Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends.
Microtubule (MT) plus end-tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.
Dragestein
Katharina A
KA
Department of Cell Biology and Genetics, Erasmus Medical Center, 3000 DR Rotterdam, Netherlands.
van Cappellen
Wiggert A
WA
van Haren
Jeffrey
J
Tsibidis
George D
GD
Akhmanova
Anna
A
Knoch
Tobias A
TA
Grosveld
Frank
F
Galjart
Niels
N
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
C522423:EB3 protein, mouse; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C076744:cytoplasmic linker protein 170