This Featured Protocol presents a cutting-edge method excerpted from Current Protocols in Cell Biology UNIT 13.1. To examine all the sections and features of a typical Unit, please refer to the forthcoming Sample Unit.

From UNIT 13.1

Microtubule/Organelle Motility Assays

This unit describes an in vitro assay that uses video-enhanced differential interference contrast (VE-DIC) microscopy to examine the motile interactions between isolated organelle fractions and microtubules (MTs; see Basic Protocol). The method can be used to dissect the molecular requirements for organelle movement and membrane trafficking. A field of axoneme-nucleated MTs, growing and shortening as they would in a living cell (dynamic MTs), is generated in a simple microscope perfusion chamber. Various combinations of isolated endoplasmic reticulum (ER) and Golgi apparatus organelles, cytosol containing motor proteins and other soluble factors, nucleotides, and specific pharmacological reagents are then added to the dynamic MT, and the motile interactions between the organelles and MTs are observed by VE-DIC microscopy.

In addition, this unit includes protocols for biochemical preparation of phosphocellulose-purified tubulin from porcine brain (see Support Protocol 3), axonemes from sea urchin sperm (see Support Protocol 2), rat liver cytosol (see Support Protocol 4), and rat liver organelle fractions (see Support Protocol 5). To ensure more reproducible results, a protocol for preparing thoroughly cleaned ("squeaky clean") coverslips and simple microscope perfusion chambers is also included (see Support Protocol 1).

STRATEGIC PLANNING

Preparation of the principal biochemical components for the motility assay is detailed in Support Protocols 2 to 5. These components must be prepared in bulk in advance, dispensed into appropriately sized aliquots, and stored at -70°C. Unopened samples can be stored for >2 years. Three different types of animal tissue must be obtained for the various preparations. The animals that are most difficult to acquire are the sea urchins, Strongylocentrotus purpuratus, used for the preparation of axonemes. Sea urchins can be obtained from early winter through mid spring from Marinus, Inc., but their availability depends on the seasonal catch. Porcine brains for the tubulin preparation must be obtained from freshly slaughtered pigs, and the tubulin preparation should begin within 3 to 4 hr after the tissue is harvested. A local butcher can supply information regarding the location of the closest slaughterhouse. Fresh rat livers are fairly easy to obtain; alternatively, flash-frozen livers can be purchased from Pel-Freez.

In contrast to the biochemistry and microscopy, setting up the motility assay in the Basic Protocol is relatively simple. Note, however, that specific brands of microscope coverslips and slides are required for the preparation of the microscope perfusion chambers (see Support Protocol 1), and the coverslips should be cleaned according to the steps outlined. Rigorous attention to the detailed instructions presented in Support Protocol 1 is crucial to the success of the assay. Inexpensive microscopy supplies are often coated with oils and dirt that can lead to spurious and inconsistent results.

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for care and use of laboratory animals.

BASIC PROTOCOL

MT/ORGANELLE MOTILITY ASSAYS

Materials

Axoneme fragments (see Support Protocol 2)

Golgi or ER membranes (see Support Protocol 5)

45 µM purified brain tubulin (see Support Protocol 3)

Rat liver cytosol (see Support Protocol 4)

PM buffer (see recipe)

PM buffer containing 1 mM GTP

20× energy regeneration system (see recipe)

15 mM MgGTP, prepared by diluting 100 mM MgGTP stock (see recipe) in PM buffer

Valap (see recipe)

Simple perfusion chambers (see Support Protocol 1)

Filter paper cut into 2-cm squares

Humid chamber made of a 90-mm glass petri dish containing moist paper towels

High-resolution VE-DIC microscope system (as described in Salmon and Tran, 1998 or equivalent)

1. Rapidly thaw and immediately place on ice one aliquot each of axonemes, Golgi or ER membranes, 45 µM purified brain tubulin, rat liver cytosol, and 20× energy regeneration system.
2. Dilute axonemes with PM buffer to the proper concentration as determined in Support Protocol 2. Prepare 6× Golgi or ER membranes by diluting organelles with PM buffer/1 mM GTP (see Support Protocol 5, step 10).
3. Prepare and place on ice a 30 µl membrane mix:


5 µl 6× Golgi or ER membranes
1.5 µl 20× energy regeneration system
10 µl 45 µM tubulin
1 µl 15 mM MgGTP
12.5 µl cytosol

4. Add axonemes to a simple perfusion chamber by slowly pipetting ~10 µl of 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.

5. Place the perfusion chamber into the humid chamber and incubate 10 min at room temperature to allow the axonemes to adhere to the glass.
6. Wash out unadhered axonemes by slowly pipetting 10 µl 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 wash two more times.
7. Dilute 5 µl of 45 µM tubulin with 10 µl PM buffer/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 VE-DIC microscope stage.


Briefly, the microscope system consists of illumination from an HBO100-W mercury arc lamp introduced into an upright microscope stand (equipped with optical components for DIC image formation) via a fiber-optic scrambler. Illumination is passed through IR reflecting and 546-nm narrow band-pass filters before being focused for Köhler illumination onto the specimen via a 1.4-NA oil-immersion condenser. The light is collected by a 100 × 1.3- or 1.4-NA objective and magnified 12.5× before being collected by a scientific-grade Newvicon tube type video camera (equivalent of Hamammatsu C2400). The video signal is processed by frame averaging, background subtraction, and contrast enhancement by a real-time image processor (equivalent to the Hamammatsu Argus 10), and then recorded in real time onto high-resolution S-VHS video tape.

8. Focus on the axonemes with the 100× objective lens. Align the slide on the stage so that one edge of the double-stick tape that forms the perfusion chamber perfectly bisects the area illuminated by the microscope condenser lens. Immerse the 100× objective lens in oil and focus on the edge of the tape. With the edge of the tape in view, back off fine focus until the very edge of the tape begins to go out of focus. Move the slide so the lens is within the area coated with axonemes, which should now be quite close to focus.


It can be 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. This procedure should make focusing on the axonemes easier.

9. Optimize the image for visualization of individual MTs by aligning the microscope for Köhler illumination. Use the real-time image processor to perform background subtraction, contrast enhancement, and frame averaging. Observe and record onto S-VHS video tape images of polymerization dynamics of individual MTs as they are nucleated off the axonemes.


Note the difference between the plus (longer, faster-growing MTs) and minus (shorter, slower-growing MTs) ends of the axonemes. For details on microscopy techniques, refer to UNIT 4.1 or Salmon and Tran (1998).

10. During the observation of MT dynamics, allow the membrane mix to warm to room temperature.
11. Perfuse 12 µl of membrane mix into the simple perfusion chamber on the microscope stage. Seal the chamber edges on both sides with a drop of melted valap. Observe and record the dynamic interactions between the organelles and MTs (see Fig. 13.1.2).


Note that often it takes up to 45 min for motility to develop. This time period is proportional to room temperature.

Pharmacological agents (see Table 13.1.1) may be added to the membrane mix prepared in step 3 to test the involvement of Golgi coat proteins and MT motor proteins in organelle movement in vitro. For review of the effects of these pharmacological agents on membrane trafficking, see Klausner et al. (1992). These agents should be added to the mix, correcting all components for concentration, and incubated 15 min at 37°C prior to being introduced into the flow chamber.

Organelles may also be pretreated to strip them of specific subsets of peripheral proteins prior to addition to the mix. This will allow examination of the involvement of these proteins in organelle movement (see Support Protocol 5, steps 12 to 14).


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Figure 13.1.1
Flow chart for performing MT/organelle motility assay.



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Figure 13.1.2
VE-DIC micrograph of a membrane/microtubule motility assay. Membrane associated with an axoneme fragment (black arrow) has extended a thin membrane tubule (black arrowheads) via a motile attachment (white arrow) to a single microtubule (white arrowheads). Many single microtubules and membrane vesicles can be seen in this field.



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Table 13.1.1 Pharmacological Agents for Addition to Membrane Mix^a

Pharmacologic agent	Final concentration	Function	Stock solution	Amount added to 30-µl membrane mix
Brefeldin A	60 µM	Removes Golgi coat proteins	Dilute 1.5 µl of a 3.6 mM Brefeldin A stock in ethanolb 1:1 into PM bufferb prior to use	3 µl
Aluminum fluoride		Activates hetero-trimeric G proteins		Add 1 µl of 30× NaFb and 1 µl of 30× AlCl3b
MgGTP-g-S	1 mM	Activates hetero-trimeric G proteins	30 mM MgGTP-g-Sb	1 <µl
MgAMP-PNP	1 mM	Inhibits kinesin-like proteinsc	150 mM MgAMP-PNPb	1 µl
Sodium ortho-vanadate	25 µM	Inhibits cytoplasmic dyneind	Dilute 1 µl of 100 mM stock into PM bufferb	1 µl

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^a Also see APPENDIX 1B.
^b See recipes for instructions on solution preparation. Abbreviations: MgAMP-PNP, 5´ adenylylimidodiphosphate magnesium salt.
^c Vale et al., 1985.
^d Shpetner et al., 1988.
 

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