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><tbody><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Acros Organics</td><td>196770010</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1042</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Acros Organics</td><td>424370025</td><td></td></tr><tr><td>Hepes</td><td>AppliChem</td><td>A3724</td><td></td></tr><tr><td>Magnesiun acetate tetrahydrate</td><td>Fluka</td><td>63047</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>AppliChem</td><td>A2948</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>10125</td><td></td></tr><tr><td>NaOH</td><td>Fluka</td><td>71690</td><td></td></tr><tr><td>Sucrose</td><td>Merck Millipore</td><td>107687</td><td></td></tr><tr><td>Leupeptin</td><td>Roche</td><td>11017101001</td><td></td></tr><tr><td>Pepstatin</td><td>Roche</td><td>10253286001</td><td></td></tr><tr><td>Aprotinin</td><td>Roche</td><td>10236624001</td><td></td></tr><tr><td>Paraformaldehide</td><td>Polysciences. Inc</td><td>380</td><td></td></tr><tr><td>Alexa Fluor 555 phalloidin</td><td>Molecular Probes</td><td>A34055</td><td></td></tr><tr><td>Actin rhodamine</td><td>Cytoskeleton. Inc</td><td>APHR-A</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma-Aldrich</td><td>81381</td><td>poly(vinyl alcohol), Mw ~31 000</td></tr><tr><td>DABCO</td><td>Sigma-Aldrich</td><td>D-2522</td><td></td></tr><tr><td>Tris-HCl</td><td>AppliChem</td><td>A1086</td><td></td></tr><tr><td>&amp;beta;-casein</td><td>Sigma-Aldrich</td><td>C6905</td><td></td></tr><tr><td>Filter 0.22 &amp;mu;m</td><td>Millex</td><td>SL6V033RS</td><td></td></tr><tr><td>Round 10 cm dishes for cell culture</td><td>Thermo Fisher Scientific</td><td>150350</td><td></td></tr><tr><td>Plastic Pasteur pipette</td><td>Assistent</td><td>569/3 40569003</td><td></td></tr><tr><td>15-mL polypropylen tube</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>Hypodermic Needle 22G Black 30mm</td><td>BD Microlance</td><td>300900</td><td></td></tr><tr><td>Sterile Luer-slip 1mL Syringes without needle</td><td>BD Plastipak</td><td>300013</td><td></td></tr><tr><td>Micro glass slides</td><td>Assistent</td><td>2406</td><td></td></tr><tr><td>18 x 18-mm glass coverslip</td><td>Assistent</td><td>1000/1818</td><td></td></tr><tr><td>SW60 centrifuge tube</td><td>Beckman coulter</td><td>344062</td><td></td></tr><tr><td>TLS-55 centrifuge tube</td><td>Beckman coulter</td><td>343778</td><td></td></tr><tr><td>200-&amp;mu;L yellow tip</td><td>Starlab</td><td>S1111-0706</td><td></td></tr><tr><td>1,000-&amp;mu;L Blue Graduated Tip</td><td>Starlab</td><td>S1111-6801</td><td></td></tr><tr><td>1.5-mL test tube</td><td>Axygen</td><td>MCT-175-C 311-04-051</td><td></td></tr><tr><td>18-mm diameter round coverslip</td><td>Assistent</td><td>1001/18</td><td></td></tr><tr><td>35-mm diameter round dish with a 20-mm glass bottom (0.16 - 0.19 mm)</td><td>In vitro Scientific</td><td>D35-20-1.5-N</td><td></td></tr><tr><td>Refractometer</td><td>Carl Zeiss</td><td>79729</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Sorvall WX80 Ultracentrifuge</td><td>Thermo Fisher Scientific</td><td>46900</td><td></td></tr><tr><td>Tabletop ultracentrifuge</td><td>Beckman coulter</td><td>TL-100</td><td></td></tr><tr><td>SW60 rotor</td><td>Beckman coulter</td><td>335649</td><td></td></tr><tr><td>TLS-55 rotor</td><td>Beckman coulter</td><td>346936</td><td></td></tr><tr><td>Confocal microscopy</td><td>Carl Zeiss</td><td>LSM-780</td><td></td></tr><tr><td>Fugene HD transfection reagent</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>Protein assay reagent A</td><td>Bio-Rad</td><td>500-0113</td><td></td></tr><tr><td>Protein assay reagent B</td><td>Bio-Rad</td><td>500-0114</td><td></td></tr><tr><td>Protein assay reagent S</td><td>Bio-Rad</td><td>500-0115</td><td></td></tr><tr><td>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><td>Refractometer</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Minimum Essential Medium Eagle (MEM)</td><td>Sigma-Aldrich</td><td>M0643</td><td></td></tr><tr><td>FCS</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (NEAA)</td><td>Thermo Fisher Scientific</td><td>11140-035</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030-024</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>pH Meter 691</td><td>Metrohm</td><td></td><td></td></tr><tr><td>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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Unit 1

The Cytoskeleton I: Actin and Microfilaments

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9.0K Views.  University of Geneva. Vroege endosome functies is afhankelijk van F-actine polymerisatie. Hier beschrijven we een microscopie gebaseerde in vitro -assay die reconstitutes de nucleatie en polymerisatie van de F-actine op vroege endosomal membranen in proefbuizen, waardoor deze complexe reeks reacties vatbaar voor biochemische en genetische manipulaties.

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

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

Biology

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Neuroscience

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

Bioengineering

Reconstitution of Actin-Based Motility with Commercially Available Proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

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

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

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

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

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

Actin Polymerization and Cell Motility

Actin is a family of globular proteins that are highly abundant in eukaryotic cells. It makes up approximately 1-5% of total cell protein concentration. Actin monomers polymerize to form a complex network of polarized filaments, the actin cytoskeleton, that plays a crucial role in many cellular processes, including cell motility, division, endocytosis, and metastasis of cancer cells.
Actin cytoskeleton dynamics can produce pushing, pulling, and resistance forces that help the cell to migrate. This is achieved by forming different types of membrane protrusions, depending on the cell type and the extracellular signals. Primarily, cells move by recurrent cycles of protrusion and attachment of the cell front to the substratum, followed by detachment and retraction at their rear end.
The human genome encodes six highly conserved actin proteins&#8212;ACTC1, ACTA1, ACTA2, ACTG1, ACTG2, and ACTB; all expressed in different cell types. Mutations in any of these six actin genes or the genes encoding actin-binding proteins can lead to disease. For example, mutations in the ACTA1 gene can lead to muscle weakness, especially in the respiratory muscles, which can cause breathing difficulties &#8212; a condition called Nemaline Myopathy. Similarly, mutations in the WAS gene, a key regulator of actin filament nucleation, which is important for cell adhesion, chemotaxis, and phagocytosis of immune cells, causes Wiskott Aldrich syndrome in humans.

Actin is a family of globular proteins that are highly abundant in eukaryotic cells. It makes up approximately 1-5% of total cell protein concentration. ...

In Vitro Polymerization of F-actin on Early Endosomes

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