局部钙离子梯度对大泡囊膜重塑的响应

We present a technique for manipulation of giant vesicles using localized calcium ion gradients. The main advantage of this technique is the entirely contactless stimulation of the vesicle's surface and direct visualization of lipid membrane remodeling upon interaction with calcium ions. Our approach introduces a means to address the details of calcium ion membrane interactions, providing new venues to study the mechanisms of cell membrane reshaping upon changes in the cell micro-environment.

To begin this procedure, prepare a solution of lipids in chloroform. The final concentration of lipids in chloroform is one milligram per milliliter, and the final mass is 0.6 milligrams. Wrap the glass vial containing the lipid mixture with metal foil to protect the contents from ambient light.

Place the metal foil-wrapped sample vial inside a glass beaker. To remove the chloroform, evaporate the solvent in a rotary evaporator for three hours with the pressure reaching seven kilopascals vacuum at 80 rpm. A dry lipid film is formed at the bottom of the glass vial after evaporation.

Slowly add 600 microliters of phosphate-buffered saline solution on top of the dried lipid film. Following the addition of the buffer, gently add six microliters of glycerol to protect the sample from complete dehydration during the formation of a giant unilamellar vesicle connected to a multilamellar vesicle, or GUV-MLV. Seal the glass vial using Parafilm.

And cover with metal foil to protect from ambient light. Store the vial at four degrees Celsius overnight. The following day, sonicate the glass vial containing the solution for one minute using an ultra-sonication bath at room temperature.

Remove the Parafilm, and pipette until a visually uniform solution of small lipid vesicles is formed. Aliquot 30 microliters of the obtained small lipid vesicle solution into individual 0.5 milliliter plastic tubes. Thaw a plastic tube containing an aliquot of the frozen suspension of small lipid vesicles at room temperature.

Vortex the tube four times for one to two seconds using a vortex mixer at maximum speed. Place five microliters of the small lipid vesicle suspension onto the surface of a glass cover slip to form a small, round droplet. Use a glass coverslip without any pre-cleaning steps.

Now, place the glass coverslip in a vacuum desiccator for 20 minutes. Store the dried lipid film at room temperature for four minutes. Then, slowly pipette 50 microliters of 10 millimolar HEPES buffer on top of the dry lipid film for rehydration.

Wait five minutes to preform the GUV-MLV complexes. Center a glass coverslip on the microscope's stage. Then, pipette 300 microliters of HEPES buffer on it and position the center of the droplet above the objective.

Transfer the 50 microliters of preformed GUV-MLV solution into the HEPES solution. Wait 25 minutes to allow sparsely formed GUV-MLV complexes to firmly adhere to the surface of the glass coverslip. Round the edges of the borosilicate glass capillaries by gently placing the capillary ends into the flame of a candle to prevent the micropipette from being broken while attaching it to the micropipette holder.

Pull at least three glass capillaries using an automatic laser puller. Use the borosilicate glass capillaries referenced in the text to achieve a micropipette with the tip opening of approximately 0.3 microns in diameter. To avoid clogging the pipette tip, filter a five millimolar calcium chloride solution in HEPES buffer solution using a 0.2 to 0.5 micron syringe filter prior to usage.

Then, back-fill each micropipette with eight microliters of the calcium chloride solution using a Microloader. Connect a micropipette holder to a micromanipulator. Mount the micropipette into the micropipette holder tightly.

Then connect the injection pump and capillary holder using the supply tube. After starting the injection pump, adjust the settings on the injection pump to a 20 hectopascal injection pressure. Also set a five hectopascal compensation pressure.

Set the microscope to bright-field mode. Configure it for differential interference contrast. Use the coarse micromanipulator to position the micropipette above the droplet containing the GUV-MLV complexes.

Locate the tip of the micropipette above the objective and bring the pipette down to the droplet using the coarse micromanipulator. Set the microscope to fluorescence mode. Position the micropipette tip at a distance of three microns from the membrane surface while injecting the calcium chloride solution from the micropipette to allow MTPs to form.

To translate the MTPS along the GUV surface, slowly move the pipette tip around the GUV surface using the fine micromanipulator. Maintain approximately the same distance between the GUV surface and the micropipette tip while continuing the calcium ion injection. To stop the microinjection, turn off the injection pump.

A representative fluorescent microscopy image of a GUV-MLV complex immobilized on the surface of a glass coverslip is shown here. The membrane tubular protrusions generated upon localized injection of calcium ions at the surface of the GUV can be seen in these images. Translation of the micropipette tip around the membrane's surface as indicated by the arrow triggers movement of the membrane tubular protrusions in the direction of the calcium ion source.

To summarize, we propose a technique which allows for contactless membrane remodeling of giant lipid vesicles which resulted in the formation of membrane tubular protrusions upon localized stimulation with calcium ions. Future applications of this method include transitioning from synthetic lipid vesicles to native biological membranes or applying this approach to polymeric systems with the purpose of developing contactless micromanipulation platforms.