Microtubule (MT)/Organelle Motility Assays

互联网2013-09-05

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Rapidly thaw and immediately place on ice one aliquot each of axonemes, Golgi or ER membranes, 45 uM tubulin, rat liver cytosol, and 20x energy regeneration system.
Dilute the axonemes with PM Buffer to the proper dilution (determined after preparation, see support protocol 2 below) and prepare 6x membranes (determined after preparation, see support protocol 4 below) by diluting organelles with PM buffer containing 1 mM GTP.

Prepare and place on ice a 30 ul Membrane Mix:
5ul 6x Golgi or ER membranes
1.5 ul of 20 x energy regeneration system
10 ul of 45 uM tubulin
1 ul of 15 mM MgGTP
12.5 ul of cytosol

Perfuse axonemes into a simple perfusion chamber by slowly pipeting ~10 ul of properly diluted axonemes against one open end of the chamber and allowing the chamber to fill.
Be careful to avoid introducing large bubbles into the chamber.

Place the perfusion chamber into the humid chamber and incubate at room temperature for 10 min to allow the axonemes to adhere to the glass.

Wash out unadhered axonemes.
Slowly pipette 10 ul of PM buffer against one end of the perfusion chamber, while simultaneously wicking excess buffer from the opposite side of the chamber with the tip of a square of filter paper. Repeat this 2 more times.

Dilute 5 ul of 45 uM tubulin with 10 ul of PM containing 1 mM MgGTP. Perfuse the diluted tubulin into the chamber containing the washed axonemes, place a drop of immersion oil on the top and bottom of the slide, and transfer it to the microscope stage.

Focus on the axonemes with the 100x objective lens.
It is difficult to focus on axonemes on the surface of the coverslip because of their very small size and the very bright illumination needed for VE-DIC. Align the slide on the stage so that an edge of the double stick tape forming the perfusion chamber perfectly bisects the area illuminated by the microscope condenser lens. Immerse the 100 x objective lens in oil, and focus on the edge of the tape. When you have brought the edge of the tape into view, back off fine focus until the very edge of the tape just begins to go out of focus. If you move the slide so the lens is within the area coated with axonemes, you should now be quite close to focus and the axonemes should be found with little difficulty.

Optimize the image for visualization of individual MTs by aligning the microscope for Koehler illumination (see unit by E.D. Salmon), and use the real time image processor to perform background subtraction, contrast enhancement, and frame averaging (see Salmon and Tran, 1998). Observe and record onto S-VHS video tape images of polymerization dynamics of individual MTs as the are nucleated off of axonemes.
Note the difference between the plus (longer, faster growing MTs) and minus (shorter, slower growing MTs) ends of the axonemes.

During the observation of MT dynamics, allow the membrane mix to warm to room temperature.

Perfuse 12 ul of membrane mix into the simple perfusion chamber on the microscope stage. Seal the chamber edges on both edges with a drop of melted valap. Observe and record the dynamic interactions between the organelles and MTs.
Note that often it takes up to 45 min for motility to develop. This time period is proportional to room temperature