page 725, Cho and Klemke describe a clever method for purifying pseudopodia that have been induced to grow or retract, and identify a signaling complex whose assembly and disassembly controls these processes.
Using a chemoattractant, the authors induced cultured mammalian cells to extend pseudopodia through 3.0-μm pores in a membrane. Under these conditions, over 90% of the cells extend a single leading pseudopodium, allowing Cho and Klemke to scrape away the cell bodies and extract the remaining pseudopodia with detergent. Because removal of the chemoattractant causes the pseudopodia to begin retracting, the system permits the purification of either growing or retracting pseudopodia for direct comparisons.
Analysis of the purified pseudopodia shows that the assembly of a p130Crk-associated substrate (CAS)/c-CrkII (Crk) scaffold leads to Rac1 translocation and activation during pseudopod growth, and Rac1 activation provides a positive feedback loop to maintain the CAS/Crk scaffold. Disassembly of the CAS/Crk scaffold is required for a decrease in Rac1 activity, which in turn induces pseudopod retraction.
The new work simultaneously defines new aspects of signaling in pseudopod growth and retraction, and describes a technique that should be broadly useful in future studies of cell migration. In addition to allowing the purification of pseudopodia for biochemical analysis, the system is quantitative and permits time-lapse imaging of pseudopodia in living cells. ▪