Preparation of microtubule-associated motor proteins from Dictyostelium using exogenous taxol-stabilized microtubules

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Procedure

1. Grow Dicty cells axenically to mid or late log phase.


2. Harvest cells by centrifugation, wash 2X in DB.


3. Resuspend cells in an equal volume of P100 pH 6.8 buffer containing freshly added:
   
   
   • 5 mM DTT
   • 10 µg/ml leupeptin
   • 20 µg/ml pepstatin
   • 100 µM PMSF
   • 1 mg/ml phenanthroline
   
   
4. Sonicate gently on ice (large tip, 10% power, setting 2) for 20 second bursts until approximately 95% lysis is reached.


5. Centrifuge at 142,000 g (35 K rpm in 45Ti) at 2°C for 1.5 h. Collect super (HSS 1).


6. To supernatant, add 3 U/ml apyrase (Sigma type V). Incubate at room temperature 15 min. Then add glycerol to 10% final concentration.


7. Centrifuge at 142,000 g at 2°C for 1.5 h. Collect super (HSS 2). Filter supernatant through a 1.2 µm filter.


8. To supernatant, add taxol to 10 µM and 200 µg/ml DEAE purified, taxol stabilized microtubules. Incubate on ice 15 min to allow motors to bind to MT.


9. Pellet microtubules by centrifugation at 38,000 g (18K in SS34) at 4°C for 30 min.


10. Wash: resuspend microtubule pellet in 1/5 of the original cell volume in P100G buffer/10 µM taxol, pH 6.8. Homogenize in glass on glass homogenizer to break up pellet. Repellet microtubules as above.


11. ATP extraction: resuspend microtubule pellet in 0.01-0.02% of original cell volume in P100G/10 mM Mg-ATP/10 µM taxol to dissociate Dicty motor proteins. Incubate RT 10 min, then 37°C 5 min.


12. Sediment microtubules by centrifugation as above. Layer this ATP extract onto a 5-20% sucrose gradient in P100 and spin 31,000 rpm, 16 h, in SW 41 rotor, 4°C. Collect fractions from the bottom. Dynein will sediment at 20S, kinesin at 9S.

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Materials

• P100 buffer
  • 100 mM PIPES, pH 6.8
  • 5 mM MgSO₄
  • 5 mM EGTA
  • 0.1 mM EDTA
  
  
• P100G buffer
  • P100 with 10% glycerol


• Protease inhibitor stocks
  • DTT (500 mM in water), dilute 1:100. Make up fresh!
  • Leupeptin (10 mg/ml quick-frozen in water), dilute 1:1000
  • Pepstatin (10 mg/ml in DMSO), dilute 1:500
  • PMSF (100 mM in isopropanol), dilute 1:100
  • Phenanthroline (100 mg/ml in ethanol), dilute 1:100
  
  
• Taxol
  • 10 mM stock in DMSO, dilute 1:1000 for 10 µM
  
  
• Taxol-stabilized microtubules
  • To thawed DEAE purified bovine tubulin (3-10 mg/ml), add taxol to 10 µM.
  • Incubate at 37°C for 5 minutes.
  • Add taxol to 100 µM, incubate an additional 10 min at 37°C.
  
  

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