We gratefully acknowledge financial support through the DFG TRR 235 (Emergence of Life, project P15), the European Research Council (grant agreement no. 694410 AEDNA), and the TUM International Graduate School for Science and Engineering IGSSE (project no. 9.05). We thank E. Falgenhauer for her help with sample preparation. We thank A. Dupin and M. Schwarz-Schilling for their help with the TX-TL system and useful discussions. We thank N. B. Holland for useful discussions.

1. Expression of Elastin-like Polypeptides
<ol>
	<li>Day 1: Preparation of a starter culture and supplies for peptide expression
	<ol>
		<li>Prepare and autoclave expression culture flasks (4 x 2.5 L) and 3 L of LB medium. For 1 L of LB medium, add 25 g of LB powder to 1 L of ultrapure water.</li>
		<li>Prepare a starter culture with 100 mL of LB medium, 50 &#181;L of sterile-filtered (0.22 &#181;m filter) chloramphenicol solution (25 mg/mL in EtOH), and 50 &#181;L of sterile-filtered (0.22 &#181;m filter) carbenicillin solution (100 mg/mL in 50% EtOH and 50% ultrapure water).</li>
		<li>Add a small streak from a pre-made bacterial stock containing E. coli strain BL21(DE3)pLysS with the pET20b(+) expression vector encoding the polypeptide sequence MGHGVGVP((GEGVP)4(GVGVP))4((GFGVP)4(GVGVP))3(GFGVP)4GWP (abbreviated as EF) to a pre-warmed 100 mL starter culture and incubate at 37 &#176;C for 16 h with 250 rpm in a shaking incubator (E. coli strains and vectors are available upon request).</li>
		<li>Pre-warm the expression culture flasks and the LB media at 37 &#176;C overnight so that they are prepared for the next day.</li>
	</ol>
	</li>
	<li>Day 2: Protein expression
	<ol>
		<li>Add 750 mL of LB medium to each expression culture flask, 375 &#181;L of sterile-filtered (0.22 &#181;m filter) chloramphenicol stock (25 mg/mL in EtOH), and 375 &#181;L of sterile-filtered (0.22 &#181;m filter) carbenicillin stock (100 mg/mL in 50% EtOH and 50% ultrapure water).</li>
		<li>After agitation to distribute the antibiotics, take 2 mL of the media as a reference sample for the optical density measurement at 600 nm (OD600).</li>
		<li>Add 7.5 mL of the starting culture to the expression flask and incubate for 1 h at 37 &#176;C with 250 rpm.</li>
		<li>After this step the OD600 of each flask will be checked every 20 min.</li>
		<li>When the optical density reaches approximately 0.8 reduce the temperature to 16 &#176;C and induce peptide expression by adding 750 &#181;L of 1 M sterile-filtered &#946;-isopropyl thiogalactoside (IPTG) in ultrapure water into each expression flask.</li>
		<li>Incubate the expression flask with the induced bacteria at 16 &#176;C for 16 h with 250 rpm.</li>
	</ol>
	</li>
	<li>Day 3: Extraction of the expressed polypeptide
	<ol>
		<li>Pre-weigh the centrifugation flask to enable determination of the mass of the cell pellet after harvesting.</li>
		<li>Harvest all the bacteria from the expression flasks by centrifugation for 20 min using a pre-cooled centrifuge at 4,000 x g and 4 &#176;C.</li>
		<li>Pour out the supernatant and blot the centrifuge bottles on a sterile paper towel.</li>
		<li>Weigh the centrifugation flasks and calculate the cell pellet mass.</li>
		<li>Resuspend the cells, which are pelleted down from 750 mL of original cell culture, in 15 mL of phosphate-buffered saline (PBS, pH 7.4) by pipetting, and centrifuge at 4,000 x g for 20 min at 4 &#176;C. Pour out the supernatant and blot the centrifuge bottles on a sterile paper towel.</li>
		<li>Resuspend the cell pellet for the lysis step in 2 mL of buffer per 1 gram of cell pellet using PBS (pH 7.4) supplemented with lysozyme (1&#8201;mg/mL), 1&#8201;mM phenylmethylsulfonyl fluoride (PMSF), 1&#8201;mM benzamidine, and 0.5&#8201;U of DNase I.</li>
		<li>Sonicate the cells for further lysis on ice with a sonicator at 8 W for 9 min with alternating 10 s sonication and 20 s pausing steps, followed by a 9 min pause during which the sample should be cooled on ice. Repeat the 9 min sonication step once more.</li>
		<li>Add 2 mL of 10% (w/v) polyethyleneimine (PEI) per 1&#8201;L of original cell culture for nucleic acid precipitation.</li>
		<li>Distribute the sample into 2 mL centrifuge tubes and incubate at 60 &#176;C for 10 min followed by an incubation for 10 min at 4 &#176;C.</li>
		<li>Centrifuge the solution at 16,000 x g for 10 min at 4 &#176;C and collect the supernatant containing the ELP.</li>
	</ol>
	</li>
	<li>Protein purification via inverse temperature cycling:
	<ol>
		<li>Adjust the supernatant to a pH of 2 to switch the hydrophilic part of the peptide to hydrophobic and induce aggregation. To adjust the pH, phosphoric acid and sodium hydroxide are used. pH stripes are sufficient to determine the pH level.</li>
		<li>To improve the yield of the purification the sample, heat the sample to 60 &#176;C and add sodium chloride up to 2 M.</li>
		<li>Subsequently, centrifuge the sample at 16,000 x g for 10 min at room temperature (RT).</li>
		<li>Discard the supernatant and re-dissolve the pellet in PBS (but only half of the initial volume is used compared to the previous step).</li>
		<li>Adjust the pH to 7 with phosphoric acid and sodium hydroxide. pH stripes are sufficiently accurate to determine the pH.</li>
		<li>Centrifuge the sample subsequently at 16,000 x g for 10 min at 4 &#176;C.</li>
		<li>Collect the supernatant and repeat steps 1.4.1&#8211;1.4.6 up to 3x total, except that in the last repetition of step 1.4.4, only water is added instead of PBS.</li>
		<li>Measure the concentration of the peptides with an absorption spectrometer. Use an extinction coefficient of 5500/M/cm at 280&#8201;nm.</li>
		<li>Adjust the concentration of the ELP to 700 &#181;M.</li>
	</ol>
	</li>
</ol>
2. Vesicle Production Using the Glass Beads Method
<ol>
	<li>Concentrate the ELP solution to 1.1 mM using a centrifugal vacuum concentrator.</li>
	<li>Mix 200 &#181;L of the concentrated ELP solution with 1,250 &#181;L of a 2:1 chloroform/methanol mixture followed by vortexing.</li>
	<li>Add 1.5 g of spherical glass beads (212&#8211;300&#8201;&#181;m in size) to a 10 mL round bottom flask.</li>
	<li>Add the solution containing ELP and the chloroform/methanol mixture to the round bottom flask and mix by gentle shaking.</li>
	<li>Connect the flask to a rotary evaporator. Adjust the speed to 150 rpm and regulate the pressure to -0.2 x 105 Pa (-200 mbar) for approximately 4 min until the liquid is evaporated at room temperature.</li>
	<li>Place the round bottom flask into a desiccator for at least 1 h to ensure that remaining chloroform and methanol are evaporated. To avoid loss of the glass beads, loosely attached aluminum foil should be wrapped around the round bottom flask opening.</li>
	<li>For a single experiment, mix 100 mg of the peptide covered glass beads with 60 &#181;L of the swelling solution, such as PBS. Incubate this sample at 25 &#176;C for 5 min.</li>
	<li>Centrifuge the sample quickly with a table-top centrifuge to sediment the glass beads.</li>
	<li>Use a pipette to collect the supernatant which contains the vesicles.</li>
</ol>
3. Transcription of RNA Aptamer dBroccoli Inside the Vesicles
<ol>
	<li>Clean the lab bench with wipes containing RNase decontamination solution to create an RNase-free working environment.</li>
	<li>Mix the ssDNA (5 &#181;M) comprising the T7 promotor and dBroccoli sequence (GAGACGGTCGGGTCCATCTGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCAGATGTCGAGTAGAGTGTGGGCTC), as well as the noncoding strand (5 &#181;M) in nuclease-free water supplemented with RNAPol reaction buffer [40&#8201;mM Tris-HCl (pH = 7.9), 6&#8201;mM MgCl2, 1&#8201;mM dithiothreitol (DTT), and 2&#8201;mM spermidine], to prepare the DNA template for the transcription reaction.</li>
	<li>Incubate the solution at 90 &#176;C for 5 min followed by a slow temperature decrease to 20 &#176;C for 30 min.</li>
	<li>Prepare a 1 mM (5Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one (DFHBI) solution by dissolving 1.26 mg of DFHBI in 5 mL of DMSO.</li>
	<li>Prepare the transcription reaction by mixing RNAPol reaction buffer [40&#8201;mM Tris-HCl (pH = 7.9), 6&#8201;mM MgCl2, 1&#8201;mM dithiothreitol (DTT), and 2&#8201;mM spermidine] with 125&#8201;mM KCl, 15&#8201;mM MgCl2, 4&#8201;mM rNTP, 10&#8201;&#181;M DFHBI, 200&#8201;nM DNA template, 0.5&#8201;U/&#181;L RNase inhibitor murine, and water.</li>
	<li>Right before the reaction is started, add 4&#8201;U/&#181;L T7 polymerase.</li>
	<li>Use this reaction mix as the swelling solution in step 2.7 and incubate at 37 &#176;C for the duration of the experiment, which is typically 1 h.</li>
</ol>
4. Transcription-translation (TX-TL) Reaction
NOTE: For the transcription-translation reaction, a crude cell extract is required as well as reaction buffer and DNA. The crude cell extract is prepared as described in Sun et al.18. For a TX-TL reaction, use the following: 33% (v/v) of the crude E. coli extract, 42% (v/v) reaction buffer, and 25% (v/v) phenol-chloroform purified DNA plus additives. The final concentrations are approximately 9 mg/mL protein, 50&#8201;mM HEPES (pH = 8), 1.5&#8201;mM ATP, 1.5 mM GTP, 0.9&#8201;mM CTP, 0.9 mM UTP, 0.2&#8201;mg/mL tRNA, 26&#8201;mM coenzyme A, 0.33&#8201;mM NAD, 0.75&#8201;mM cAMP, 68&#8201;mM folinic acid, 1&#8201;mM spermidine, 30&#8201;mM PEP, 1&#8201;mM DTT, 2% PEG-8000, 13.3 mM maltose, 1 U of T7 RNA polymerase, and 50 nM plasmid DNA in ultrapure water.
<ol>
	<li>Phenol-chloroform purification of the DNA template
	<ol>
		<li>Prepare six overnight cultures with 5 mL of LB medium and 5 &#181;L of sterile-filtered (0.22 &#181;m filter) carbenicillin solution (100 mg/mL in 50% EtOH and 50% ultrapure water).</li>
		<li>Add a small streak from a pre-made bacterial stock containing DH5&#945; E. coli with the pET20b(+) expression vector to each pre-warmed 5 mL overnight culture and incubate at 37 &#176;C for 16 h with 250 rpm. Depending on which peptide (EF) or protein (YPet or mVenus) is to be expressed, a specific encoding plasmid is used.</li>
		<li>Extract the plasmid with a mini-prep kit as described in the user manual or by following the protocol provided by Zhang et al.24.</li>
		<li>Prepare 100 &#181;L of the pre-purified plasmid DNA (concentration can range from 200&#8211;600 ng/&#181;L) and mix with 100 &#181;L of Roti-phenol/chloroform/isoamyl alcohol (pH = 7.5&#8211;8.0) in a microcentrifuge tube to enable better phase separation.</li>
		<li>Gently invert the tube up to 6x and centrifuge for 5 min at 16,000 x g at RT.</li>
		<li>Add 200 &#181;L of chloroform to the upper phase of the tube and invert the tube up to 6x.</li>
		<li>Centrifuge the sample at 16,000 x g for 5 min at RT.</li>
		<li>Pipet the supernatant to a separate tube and add 10 &#181;L of 3 M of sodium acetate for ethanol precipitation.</li>
		<li>Add 1 mL of -80 &#176;C cold ethanol and store the sample at -80 &#176;C for 1 h.</li>
		<li>Centrifuge the sample at 16,000 x g for 15 min at 4 &#176;C.</li>
		<li>Decant the supernatant and add 1 mL of -20 &#176;C cold 70% (v/v) ethanol.</li>
		<li>Centrifuge at 16,000 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the liquid by pipetting. Be careful to not disturb the DNA pellet.</li>
		<li>Store the sample at RT for approximately 15 min to evaporate the remaining ethanol.</li>
		<li>Add ultrapure water to the sample to adjust the sample concentration to approximately 300 nM, which is measured by absorption at 260 nm.</li>
	</ol>
	</li>
	<li>Preparation of the TX-TL reaction
	<ol>
		<li>Thaw the prepared crude cell extract and the reaction buffer on ice.</li>
		<li>For a 60 &#181;L reaction mix, add the plasmid DNA (phenol-chloroform purified) to 37.5 &#181;L of the reaction buffer [50&#8201;mM HEPES (pH = 8), 1.5&#8201;mM ATP, 1.5 mM GTP, 0.9&#8201;mM CTP, 0.9 mM UTP, 0.2&#8201;mg/mL tRNA, 26&#8201;mM coenzyme A, 0.33&#8201;mM NAD, 0.75&#8201;mM cAMP, 68&#8201;mM folinic acid, 1&#8201;mM spermidine, 30&#8201;mM PEP, 1&#8201;mM DTT, 2% PEG-8000, and 13.3 mM maltose), followed by the addition of 28.7 &#181;L of crude cell extract. Fill to a final volume with ultrapure water to 58.8 &#181;L.</li>
		<li>Right before the reaction starts, add 1.2 &#181;L of the T7 RNA polymerase solution and mix the sample by pipetting up and down.</li>
		<li>Use this reaction mix as a swelling solution in step 2.7 and incubate at 29 &#176;C for the duration of the experiment, which is typically 4&#8211;8 h.</li>
	</ol>
	</li>
</ol>
5. Conjugation of Elastin-like Polypeptides with Fluorophores via Copper Catalyzed Azide-alkyne Huisgen Cycloaddition
<ol>
	<li>Prepare a 20 mM NHS-azide linker (&#947;-azidobutyric acid N-hydroxysuccinimide ester) solution in DMSO.</li>
	<li>Mix 250 &#181;L of the ELP solution (600 &#181;M) with PBS (8&#8201;g/L NaCl, 0.2&#8201;g/L KCl, 1.42&#8201;g/L Na2HPO4, 0.27&#8201;g/L K2HPO4, pH = 7.2) and NHS-azide (1.2 mM) and incubate for 12 h at RT.</li>
	<li>Load the sample in a 10 kDa dialysis cassette and store it at 4 &#176;C for 12 h in 1 L of ultrapure water to remove the salts and residual NHS-azide.</li>
	<li>Mix the activated ELP (500 &#181;M) with the alkyne-conjugated dye (500 &#181;M), Cy5-alkyne, or Cy3-alkyne.</li>
	<li>Mix this sample with 1&#8201;mM TBTA (tris(benzyltriazolylmethyl)amine), 10&#8201;mM TCEP (tris(2-carboxyethyl)-phosphine hydrochloride) and 10&#8201;mM CuSO4 and incubate at 4 &#176;C for 12 h.</li>
	<li>Load the sample in a 10 kDa dialysis cassette and store it at 4 &#176;C for 12 h in 1 L of ultrapure water, to remove the salts and residual alkyne-conjugated dyes.</li>
	<li>These dye-modified ELPs are used in a 1:1 mixture of the Cy3 labeled ELPs and the Cy5 labeled ELPs analogous to the peptides in part two.</li>
</ol>

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The authors declare no competing financial interests.

Film rehydration is a common procedure for the creation of small unilamellar vesicles. The main source of failure is the wrong handling of the materials used in the procedure.

Initially, the ELPs are produced by E. coli cells. The yield after ELP purification can vary significantly depending on how carefully the protocol is conducted during its crucial steps. These are the inverse temperature cycling (ITC) step and the resuspension of the ELPs after precipitation. For purification, we triggered the hydrophobic collapse of the peptide through the addition of phosphoric acid, which changes the pH to acidic and leads to protonation of the glutamic acid side chains in the hydrophilic block. After this step, both blocks are hydrophobic at RT. It is preferable to use an acid whose salt is already in the solution to keep the ionic strength constant, but other acids such as hydrochloric acid can be used, as well. To increase the yield of ELP production, additional salts can be added to enhance the coacervation process, because salt decreases the transition temperature Tt and makes the ELP more hydrophobic. Usually sodium chloride is used, but kosmotropic salts such as sodium sulphate are far better, whereas the salt concentration can be close to its solution limit. Additional heating using a water bath can increase the yield even further.

Another critical step is the redissolution of the ELP pellet. For maximum ELP solubility, the solution should be pre-cooled, and a quick pH adjustment can help to dissolve the pellet faster. The pH may need to be readjusted during this process several times. For further improvement, the sample can be stored in the fridge at 4 &#176;C and shaking of the sample should be avoided. To exchange salts, the described dialysis step at the end of the purification protocol should be preferred. In principle, ITC can be used to separate the peptides from the salt-containing supernatant. But depending on the previous salt concentration, residual salt can be still found in this pellet. Furthermore, dialysis can be used to concentrate the peptides needed for the experiments without losses as mentioned before.

The next crucial step is the evaporation of the chloroform/methanol mixture. During evaporation at reduced pressures, a delay in boiling can cause splatters or turbulences, which may result in large loss of glass beads. This can be prevented using either filters or tissues. The same problem arises when the desiccator is used, but aluminum foil at the opening of the round bottom flask can be used to prevent this. For the handling of the ELP-coated glass beads, it is recommended to use disposable antistatic micro-spatula, which also reduces losses and simplifies the procedure.

The swelling of the vesicles using various swelling solutions such as PBS and transcription mix is straightforward and less error prone. However, for the TX-TL system, variations from batch-to-batch of up to 10% can occur. To minimize this problem, all experiments should be performed with one batch of cell-free extract only, which can be used for up to 3,000 reactions. After rehydration, the vesicle content and outer solution are the swelling solution. A purification of the outside solution should not be performed, because this may change the osmotic pressure difference between vesicle inside and outside. The result would be an uncontrolled size change or even bursting of the vesicles. Therefore, possible reactions at the outside should be only suppressed. Depending on the reaction, this can be done, for instance, by the addition DNase I to digest present DNA or by adding antibiotics such as kanamycin, which inhibits translation.

Film rehydration from glass beads has several advantages when compared to other preparation methods. In contrast to solvent injection, emulsion phase transfer or microfluidics displacement of solvent is not required, since the initial solvent is completely evaporated, and the peptides are rehydrated in aqueous solution. Therefore, the glass beads method is particularly suitable for sensitive samples such as TX-TL, which can be significantly affected or even destroyed by residual organic solvents used in other methods. Furthermore, the protocol is straightforward, less error prone, and able to be easily scaled up. Because of the large surface to volume ratio only small amounts of glass beads are needed and allow a high degree of parallelization with high throughput.

As its main disadvantage, the glass beads method is only useful for the creation of small unilamellar vesicles, which cannot be observed via fluorescence microscopy. The described method is somewhat limited when microscopic observation of the dynamics of single vesicles is desired, which is sometimes necessary (e.g., when observing potential vesicle division processes). Furthermore, the small size of SUVs limits the encapsulation of low-concentrated components such as the peptide-encoding plasmid, which explains the relatively low expression of the peptides. The underlying encapsulation process is a Poisson process, and thus the concentration of any particular component must be at least 150&#160;nM to guarantee an encapsulation probability of 95%. Therefore, it is quite remarkable that it is possible to observe such a large increase in vesicle size in these experiments.

The protocol presented here will enable researchers to create peptide-based vesicles from elastin-like polypeptides. It also opens up opportunities to produce artificial cell-like structures encapsulated by peptide membranes, which represent attractive alternatives to classical compartments made from fatty acids or phospholipids. Peptides are advantageous in that they can be easily expressed in a cell-free environment (for instance, in the TX-TL system used here), which allows coupling of membrane growth directly to a transcription-translation process inside the vesicle. Furthermore, ELPs can be designed and adjusted to specific physicochemical parameters such as contour length, hydrophobicity/hydrophilicity of the used deblock, and sensitivity to stimuli such as pH or ionic strength.

The creation of synthetic living cellular systems is usually approached from two different directions. In the top-down method, the genome of a bacterium is reduced to its essential components, ultimately leading to a minimal cell. In the bottom-up approach, artificial cells are assembled de novo from molecular components or cellular subsystems, which need to be functionally integrated into a consistent cell-like system.
In the de novo approach, compartmentalization of the necessary biochemical components is usually achieved using membranes made from phospholipids or fatty acids1,2,3,4. This is because &#34;modern&#34; cell membranes mainly consist of phospholipids, while fatty acids are regarded plausible candidates of prebiotic membrane enclosures5,6. For the formation of new membranes or to facilitate membrane growth, amphiphilic building blocks must be provided from the exterior7 or ideally through production within a membranous compartment using the corresponding anabolic processes4,8.
While lipid synthesis is a relatively complex metabolic process, peptides can be produced quite readily using cell-free gene expression reactions9,10. Hence, peptide membranes formed by amphiphilic peptides represent an interesting alternative to lipid membranes as enclosures for artificial cell mimics that are able to grow11.
Amphiphilic elastin-like di-block copolymers (ELPs) are an attractive class of peptides, which can serve as the building block for such membranes12. The basic amino acid sequence motif of ELPs is (GaGVP)n, where &#8220;a&#8221; can be any amino acid except for proline and &#8220;n&#8221; is the number of motif repeats13,14,15,16,17. ELPs have been created with a hydrophobic block containing mainly phenylalanine for a and a hydrophilic block mainly composed of glutamic acid11. Depending on a and solution parameters, such as pH and salt concentration, ELPs exhibit a so-called inverse temperature transition at temperature Tt, where the peptides undergo a fully reversible phase transition from a hydrophilic to hydrophobic state. The synthesis of the peptides can be easily implemented inside vesicles using the &#8220;TX-TL&#8221; bacterial cell extract11,18,19,20,21, which provides all necessary components for coupled transcription and translation reactions.
The TX-TL system was encapsulated together, with the DNA template encoding the ELPs into ELP vesicles utilizing dehydration-rehydration from glass beads as a solid support. The formation of vesicles occurs through rehydration of the dried peptides from the bead surface11. Other methods22 for vesicle formation can be used, which potentially show lower polydispersity and larger vesicle sizes (e.g., electro-formation, emulsion phase transfer, or microfluidics-based methods). To test the viability of the encapsulation method, transcription of the fluorogenic aptamer dBroccoli23 can alternatively be used11, which is less complex than gene expression with the TX-TL system.
Due to the expression of the membrane building blocks in vesiculo and their subsequent incorporation into the membrane, the vesicles start to grow11. Membrane incorporation of the ELPs can be demonstrated through a FRET assay. To this end, the ELPs used for formation of the initial vesicle population are be conjugated with fluorescent dyes in equal shares constituting a FRET pair. Upon expression of non-labeled ELPs in vesiculo and their incorporation into the membrane, the labeled ELPs in the membrane are diluted and consequently the FRET signal decreases11. As a versatile and common method for conjugation, copper catalyzed azide-alkyne cycloaddition is used. With the use of a stabilizing ligand such as tris(benzyltriazolylmethyl)-amine, the reaction can be carried out in an aqueous solution at a physiological pH without the hydrolysis of reactants11, which is appropriate for conjugation reactions involving peptides.
The following protocol presents a detailed description of the preparation for growing ELP-based peptidosomes. The expression of the peptides and vesicle formation using the glass beads method are described. Furthermore, it is described how to implement transcription of the fluorogenic dBroccoli aptamer and the transcription-translation reaction for protein expression inside the ELP vesicles. Finally, provided is a procedure for the conjugation of ELPs with fluorophores, which can be used to prove vesicle growth through a FRET assay11.

<table><tbody><tr><td>2xYT</td><td>MP biomedicals</td><td>3012-032</td><td></td></tr><tr><td>3-PGA</td><td>Sigma-Aldrich</td><td>P8877</td><td></td></tr><tr><td>5PRIME Phase Lock GelTM tube</td><td>VWR</td><td>733-2478</td><td></td></tr><tr><td>alkine-conjugated Cy3</td><td>Sigma-Aldrich</td><td>777331</td><td></td></tr><tr><td>alkine-conjugated Cy5</td><td>Sigma-Aldrich</td><td>777358</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A8937</td><td></td></tr><tr><td>Benzamidin</td><td>Carl Roth</td><td>CN38.2</td><td></td></tr><tr><td>BL21 Rosetta 2 &lt;em&gt;E. coli&lt;/em&gt; strain</td><td>Novagen</td><td>71402</td><td></td></tr><tr><td>Bradford BSA Protein Assay Kit</td><td>Bio-rad</td><td>500-0201</td><td></td></tr><tr><td>cAMP</td><td>Sigma-Aldrich</td><td>A9501</td><td></td></tr><tr><td>Carbenicillin</td><td>Carl Roth</td><td>6344.2</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C1919</td><td></td></tr><tr><td>Chloramphenicol</td><td>Carl Roth</td><td>3886.3</td><td></td></tr><tr><td>Chloroform</td><td>Carl Roth</td><td>4432.1</td><td></td></tr><tr><td>CoA</td><td>Sigma-Aldrich</td><td>C4282</td><td></td></tr><tr><td>CTP</td><td>USB</td><td>14121</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth</td><td>P024.1</td><td></td></tr><tr><td>DFHBI</td><td>Lucerna Technologies</td><td>410</td><td></td></tr><tr><td>DMSO</td><td>Carl Roth</td><td>A994.1</td><td></td></tr><tr><td>DNase I</td><td>NEB</td><td>M0303S</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth</td><td>9065.2</td><td></td></tr><tr><td>Folinic acid</td><td>Sigma-Aldrich</td><td>F7878</td><td></td></tr><tr><td>Glass beads, acid-washed</td><td>Sigma-Aldrich</td><td>G1277</td><td></td></tr><tr><td>GTP</td><td>USB</td><td>16800</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H6147</td><td></td></tr><tr><td>IPTG (&amp;beta;-isopropyl thiogalactoside )</td><td>Sigma-Aldrich</td><td>I6758</td><td></td></tr><tr><td>KCl</td><td>Carl Roth</td><td>P017.1</td><td></td></tr><tr><td>K-glutamate</td><td>Sigma-Aldrich</td><td>G1149</td><td></td></tr><tr><td>LB Broth</td><td>Carl Roth</td><td>X968.2</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>Methanol</td><td>Carl Roth</td><td>82.2</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Carl Roth</td><td>KK36.3</td><td></td></tr><tr><td>Mg-glutamate</td><td>Sigma-Aldrich</td><td>49605</td><td></td></tr><tr><td>Micro Bio-Spin Chromatography Columns</td><td>Bio-Rad</td><td>732-6204</td><td></td></tr><tr><td>NAD</td><td>Sigma-Aldrich</td><td>N6522</td><td></td></tr><tr><td>NHS-azide linker (y-azidobutyric acid oxysuccinimide ester)</td><td>Baseclick</td><td>BCL-033-5</td><td></td></tr><tr><td>PEG-8000</td><td>Carl Roth</td><td>263.2</td><td></td></tr><tr><td>pH stripes</td><td>Carl Roth</td><td>549.2</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride</td><td>Carl Roth</td><td>6367.2</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>VWR</td><td>76180-684</td><td></td></tr><tr><td>Phosphoric acid</td><td>Sigma-Aldrich</td><td>W290017</td><td></td></tr><tr><td>Polyethyleneimine</td><td>Sigma-Aldrich</td><td>408727</td><td></td></tr><tr><td>Potassium phosphate dibasic solution</td><td>Sigma-Aldrich</td><td>P8584</td><td></td></tr><tr><td>Potassium phosphate monobasic solution</td><td>Sigma-Aldrich</td><td>P8709</td><td></td></tr><tr><td>Qiagen Miniprep Kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>RNAPol reaction buffer</td><td>NEB</td><td>B9012</td><td></td></tr><tr><td>RNase inhibitor murine</td><td>NEB</td><td>M0314S</td><td></td></tr><tr><td>RNaseZap Wipes</td><td>ThermoFisher</td><td>AM9788</td><td></td></tr><tr><td>rNTP</td><td>NEB</td><td>N0466S</td><td></td></tr><tr><td>Roti-Phenol/Chloroform/Isoamyl alcohol</td><td>Carlroth</td><td>A156.1</td><td></td></tr><tr><td>RTS Amino Acid Sampler</td><td>5 Prime</td><td>2401530</td><td></td></tr><tr><td>Slide-A-Lyzer Dialysis Cassettes, 10k MWCO (Kit)</td><td>Thermo-Scientific</td><td>66382</td><td></td></tr><tr><td>Sodium chloride</td><td>Carl Roth</td><td>9265.1</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Carl Roth</td><td>8655.1</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich</td><td>85558</td><td></td></tr><tr><td>Sterile-filtered (0.22 &amp;micro;m filter)</td><td>Carl Roth</td><td>XH76.1</td><td></td></tr><tr><td>T7 polymerase</td><td>NEB</td><td>M0251S</td><td></td></tr><tr><td>TBTA (tris(benzyltriazolylmethyl)amine)</td><td>Sigma-Aldrich</td><td>678937</td><td></td></tr><tr><td>TCEP (tris(2-carboxyethyl)-phosphine hydrochloride)</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Tris base</td><td>Fischer</td><td>BP1521</td><td></td></tr><tr><td>tRNA (from E. coli)</td><td>Roche Applied Science</td><td>MRE600</td><td></td></tr><tr><td>UTP</td><td>USB</td><td>23160</td><td></td></tr></tbody></table>

in vesiculo synthesis of peptide membrane precursors for autonomous vesicle growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Vesicle production 
Figure 1 shows transmission electron microscopy (TEM) images of vesicles prepared with different swelling solutions and the glass beads method (also see Vogele et al.11). For the sample in Figure 1A, only PBS was used as swelling solution to prove the formation of vesicles and to determine their size. When TX-TL was used as swelling solution (Figure 1B), the vesicles also formed. Dynamic light scattering (DLS) measurements were performed to show that the glass beads method has an effect on vesicle formation. Figure 2 depicts the measured intensity autocorrelation curves of an EF sample prepared without glass beads in PBS (blue) and an EF sample prepared with the glass beads method with PBS as the swelling solution (red). The sizes were calculated from a cumulant fit25 and resulted in a diameter of 134 nm with a polydispersity of 25% when the vesicles were prepared without the glass beads method. When the glass beads were used, the cumulant fit resulted in a diameter of 168 nm with a polydispersity of 21%.
In vesiculo transcription11 
Figure 3 shows the fluorescence intensity profile over time for the transcription of the dBroccoli aptamer inside the EF vesicles (green). As a negative control, DNase I was added to the swelling solution and thus also incorporated during vesicle formation (blue). The measurements were performed in fluorescence plate reader with excitation at 480 nm and emission at 520 nm.
In vesiculo protein expression11 
Figure 4 shows the fluorescence intensity of two fluorescent proteins which were expressed inside the ELP vesicles using a fluorescence plate reader. Excitation was carried out 500 nm and emission at 520 nm. Transcription-translation of mVenus (Figure 4A) was performed to investigate expression dynamics and expression level of proteins in the ELP vesicle. It is important to note that after vesicle formation, the contents of the vesicles and outer solution are the same. Hence, to suppress protein expression outside of the vesicle, the antibiotic kanamycin was added to the exterior solution. As a control, kanamycin was also added to the swelling solution, in which case protein expression inside of the vesicle was suppressed. This further indicates that the small molecule kanamycin stays inside the vesicle and suggests that the membrane is not permeable for this molecule over the time scale of these experiments. If kanamycin diffused through the membrane, the mVenus expression would not have been suppressed and at 5 h, the fluorescence would be higher. In the second experiment, the expression of YPet (Figure 4B) was carried out. Both proteins were chosen because they exhibit a faster maturation time than, for instance, GFP. Furthermore, the T7 promoter was used for mVenus transcription and a constitutive promoter used for YPet transcription to show that both inducible and continuous expression are possible.
FRET assay11 
Figure 5A shows the results of the FRET assay performed to demonstrate ELP incorporation into the membrane. Therefore, the vesicles were produced using a mixture of two equally concentrated fluorophore-peptide constructs. These were Cy3-EF (donor) and Cy5-EF (acceptor). At time t = 0, the starting FRET level was measured. The signals of the donor and the FRET signal depend on the mean distance between the dyes in the membrane. Upon expression of the membrane ELPs, additional peptides incorporate into the membrane, which increases the average distance between the FRET pairs. The latter was measured through the increase in donor fluorescence and a decrease of the FRET signal at time t = 2.5 h. Figure 5B shows the negative control. Here, kanamycin was added to the swelling solution before vesicle formation. Kanamycin suppresses protein expression; therefore, no change in FRET was visible. It is important to note that this assay only shows EF incorporation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59831/59831fig1.jpg" /> 
Figure 1: Representative TEM images. (A) EF vesicles formed in the swelling solution PBS. (B) EF vesicles formed in the swelling solution TX-TL. Scale bars = 200 nm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59831/59831fig1large.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/59831/59831fig2.jpg" /> 
Figure 2: Representative DLS data. Intensity autocorrelation curves measured by DLS. The blue curve depicts EF peptides in PBS prepared without the glass beads method. The red curve depicts an EF solution produced with the glass beads method and PBS as the swelling solution. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59831/59831fig2large.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/59831/59831fig3.jpg" /> 
Figure 3: dBroccoli transcription. Representative data of the transcription of the dBroccoli aptamer inside EF vesicles. The green curve depicts the fluorescence signal and the blue curve the control measurement with encapsulated DNase I. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59831/59831fig3large.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/59831/59831fig4.jpg" /> 
Figure 4: Protein expression inside peptide vesicles. (A) A plasmid encoding mVenus and the TX-TL expression mix were encapsulated in the ELP vesicles. After approximately 5 h, the fluorescence saturated. When the antibiotic kanamycin was encapsulated as well, no fluorescence was observed. (B) Similar results were obtained upon encapsulation of a YPet plasmid. Error bars represent the standard deviation of the measured values at the indicated time during a time frame of 15 min. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59831/59831fig4large.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/59831/59831fig5.jpg" /> 
Figure 5: FRET assay. (A) Starting fluorescence levels at time t = 0 for donor emission and FRET signal (acceptor emission). At t = 2.5 h, the donor showed increased emission, and the FRET signal decreased. (B) When only hydrophilic ELP were expressed inside the vesicles, the FRET signal and donor emission stayed constant. Error bars represent the standard deviation of the measured values at the indicated time during a time frame of 15 min. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59831/59831fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File: Contains all plasmid sequences resp. gene sequences. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59831/SI_Vogele_JoVE.pdf">Please click here to download this file.</a>

Watch this Scientific Journal Video about In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth at JoVE.com

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

in-vesiculo-synthesis-peptide-membrane-precursors-for-autonomous

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

5.7K Views.  Technische Universität München. Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Video: In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Chemistry

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

Biochemistry

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Biology

Pinching-off of Coated Vesicles

Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming the coated vesicle.
COP-coated vesicles bud off their organelle membrane with the help of several small GTPases such as SEC, SAR, and ARF proteins. On the other hand, Clathrin-coated vesicles require additional proteins, the most important of which is Dynamin. Dynamin is a GTPase of about 100-kDa that assembles as helical spirals at the necks of vesicle buds. It undergoes a GTP hydrolysis-driven conformational change that results in a twisting motion, which detaches the clathrin-coated vesicle from the membrane.
Once a vesicle buds, it immediately sheds its coat. The energy required for uncoating the clathrin coat is derived through ATP hydrolysis by Hsp70 chaperone protein, an ATPase activated by a co-chaperone named auxin. Auxilin binds Hsp70 and guides it to appropriate locations on the clathrin lattice to stimulate ATP hydrolysis. Auxilin binding creates a detectable distortion in the clathrin coat. It simultaneously contacts another clathrin at its terminal domain, recruiting more Hsc70 to the neighborhood, thus uncoating the entire vesicle. Tight control of vesicular coat formation and shedding facilitates the timing of vesicle budding and fusion with the target membrane.

Szczypanie powlekanych pęcherzyków

Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, ...

Education

JoVE Core

Cell Biology

Unit 1

Intracellular Membrane Traffic

KEY TERMS AND CONCEPTS

Intralumenal Vesicles and Multivesicular Bodies

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing numerous ILVs is called a multivesicular body (MVB). ILVs contain internalized molecules such as antigens, nucleic acids, proteins, and metabolites. Some of these molecules are released from the MVBs inside exosomes and are transported to other cells. Other MVBs contain molecules that are retained in the ILVs and are later degraded within the cell.
MVBs were first observed in neuronal cells in 1955 using electron microscopy. MVBs have a diameter of 400-500 nm, are spherical, and were initially thought to be a form of the late endosome; however, recent studies have shown that MVBs are an intermediate form of these organelles between the early and the late endosomes.
ILV formation takes place when a ubiquitin-tagged protein enters the cell in an endocytic vesicle and fuses with the early endosome. As the early endosome matures, the endosomal membrane bends inwards to form ILVs. An ESCRT protein complex mediates ILV formation. Phosphatidylinositol 3-phosphate (PtdIns3P) is also required for ILV formation. PtdIns3P acts as a docking site for ESCRTs for the bending of the endosomal membrane.
The ESCRT complex and the ATPase, Vps4, required for ILV formation can be hijacked by enveloped retroviruses that infect host cells. Normally, ILVs are formed inside maturing endosomes with the help of ESCRTs within the cell, and the ubiquitin-tagged proteins are recognized and sorted into ILVs. In contrast, retroviruses use the ESCRTs to be released outside the cell. For example, the human immunodeficiency virus (HIV) codes for proteins, such as Gag, NC-p2, and Tat, that require ubiquitination for export. This ubiquitination is essential because ESCRTs and Vps4 recognize and sort only ubiquitin-tagged proteins into viral buds. Once the viral particles bud off from an infected cell, they are released into the extracellular space to infect other host cells.

Pęcherzyki śródświetlne i ciałka wielopęcherzykowe

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing ...

Endocytosis and Exocytosis

Vesicular Tubular Clusters

After budding out from the ER membrane, some COPII vesicles lose their coat and fuse with one another to form larger vesicles and interconnected tubules called vesicular tubular clusters or VTCs. These clusters constitute a compartment at the ER-Golgi interface known as ERGIC (Endoplasmic Reticulum Golgi Intermediate Compartment). The ERGIC is a mobile membrane-bound cargo transport system that sorts proteins secreted from ER and delivers them to the Golgi.
With the help of motor proteins such as kinesin and dynein, the VTCs move along microtubule tracks to unload their cargo at the appropriate destination&#8211; the Golgi apparatus. As the VTC moves along the tracks, COPI vesicles bud off, carrying escaped ER proteins that need to be taken back to the ER. The composition of the VTC gradually changes as it moves towards the Golgi. The cisternal maturation model, which explains protein traffic through the Golgi, hypothesizes that such VTCs become the cis Golgi network as they arrive from the ER. Eventually, they mature into medial cisterna and finally the trans-cisterna.

Pęcherzykowe klastry rurkowe

After budding out from the ER membrane, some COPII vesicles lose their coat and fuse with one another to form larger vesicles and interconnected tubules ...

Overview of Secretory Vesicles

Secretory vesicles, also known as dense core vesicles (DCVs), are membrane-bound vesicles that transport secretory proteins, such as hormones or neurotransmitters. Regulated secretory vesicles transport proteins from the trans-Golgi network to the exterior of the cell. Proteins present in regulated secretory vesicles are required to be rapidly exocytosed in large amounts upon a specific stimulus.
Various proteins regulate the aggregation of molecules inside the secretory vesicles. Chromogranins (Cgs) A and B are proteins that bind to soluble molecules present in secretory vesicles. This binding aggregates the soluble molecules, forming DCVs. Cgs function optimally at low pH and high intracellular calcium concentration. The low pH is maintained by the continuous pumping of protons by the V-type ATPases. A high concentration of calcium is present in the endoplasmic reticulum and Golgi complex of most mammalian cells. Hence, the secretory vesicles that bud off from the trans-Golgi network are rich in calcium. Calcium induces pH-dependent conformational changes in Cgs A and B. These conformational changes lead to the binding and aggregation of CgsA and B among other vesicular matrix proteins with the vesicular membrane. Aggregation is important for specific proteins to be sorted into regulated secretory vesicles.
The yeast Saccharomyces cerevisiae is a model system for studying the regulation of secretory vesicle-mediated transport. In 1979, Randy Schekman and Peter Novik generated temperature-sensitive mutants of S. cerevisiae. These mutants expressed proteins that were functional only at low temperatures but not at 37&#176;C. Schekman and Novik observed that these mutants accumulated vesicles and internal membranes. These mutants were also defective in different stages of protein secretion and therefore called them sec mutants. Some sec mutants contained vesicles that were unable to fuse with the plasma membrane, while other mutants could not transport proteins to other organelles. Further, molecular analysis of these mutants revealed 23 sec genes that produced proteins with regulatory roles in different transport stages of secretory vesicles.

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

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