Biophysicists reveal how three proteins interact to fine-tune cellular movement EmoryUniversity NatureComms
. F-actin was incubated for 2 h at room temperature with a fivefold molar excess of Alexa-488 NHS ester dye . F-actin was then pelleted by centrifugation at 450,000 ×for 40 min at room temperature, and the pellet was resuspended in G-buffer, homogenized with a Dounce, and incubated on ice for 2 h to depolymerize the filaments. The monomeric actin was then re-polymerized on ice for 1 h by the addition of 100 mM KCl and 1 mM MgClat 4 °C.
. Cells were grown in Terrific Broth to log phase at 37 °C. Expression was induced overnight at 18 °C by the addition of 1 mM IPTG. Cells were harvested by centrifugation at 11,200 ×for 15 min and the cell pellets were stored at −80 °C. For purification, frozen pellets were thawed and resuspended in 35 mL lysis buffer ). Cells were lysed using a tip sonicator while being kept on ice. The cell lysate was then centrifuged at 120,000 ×for 45 min at 4 °C.
For 649-mDia1, 549-CP, and 488-actin experiments , 0.5 µM G-actin , 1 µM profilin, along with 50 pM 649-mDia1 in TIRF buffer were introduced into the flow cell and filaments were allowed to grow for 2 to 3 min. The flow cell was then rinsed with TIRF buffer to remove free formins, and the solution was replaced with profilin-actin and 10 nM 549-CP .
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