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    Sea Urchin Axonemes

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    629

    实验试剂

     

    1. 4 Male S. purpuratus sea urchins. (It is impossible to tell what sex sea urchins are until they shed gametes from the five pores on the top center of the animal, thus order twice as many animals as needed.)

    2. 0.55 M KCl

    3. Artificial sea water  (salt mixes available from aquarium supply stores, mix as per manufacturers instructions)

    4. 20% (W/V) sucrose in distilled water

    5. Isolation Buffer (add 7mM 2-mercaptoethanol just prior to use)

    6. High Salt Buffer (add 7mM 2-mercaptoethanol and 1mM dithiothreitol just prior to use)

    7. Isolation Buffer plus 50% (V/V) glycerol 

    实验设备

     

    Tabletop clinical centrifuge Refrigerated superspeed centrifuge (Sorvall RC-5B) with equivalent of Sorvall SS-34 rotor

    10 and 50 ml polycarbonate centrifuge tubes

    50 ml Dounce glass homogenizer with "A" and "B" type pestles

    实验步骤

     

    1. Sacrifice the sea urchins to collect sperm.

    2. Often upon arrival from shipment the urchins will have shed some gametes; allowing easy sex identification; sperm is white, while eggs are peach to orange colored. The urchins must be injected and sacrificed to induce their gametes to shed.  Fill a 60 ml syringe with 0.55 M KCl, pierce the urchin from the bottom in the center of the animal with a 18 ga needle, insert the needle to ~1 in depth, and inject to fill the body cavity with fluid until resistance is felt.  Place the animal upright on paper toweling.

    3. Collect sperm.

    4. Several minutes after injection, gametes will begin to slowly exude from the five pores on the top of the urchin.  Do not collect orange eggs!  Use a glass Pasteur pipette with a rubber bulb to collect white sperm dropwise as it is exuded, pool sperm from all the animals into a test tube on ice.  Expect to get 3-4 ml of sperm per animal over a ~20 min collection period.

    5. Dilute the sperm with 3 volumes of artificial sea water, let stand 20 min on ice.

    6. Centrifuge at 500 rpm for 5 min in a clinical centrifuge to pellet debris.

    7. Collect the supernatant, transfer it to 50 ml polycarbonate centrifuge tubes, and pellet the sperm by centrifuging at 3000 x g (5000 rpm) at 4℃ in a SS-34 rotor for 5 min.

    8. Remove and discard the supernatant.  Resuspend the pellet by trituration in 5 volumes of 20% sucrose to osmotically remove the plasma membranes.

    9. Transfer the demembranated sperm to a  Dounce glass homogenizer partially immersed in slushy ice, and homogenize with 15 rapid passes of a type "B" pestle to break the sperm heads from the tails.

    10. The pestle should be moved from the top of the liquid to the very bottom of the homogenizer, and upon return to the top, the pestle should not break the surface of the liquid.

    11. Transfer the homogenate to 50 ml centrifuge tubes, pellet sperm heads by centrifugation at 12,000 x g (10,000 rpm) for 10 min at 4℃ in a SS-34 rotor.

    12. Collect the supernatant containing the demembranated tails into new 50 ml tubes and pellet the tails by centrifugation at 20,000 x g (13,000 rpm) for 15 min at 4℃ in an SS-34 rotor.

    13. Discard the supernatant.  The pellet will be stratified into a top white layer that contains the demembranated tails and a bottom yellow layer that contains heads and debris.  Collect only the white layer with a metal weighing spatula and Pasteur pipette; resuspend it by trituration in 4 volumes isolation buffer (7mM BME added just prior to use).  Transfer the resuspended tails to a Dounce glass homogenizer on ice, and homogenize by 5 rapid passes with a type "A" pestle to break the tails into fragments.  Transfer the fragment suspension into 50 ml centrifuge tubes.

    14. Pellet the tail fragments in 50 ml tubes by centrifugation at 12,000 x g (10,000 rpm) for 10 min at 4℃ in an SS-34 rotor.  This again will result in a two-layer pellet.

    15. Discard the supernantant and collect and resuspend only the top white layer by trituration in 4 volumes of isolation buffer, transfer to new tubes, and centrifuge as in step 11.  Repeat this cycle of resuspension and centrifugation (maybe 1-2 more times) to completely separate the tail fragments from heads and debris until the pellet is a single layer of pure white.

    16. Discard the supernatant and resuspend the white pellet in 4 volumes of extraction buffer (7mM BME and 1mM DTT added just prior to use), transfer to a Dounce glass homogenizer on ice , homogenize by 5 passes with a type "A" pestle, and incubate on ice for 45 min to extract dyneins and central pair MTs from the tail fragments.

    17. Transfer the extraction suspension to 50 ml centrifuge tubes and separate the extracted axonemes from soluble proteins by centrifuging them at 20,000 x g (13,000 rpm) for 15 min at 4oC in an SS-34 rotor.

    18. Discard the supernatant and resuspend the axoneme pellet by trituration in 4 volumes of extraction buffer, transfer to a new 50 ml centrifuge tube, and re-extract the axoneme fragments by incubation on ice for 15 min.

    19. Pellet the extracted axonemes by centrifugation at 20,000 x g (13,000 rpm) for 15 min at 4oC in an SS-34 rotor.

    20. Discard the supernatant and resuspend the extracted axoneme pellet by trituration in 1/3 the original volume of sperm in isolation buffer containing 50% glycerol.

    21. Distribute the axonemes into 20 ul aliquots in 0.6 ml eppendorf tubes and freeze by immersion of the tubes in liquid nitrogen.

    22. Determine the proper axoneme dilution for use in the MT/Organelle motility assay.  Thaw an aliquot of axonemes and observe various dilutions by VE-DIC (at the same magnification as will be used in the assay) in a simple perfusion chamber (made as described below).  Note the dilution required so that there are 2-3 axonemes visible per ~30 um2 microscopic field.

    23. Store frozen axonemes at -80℃ until use.

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