EB1 is required for primary cilia assembly in fibroblasts.
EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and different cellular organelles [1, 2]. EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network [3, 4]. We previously showed that EB1 localizes to the flagellar tip and proximal region of the basal body in Chlamydomonas[5], but the function of EB1 in the cilium/flagellum is unknown. We depleted EB1 from NIH3T3 fibroblasts by using siRNA and found that EB1 depletion causes a approximately 50% reduction in the efficiency of primary cilia assembly in serum-starved cells. Expression of dominant-negative EB1 also inhibited cilia formation, and expression of mutant dominant-negative EB1 constructs suggested that binding of EB1 to p150(Glued) is important for cilia assembly. Finally, expression of a C-terminal fragment of the centrosomal protein CAP350, which removes EB1 from the centrosome but not MT plus ends [6], also inhibited ciliogenesis. We conclude that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts.
Schrøder
Jacob M
JM
Department of Molecular Biology, Section of Cell and Developmental Biology, The August Krogh Building, University of Copenhagen, Universitetsparken 13, Copenhagen, Denmark.
Schneider
Linda
L
Christensen
Søren T
ST
Pedersen
Lotte B
LB
D000818:Animals; D000941:Antigens / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002502:Centrioles / Q000378:metabolism; D002923:Cilia / Q000378:metabolism*; D000072159:Dynactin Complex; D005347:Fibroblasts / Q000378:metabolism* / Q000502:physiology; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D041681:NIH 3T3 Cells; D034741:RNA, Small Interfering
D000941:Antigens; C000606364:Dctn1 protein, mouse; D000072159:Dynactin Complex; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C086178:pericentrin; D049452:Green Fluorescent Proteins