- 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
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
Note that often it takes up to 45 min for motility to develop.
This time period is proportional to room temperature
*=As published in...
Waterman-Storer, C.M. (In press) Microtubule/organelle
motility assays. In: Current Protocols in Cell Biology, J.S.
Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M.
Yamada, eds. John Wiley, NY.