Support was received from the Swiss National Science Foundation; the Swiss Sinergia program; the Polish-Swiss Research Programme (PSPB-094/2010); the NCCR in Chemical Biology; and LipidX from the Swiss SystemsX.ch initiative, evaluated by the Swiss National Science Foundation (to J. G.). O. M. was supported by an EMBO long-term fellowship (ALTF-516-2012).

1. Solutions and Preparations
NOTE: All buffers and solutions should be prepared in double-distilled (dd) H2O. Because the hydration state of sucrose varies, the final concentration of all sucrose solutions must be determined using a refractometer.
<ol>
	<li>Prepare phosphate-buffered saline without divalent cations (PBS-): 137 mM NaCI, 2.7 mM KCl, 1.5 mM KH2PO4, and 6.5 mM Na2HPO4. Adjust the pH to 7.4. Sterilize by autoclaving (1 cycle at 121 &#176;C for 15 min) and store at room temperature.</li>
	<li>Prepare stock solution of 300 mM imidazole: 0.204 g of imidazole in 10 mL of ddH2O.Adjust the pH to 7.4 at 4 &#176;C using HCl. Filter-sterilize using a 0.22 &#956;m pore size filter and store at 4 &#176;C.</li>
	<li>Prepare homogenization buffer (HB; prepare approximately 100 mL): 250 mM sucrose in 3 mM imidazole. Adjust the pH to 7.4 at 4 &#176;C using a pH-meter. Filter-sterilize using a 0.22 &#956;m pore size filter and store at 4 &#176;C. Complement the HB just before use with a cocktail of protease inhibitors at final concentrations corresponding to 10 mM leupeptin, 1 mM pepstatin A, and 10 ng/mL aprotinin.</li>
	<li>For the step floatation gradients, prepare 62%, 39%, and 35% sucrose solutions in 3 mM imidazole, pH 7.4, as described below. Precisely determine the final concentration of all sucrose solutions using a refractometer.
	<ol>
		<li>Prepare 100 mL of 62% sucrose solution in 3 mM imidazole, pH 7.4. Dissolve by stirring 80.4 g of sucrose in ddH2O pre-warmed to 35 &#176;C. Add 1 mL of 300 mM imidazole stock solution and fill to 100 mL with ddH2O. Adjust the pH to 7.4 at 4 &#176;C. Filter-sterilize using a 0.22 &#956;m pore size filter and store at 4 &#176;C.</li>
		<li>Prepare 100 mL of 35% sucrose solution in 3 mM imidazole, pH 7.4. Dissolve by stirring 40.6 g of sucrose in ddH2O. Add 1 mL of 300 mM imidazole stock solution and fill to 100 mL with ddH2O. Adjust the pH to 7.4 at 4 &#176;C. Filter-sterilize using a 0.22 &#956;m pore size filter and store at 4 &#176;C.</li>
		<li>Prepare 50 mL of 39% sucrose in 3 mM imidazole, pH 7.4. Dilute the 62% sucrose solution with the 35% sucrose solution. Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Dissolve 3 g of paraformaldehyde (PFA) by stirring in 100 mL of PBS- pre-warmed to 60 &#176;C while adding 1 M NaOH dropwise; adjust the final pH to 7.4 using NaOH. Filter-sterilize using a 0.22-&#956;m pore size filter and store at -20 &#176;C. 
	CAUTION: 3% PFA is a toxic reagent.</li>
	<li>Prepare 1 M KCl stock solution. Dissolve 3.73 g of KCl by stirring in 50 mL of ddH2O. Filter-sterilize using a 0.22-&#956;m pore size filter and store at room temperature.</li>
	<li>Prepare 50X concentrated stock solution containing 0.625 M Hepes at pH 7.0 and 75 mM MgOAc2in ddH2O. Aliquot and store at -20 &#176;C.</li>
	<li>Prepare mounting medium. Mix by stirring 2.4 g of poly(vinyl alcohol) Mw ~31,000 (PVA) with 6 g of glycerol. Add 6 mL of ddH2O and leave for at least 2 h at room temperature. Add 12 mL of 0.2 M Tris-Cl (pH 8.5) and heat to approximately 53 &#176;C with occasional stirring until the PVA has dissolved.</li>
	<li>Clarify the solution by centrifugation at 5,000 x g for 20 min at room temperature. Collect the supernatant and add 2.5% (w/v) 1,4-diazabicyclo-[2.2.2]octane (DABCO) to prevent photobleaching during fluorescence detection. Vortex for about 30 s to dissolve. Aliquot into 1.5-mL plastic tubes and store at -20 &#176;C.</li>
</ol>
2. Cell Culture
<ol>
	<li>Culture HeLa cells in Dulbecco&#39;s Modified Eagle Medium supplemented with 10% fetal calf serum, 10% non-essential amino acids, 10% L-glutamine, and 1% penicillin-streptomycin. Maintain them at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Transfect the cells 24 h before the experiment with GFP-RAB510, using commercial transfection reagent according to the manufacturer&#39;s instructions.</li>
	<li>When needed, prepare endosomal fractions and cytosol from cells depleted of a protein of interest (i.e., using RNAi or CRISPR/Cas9) and/or overexpressing a wildtype or mutant form of the protein of interest.</li>
</ol>
3. Endosomal Fraction Preparation
NOTE: This protocol describes the straightforward preparation of subcellular fractions containing endosomes and other light membranes. If needed, purified endosome fractions can also be used11. Prepare 2 Petri dishes (10-cm outer diameter; 57 cm2) of confluent cells expressing GFP-RAB5 as starting material. The total number of confluent HeLa cells in 2 Petri dishes corresponds roughly to 2.5 x 107 cells. When needed, the endosomes can also be prepared from cells depleted of a protein of interest (i.e., using RNAi or CRISPR/Cas9) and/or overexpressing a wildtype or mutant form of the protein of interest, always expressing GFP-RAB5.
CAUTION: All steps of the fractionation protocol should be performed on ice.
<ol>
	<li>Place the Petri dishes onto a wet metal plate in a flat ice bucket and immediately wash the cells twice with 5 mL of ice-cold PBS-.</li>
	<li>Remove all PBS- from the last wash and add 3 mL of ice-cold PBS- per dish. Cells should not dry during handling: if necessary, place the ice bucket on a rocking platform to adequately cover cells with fluid.</li>
	<li>Mechanically remove the cells in PBS from the Petri dishes using a flexible rubber policeman (i.e., homemade cell scraper). Scrape the cells off the dishes first in a quick, circular motion around the outside of the dish, followed by a downward motion in the middle of the dish. Scrape gently to obtain &#34;sheets&#34; of attached cells. Gently transfer the cells using a plastic Pasteur pipette with a wide opening to a 15-mL conical polypropylene tube on ice.</li>
	<li>Centrifuge for 5 min at 175 x g and 4 &#176;C.</li>
	<li>Gently remove the supernatant (SN) and add 1 - 3 mL of HB to the pellet. Using a plastic Pasteur pipette with a wide opening, gently pipette up and down once to resuspend the cells.</li>
	<li>Centrifuge for 7 - 10 min at 1,355 x g and 4 &#176;C. Gently remove the SN.</li>
	<li>Perform cell homogenization. 
	NOTE: Conditions should be gentle so that the plasma membranes of the cells are broken but the endosomes remain intact.
	<ol>
		<li>Add a known volume of HB with protease inhibitors onto each cell pellet (approximately 100 &#956;L per cell pellet). Using a 1,000-&#181;L micropipette, gently pipette up and down until the cells are resuspended. Do not introduce air bubbles.</li>
		<li>Prepare a 1-mL tuberculin syringe with a 22G needle pre-rinsed with HB and free of air or bubbles.</li>
		<li>Fill the syringe with the cell suspension relatively slowly (to avoid creating air bubbles), place the beveled tip of the needle against the wall of the tube, and expel the cell suspension rapidly to shear off the plasma membranes of the attached cells.</li>
		<li>Take a small aliquot of the homogenate (approximately 3 &#181;L) and dilute it into 50 &#181;L of HB on a glass slide. Mix and cover with a glass coverslip. Inspect the homogenate under a phase-contrast microscope equipped with a 20X or 40X objective. 
		NOTE: Under optimal conditions, most cells are broken, but nuclei, which appear as dark grey and round structures, are not (Figure 1).</li>
		<li>Repeat step 3.7.3 and 3.7.4 until most cells are broken; usually, 3 - 6 up-and-down strokes through the needle are necessary. Keep a small aliquot (10 - 30 &#181;L) of the homogenate for protein determination.</li>
	</ol>
	</li>
	<li>Centrifuge homogenate for 7 min at 1,355 x g and 4 &#176;C. 
	NOTE: After centrifugation, the pellet (defined as the nuclear pellet; NP) contains the nuclei and the supernatant (defined as the postnuclear supernatant; PNS) contains the cytosol and the organelles released upon homogenization and in free suspension.</li>
	<li>Carefully collect the PNS. Keep small aliquots (10 - 30 &#181;L) of the PNS and NP for protein determination and discard the NP.</li>
	<li>Adjust the PNS to 40.6% sucrose by diluting the 62% sucrose solution with PNS using a 1:1.1 ratio of PNS:62% sucrose. Mix gently but thoroughly, without creating air bubbles. Check the sucrose concentration using the refractometer.</li>
	<li>Place the PNS in 40.6% sucrose at the bottom of an ultracentrifugation tube. Overlay carefully with 2.5 mL of the 35% sucrose solution and fill the centrifuge tube with HB. 
	NOTE: The sucrose solutions should be layered so that interfaces are clearly visible.</li>
	<li>Ultracentrifuge the gradients for 1 h at&#160; &#820;165,000 x g and 4 &#176;C.</li>
	<li>Carefully remove the white layer of lipids on top of the gradient. Next, collect the interface containing endosomes, between the HB and 35% sucrose, using a 200-&#956;L micropipette with a cut tip. 
	NOTE: The endosomal fractions can be used directly in the in vitro actin polymerization assay or can be aliquoted on ice, flash-frozen in liquid nitrogen, and stored at -80 &#176;C.</li>
	<li>Determine the protein concentration of the PNS and endosomal fractions. 
	NOTE: This concentration should be at least 100 &#181;g/mL. Approximately 2.5 x 107 cells grown in 2 Petri dishes are needed to obtain a PNS with at least 100 &#181;g/mL (see the Note above).</li>
</ol>
4. Cytosol Preparation
NOTE: Prepare 2 Petri dishes (10 cm diameter; 57 cm2) of confluent HeLa cells as starting material.When needed, the cytosol can also be prepared from cells depleted of a protein of interest (i.e., using RNAi or CRISPR/Cas9) and/or overexpressing a wildtype or mutant form of the protein of interest. As for the endosome fractionation protocol, all steps should be performed on ice.
<ol>
	<li>Repeat steps 3.1 - 3.8.</li>
	<li>Place the PNS in a centrifuge tube and ultracentrifuge for 45 min at &#820;250,000 x g and 4 &#176;C.</li>
	<li>Carefully remove the white layer on top and collect the SN (cytosol fraction) without disturbing the microsome pellet. Keep an aliquot of the cytosol fraction for protein determination. 
	NOTE: The cytosol can be used directly in the in vitro actin polymerization assay or can be aliquoted on ice, flash-frozen in liquid nitrogen, and stored at -80 &#176;C.</li>
	<li>Determine the protein concentration of the cytosol. 
	NOTE: It should be at least 3 mg/mL.</li>
</ol>
5. Assay Measuring Endosome-dependent Actin Polymerization In Vitro
NOTE: Endosome-dependent actin polymerization can be performed using two alternative approaches. In the first approach, the materials are mixed in a test tube, fixed, and transferred to a coverslip and analyzed. In the second approach, described here, the assay can be carried out directly in the imaging chamber and can be fixed and analyzed without mechanical perturbation (Figure 2C). In this second approach, the assay mixture is not transferred from a test tube to a coverslip and thus remains unperturbed, decreasing the danger of F-actin networks being physically perturbed during the transfer. In addition, this second approach is compatible with a time-lapse analysis of F-actin polymerization. It should be noted that no difference in the analysis of F-actin polymerization was observed with either approach. 
NOTE: Use cut tips throughout the protocol.
<ol>
	<li>In a test tube
	<ol>
		<li>Gently mix ice-cold purified GFP-RAB5 endosomes (step 3) with cytosol (step 4) at a ratio of 1:10 (protein concentration) in an ice-cold 1.5-mL conical test tube. 
		NOTE: A typical experiment is carried out with approximately 40 - 50 &#181;L.</li>
		<li>Adjust the ice-cold reaction mixture to final concentrations of 125 mM KCl (using the 1 M KCl stock solution), 12.5 mM Hepes, and 1.5 mM MgOAc2 (using the 50x stock solution). Then, adjust the mixture with protease inhibitors to final concentrations of 10 mM leupeptin, 1 mM pepstatin A, and 10 ng/mL aprotinin. Gently mix all components.</li>
		<li>Place the test tube containing the reaction mixture at 37 &#176;C, without shaking, and incubate for the desired time. 
		NOTE: Typically, it will take approximately 3 min to detect actin polymerization on endosomes and 30 min for the formation of an extensive network.</li>
		<li>At the desired time (i.e., 3 min or 30 min), stop the reaction by placing the test tube on ice and add 1/10 (vol:vol) of the precooled 3% PFA solution.</li>
		<li>Add 0.3:10 (vol:vol) of 200 units/mL (~6.6 &#956;M) phalloidin conjugated to red-orange dye to stain the polymerized actin.</li>
		<li>Place 12 &#956;L of the mixture onto 12 &#956;L of PVA mounting medium on a micro glass slide. Put an 18 x 18 mm2 glass coverslipon top.</li>
		<li>Analyze the sample with confocal microscopy.</li>
	</ol>
	</li>
	<li>In the imaging chamber
	<ol>
		<li>To make microscopic imaging chambers, use plasma cleaner for 1 min to clean a round 18-mm diameter coverslip and a round 35 mm diameter Petri dish equipped with a 20 mm glass bottom (0.16-0.19 mm).</li>
		<li>Incubate the cleaned surfaces with 1% &#946;-casein for 20 min to minimize protein binding to the glass. Wash twice with 3 mM imidazole.</li>
		<li>Add endosome and cytosol, in the same assay mixture as in steps 5.1.1 and 5.1.2, to a precooled 1.5 mL test tube and complement the assay mixture with 0.1 &#956;g/&#956;L rhodamine-actin.</li>
		<li>Adjust the final refractive index of the mixture to 1.375 (26.5% sucrose) using 39% sucrose solution. 
		NOTE: Typically, the same volume is added; however, this has to be empirically re-adjusted depending upon the sucrose concentration of the collected fraction, determined using a refractomer, since the sucrose concentration may slightly change from experiment to experiment.</li>
		<li>Place the mixture on the pretreated 35-mm dish (step 5.2.1) and cover it with the pretreated 18-mm coverslip (step 5.2.1).</li>
		<li>Place the chamber at 37 &#176;C, without shaking.</li>
		<li>Analyze the sample using fluorescence confocal microscopy.</li>
	</ol>
	</li>
</ol>
6. Image Acquisition and Analysis of Actin Network
<ol>
	<li>Image acquisition
	<ol>
		<li>Acquire the images on an inverted scanning confocal microscope. 
		NOTE: Image acquisition settings were optimized for an inverted scanning confocal microscope with a 63X 1.25 NA oil objective. Adjust the laser power (usually around 50%) to achieve a good signal-to-noise ratio.</li>
	</ol>
	</li>
	<li>Quantification of the actin network
	<ol>
		<li>Analyze the fluorescent micrographs. Use the opensource software CellProfiler (v2.1.1)12 to measure the amount of actin per endosome (alternative software can be used).
		<ol>
			<li>First, identify endosomes as the primary object using the GFP-RAB5 signal. Then, quantify the F-actin associated with individual endosomes using the propagation of the actin signal (red-orange dye conjugated Phalloidin) as a secondary object.</li>
			<li>Average and normalize the number of associated material for each object to the control condition.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Actin plays a crucial role in endosome membrane dynamics4,14. We previously reported that actin nucleation and polymerization occur on early endosomes, forming small F-actin patches or networks. These F-actin networks are absolutely required for membrane transport beyond early endosomes along the degradation pathway. Interfering at any step of this nucleation and polymerization process prevents endosome maturation and thus downstream transport towards late endoso...

In higher eukaryotic cells, proteins and lipids are internalized into early endosomes where sorting occurs. Some proteins and lipids, which are destined to be reutilized, are incorporated into tubular regions of the early endosomes and then transported to the plasma membrane or to the trans-Golgi network (TGN)1,2. By contrast, other proteins and lipids are selectively packaged into regions of the early endosomes that exhibit a multivesicular appearance. These regions expand and, upon detachment from early endosomal membranes, eventually mature into free endosomal carrier vesicles or multivesicular bodies (ECV/MVBs), which are responsible for cargo transport towards late endosomes1,2.
Actin plays a crucial role in the membrane remodeling process associated with endosomal sorting capacity and endosome biogenesis. Protein sorting along the recycling pathways to the plasma membrane or to the TGN depends on the retromer complex and the associated proteins. This sorting machinery seems to be coupled to the formation of the recycling tubules via interactions of the retromer complex, with the WASP and Scar homologue (WASH) complex and branched actin3,4,5. In contrast, molecules destined for degradation, particularly activated signaling receptors, are sorted into intraluminal vesicles (ILVs) by the endosomal sorting complexes required for transport (ESCRT)2,6,7. While the possible role of actin in the ESCRT-dependent sorting process is not known, F-actin plays an important role in the biogenesis of ECV/MVBs and in transport beyond early endosomes. In particular, we found that annexin A2 binds cholesterol-enriched regions of early endosome, and together with spire1, nucleates F-actin polymerization. The formation of the branched actin network observed on endosomes requires the branching activity of the actin-related protein (ARP) 2/3 complex, as well as the ERM protein moesin and the actin-binding protein cortactin8,9.
Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes. This assay has been used previously to investigate the role of annexin A2 in F-actin nucleation and of moesin and cortactin in the formation of endosomal actin networks8,9. With this in vitro protocol, the complex series of reactions that occur on endosomes during actin polymerization become amenable to the biochemical and molecular analysis of the sequential steps of the process, including actin nucleation, linear polymerization, branching, and crosslinking.

<table border="0" cellpadding="0" cellspacing="0" width="1287">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:477px;">NaCl</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:145px;">71380</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Acros Organics</td>
			<td>196770010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>AppliChem</td>
			<td>A1042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Acros Organics</td>
			<td>424370025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes</td>
			<td>AppliChem</td>
			<td>A3724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesiun acetate tetrahydrate</td>
			<td>Fluka</td>
			<td>63047</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td>AppliChem</td>
			<td>A2948</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>10125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fluka</td>
			<td>71690</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Merck Millipore</td>
			<td>107687</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Roche</td>
			<td>11017101001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Roche</td>
			<td>10253286001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Roche</td>
			<td>10236624001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehide</td>
			<td>Polysciences. Inc</td>
			<td>380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 555 phalloidin</td>
			<td>Molecular Probes</td>
			<td>A34055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Actin rhodamine</td>
			<td>Cytoskeleton. Inc</td>
			<td>APHR-A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>poly(vinyl alcohol), Mw ~31 000&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DABCO</td>
			<td>Sigma-Aldrich</td>
			<td>D-2522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1086</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-casein</td>
			<td>Sigma-Aldrich</td>
			<td>C6905</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter 0.22um</td>
			<td>Millex&#160;</td>
			<td>SL6V033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round 10cm dishes for cell culture</td>
			<td>Thermo Fisher Scientific</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Pasteur pipette</td>
			<td>Assistent</td>
			<td>569/3 40569003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15-ml polypropylen tube&#160;</td>
			<td>TPP</td>
			<td>91015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hypodermic Needle 22G Black 30mm&#160;</td>
			<td>BD Microlance</td>
			<td>300900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Luer-slip 1ml Syringes without needle</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro glass slides&#160;</td>
			<td>Assistent</td>
			<td>2406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18X18-mm glass coverslip</td>
			<td>Assistent</td>
			<td>1000/1818</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>344062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200-&#956;l yellow tip</td>
			<td>Starlab</td>
			<td>S1111-0706</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1000-&#956;l Blue Graduated Tip</td>
			<td>Starlab</td>
			<td>S1111-6801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5-ml test tube</td>
			<td>Axygen</td>
			<td>MCT-175-C 311-04-051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18-mm diameter round coverslip&#160;</td>
			<td>Assistent</td>
			<td>1001/18</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35-mm diameter round dish with a 20-mm glass bottom (0.16-0.19&#160;mm)&#160;</td>
			<td>In vitro Scientific</td>
			<td>D35-20-1.5-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td>79729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sorvall WX80 Ultracentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>46900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>TL-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 rotor</td>
			<td>Beckman coulter</td>
			<td>335649</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 rotor</td>
			<td>Beckman coulter</td>
			<td>346936</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscopy</td>
			<td>Carl Zeiss</td>
			<td>LSM-780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fugene HD transfection reagent</td>
			<td>Promega</td>
			<td style="width:145px;">E2311</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent A</td>
			<td>Bio-Rad</td>
			<td>500-0113</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent B</td>
			<td>Bio-Rad</td>
			<td>500-0114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent S</td>
			<td>Bio-Rad</td>
			<td>500-0115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell scraper</td>
			<td>Homemade</td>
			<td></td>
			<td>Silicone rubber piece of about 2 cm, cut at a very sharp angle and attached to a metal bar or held with forceps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimum Essential Medium Eagle (MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M0643</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids (NEAA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Meter 691</td>
			<td>Metrohm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software</td>
			<td></td>
			<td></td>
			<td>NIH, Bethesda MD</td>
		</tr>
	</tbody>
</table>

in vitro polymerization of f-actin on early endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

To gain insights into the formation of F-actin patches on early endosome membranes, we followed the protocol outlined in Figure 2. Briefly, cells were transfected with GFP-RAB5 and then early endosomes were prepared by subcellular fractionation. These purified early endosomes were incubated with cytosol in order to provide actin itself as well as other factors possibly involved in the reaction. At the end of the incubation period, the reaction was stopped by ...

Watch this Scientific Journal Video about In Vitro Polymerization of F-actin on Early Endosomes at JoVE.com

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

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8.9K Views.  University of Geneva. Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

Video: In Vitro Polymerization of F-actin on Early Endosomes

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

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

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

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

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

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

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.