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¹ Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611
² Department of Cell Biology and Genetics, Erasmus University, 3015 GE Rotterdam, Netherlands

Address correspondence to Yulia Komarova, Dept. of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611-3008. Tel.: (312) 503-2854. Fax: (312) 503-7912. E-mail: y-komarova{at}northwestern.edu

The role of plus end–tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150^Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150^Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150^Glued was not recruited back to MT plus ends. Expression of p150^Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin.

Key Words: plus end–tracking proteins; p150^Glued; cytoskeleton; dynamics; mammalian cell culture

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