This
protocol should yield enough axoneme fragments to perform thousands
of motility assays.
Solutions and reagents
- 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.)
- 0.55 M KCl
- Artificial sea water
(salt mixes available from aquarium supply stores, mix as per manufacturers
instructions)
- 20% (W/V) sucrose in distilled
water
- Isolation Buffer (add 7mM
2-mercaptoethanol just prior to use)
- High Salt Buffer (add 7mM
2-mercaptoethanol and 1mM dithiothreitol just prior to use)
- Isolation Buffer plus 50%
(V/V) glycerol
Equipment:
Tabletop clinical centrifuge Refrigerated superspeed centrifuge (Sorvall
RC-5B) with equivalent of Sorvall SS-34 rotor ~10 50 ml polycarbonate
centrifuge tubes (Sorvall # 03146) 50 ml Dounce glass homogenizer
with "A" and "B" type pestles
Prepare Sea Urchin Sperm Axonemes
- Sacrifice the sea urchins
to collect sperm.
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.
- Collect sperm.
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.
- Dilute the sperm with 3 volumes
of artificial sea water, let stand 20 min on ice.
- Centrifuge at 500 rpm for
5 min in a clinical centrifuge to pellet debris.
- Collect the supernatant, transfer
it to 50 ml polycarbonate centrifuge tubes, and pellet the sperm by
centrifuging at 3000 x g (5000 rpm) at 4oC in a SS-34 rotor for 5
min.
- Remove and discard the supernatant.
Resuspend the pellet by trituration in 5 volumes of 20% sucrose
to osmotically remove the plasma membranes.
- 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.
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.
- 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 4oC in a SS-34 rotor.
- 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 4oC in
an SS-34 rotor.
- 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.
- Pellet the tail fragments
in 50 ml tubes by centrifugation at 12,000 x g (10,000 rpm) for 10
min at 4oC in an SS-34 rotor. This again will result in a two-layer
pellet.
- 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.
- 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.
- 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.
- 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.
- Pellet the extracted axonemes
by centrifugation at 20,000 x g (13,000 rpm) for 15 min at 4oC in
an SS-34 rotor.
- 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.
- Distribute the axonemes into
20 ul aliquots in 0.6 ml eppendorf tubes and freeze by immersion of
the tubes in liquid nitrogen.
- 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.
- Store frozen axonemes at -80oC
until use.
*=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.
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