Cytoplasmic linker proteins promote microtubule rescue in vivo.
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 p150Glued 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 p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued 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.
Komarova
Yulia A
YA
Department of Cell and Molecular Biology, N University Medical School, Chicago, IL 60611, USA. y-komarova@northwestern.edu
Akhmanova
Anna S
AS
Kojima
Shin-Ichiro
S
Galjart
Niels
N
Borisy
Gary G
GG
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D000072159:Dynactin Complex; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009363:Neoplasm Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000072159:Dynactin Complex; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C076744:cytoplasmic linker protein 170