovery). The figure is adapted from Di Iorio et al.8. <a href="https://www.jove.com/files/ftp_upload/66531/66531fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Author Spotlight: Photo Switchable Protein Recruitment for Reversible Patterning in Artificial Cellular Systems at JoVE.com

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).

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 ...

dynamic-light-induced-protein-patterns-at-model-membranes

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JoVE Journal

Bioengineering

1.0K Views.  University of Münster. 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).

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is essential to determine the effect of high ground impact forces on lower-body bone deformation to better understand the mechanisms of bone stress injuries. Conventional strain gauge measurement has been used to study in vivo tibia deformation. This method is associated with limitations including invasiveness of the procedure, involvement of few human subjects, and limited strain data from small bone surface areas. The current study intends to introduce a novel approach to study tibia bone strain under high impact loading conditions. A subject-specific musculoskeletal model was created to represent a healthy male (19 years, 80 kg, 1,800 mm). A flexible finite element tibia model was created based on a computed tomography (CT) scan of the subject&#39;s right tibia. Laboratory motion capture was performed to obtain kinematics and ground reaction forces of drop-landings from different heights (26, 39, 52 cm). Multibody dynamic computer simulations combined with a modal analysis of the flexible tibia were performed to quantify tibia strain during drop-landings. Calculated tibia strain data were in good agreement with previous in vivo studies. It is evident that this non-invasive approach can be applied to study tibia bone strain during high impact activities for a large cohort, which will lead to a better understanding of injury mechanism of tibia stress fractures.

During landing, lower-body bones experience large mechanical loads and are deformed. It is essential to measure bone deformation to better understand the mechanisms of bone stress injuries associated with impacts. A novel approach integrating subject-specific musculoskeletal modeling and finite element analysis is used to measure tibial strain during dynamic movements.

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...