The overall goal of this microscopy based in vitro assay is to study actin polymerization from endosomes in vitro. This method can help answer key questions in the membrane trafficking and actin fields, such as the world of actin in the acidic pathway. And the main advantage of this technique is to reconstitute the nucleation polymerization of F-actin on early endosomal membranes in the test tube.

That's rendering this complex series of reactions amenable to my chemical manipulations. Demonstrating the procedure will be Jorge Larios and Olivia Muriel from the lab. Begin by placing dishes of hela cells onto a wet metal plate in a flat ice bucket.

And washing the cells twice with five milliliters of ice cold PBS. After removing the PBS from the last wash, add three milliliters of ice cold PBS per dish. Then use a flexible rubber policeman to scrape the cells off the dishes 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 sheets of attached cells. Next use a plastic pasteur pipette with a wide opening to gently transfer the cells to 15 milliliter conical polypropylene tubes on ice.

Centrifuge for five minutes at 175 times G and four degrees celsius. Gently remove the supernatant and add one to three milliliters of homogenization buffer to the pellets. Using a plastic pasteur pipette with a wide opening, gently pipette up and down once to re suspend the cells.

Then centrifuge for seven to 10 minutes at 1, 355 times G and four degrees celsius. After removing the supernatant, add a known volume of homogenization buffer with protease inhibitors to each cell pallet. And gently pipette up and down without introducing air bubbles until the cells are re suspended.

Rinse a one milliliter tuberculin syringe with homogenization buffer and ensure that it is free of air or bubbles. Then slowly fill the syringe with the cell suspension to avoid creating air bubbles. Place the bevel 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.

Then take a small aliquot of each homogenate, dilute it into 50 microliters of homogenization buffer on a glass slide and mix. Then cover it with a glass cover slip, inspect the homogenates under a phase contrasting microscope equipped with a 20 times or 40 times objective. Under optimal conditions, most cells are broken, but nuclei which appear dark gray are round and are not.

Repeat the shearing step until most cells are broken, usually three to six up and down strokes through the needle are necessary. Keep a small aliquot of each homogenate for protein determination. After centrifuging as before, carefully collect the post nuclear supernatant.

Reserve small aliquots of each post nuclear supernatant and nuclear pellet for protein determination and then discard the nuclear pellets. Adjust the post nuclear supernatants to 40.6 percent sucrose by adding post nuclear supernatant to 62 percent sucrose solution at a ratio of one to one point one. Mix gently but thoroughly without creating air bubbles.

Transfer the post nuclear supernatants in 40.6 percent sucrose to ultra centrifugation tubes. Overlay carefully with two point five milliliters of 35 percent sucrose solution, and fill the centrifuge tube with homogenization buffer. Ultra centrifuge the gradients for one hour at approximately 165, 000 times G and four degrees celsius.

Following ultra centrifugation, carefully remove the white layer of lipids on top of the gradients. Next, use a 200 microliter micro pipette with a cut tip to collect the interface containing endosomes which is between the homogenization buffer and 35 percent sucrose. To collect the cytosol, pipette the post nuclear supernatant into an ultra centrifuge tube.

And ultra centrifuge for 45 minutes at approximately 250, 000 times G and four degrees celsius. After, carefully remove the white layer on top, and collect the supernatant which is the cytosol fraction without disturbing the microsome pellet. Begin the assay by gently mixing ice cold purified GFP wrapped five endosomes with cytosol at a ratio of one to 10 protein concentration in an ice cold one point five milliliter conical test tube.

Next, use potassium chloride stock solution to adjust the reaction mixture to final concentrations of 125 millimolar potassium chloride. 12.5 millimolar hepes and 1.5 millimolar magnesium acetate. Then adjust the mixture with protease inhibitors to final concentrations of 10 millimolar of leupeptin one millimolar of pepstatin a and 10 nanograms per milliliter aprotinin.

Place the test tube containing the reaction mixture at 37 degrees celsius without shaking and incubate for the desired time. After the incubation period, stop the reaction by placing the test tube on ice and adding pre cooled three percent PFA solution at a volumetric ratio of one to 10. Add 200 micrograms per milliliter phalloidin conjugated to red orange dye at a volumetric ratio of zero point three to 10 to stain the polymerized actin.

Pipette 12 microliters of the mixture onto 12 microliters of PVA mounting medium on a micro glass slide. Put an 18 by 18 milliliter glass cover slip on top. Then analyze the sample using fluorescence confocal microscopy.

Begin by incubating the clean surface of microscopic imaging chambers with one percent beta casein for 20 minutes on ice to minimize protein binding to the glass. Following the incubation, wash twice with three millimolar imidazole. Add the rhodamine actin into a pre cooled one point five milliliter test tube containing the endosomes and cytosol mixture for the assay as previously demonstrated.

Adjust the final refractive index of the mixture to one point three seven five by adding 39 percent sucrose solution to a final concentration of 26.5 percent. Dispense the mixture onto the pre treated 35 millimeter dish and cover it with the pre treated 18 millimeter cover slip. Place the chamber at 37 degrees celsius for three or thirty minutes as before.

Then analyze by fluorescence confocal microscopy and quantify the actin network using the recommendations in the written protocol. The following images show early endosomes purified from cells expressing GFP-RAB5 that were incubated with cytosol as a source of actin. The assay mixture was fixed, sampled and placed on a microscopic slide and f actin was visualized with phalloidin conjugated to a red flora for.

The actin nucleation and polymerization process was rapid since f actin could already be detected on some endosomes after one minute. These short actin structures rapidly grew in length over a period of 30 minutes and became interconnected and branched, eventually forming a filamentous network. Presumably reflecting unbalanced actin dynamics in vitro.

These images were obtained when the assay was carried out with purified rhodamine labeled actin in addition to cell cytosol in glass bottomed dishes. So that structures could be imaged directly In the absence of any perturbation. As seen here, similar nucleation and polymerization rates were observed.

In the assay described here, early endosomes nucleate de novo actin polymerization selectively. Since no actin polymerization is detected when either the endosome or the cytosol is omitted. These observations demonstrate that early endosomes had the intrinsic and specific capacity to trigger actin nucleation and polymerization.

When attempting this procedure, it's important to remember that conditions of relatively low level of RAP5 expression in vivo are preferred. And that cellular membranes are relatively fragile and sensitive to biochemical or chemical shocks. Similarly, the actin network from in vitro is also very fragile and should be handled delicately to avoid collapse or breakage.

After watching this video, you should have a good understanding of how to reconstitute the nucleation polymerization of f actin on early endosomal membranes in the test tube. With this technique you will be able to study the world of factors involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of f actin filaments.