Jacob Morville Schrøder:
EB1 is a small microtubule (MT)‐binding protein that associates preferentially with MT plus ends. EB1 plays a role in regulating MT dynamics, localizing other MT‐associated proteins to the plus end, and in regulating interactions of MTs with the cell cortex, mitotic kinetochores and different cellular organelles (Lansbergen and Akhmanova, 2006). EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network (Askham et al., 2002). Further, EB1 has been shown to localize to the flagellar tip and proximal region of the basal bodies in the biflagellate green alga Chlamydomonas (Pedersen et al., 2003), and is required for ciliogenesis in mouse fibroblasts (Schroder et al., 2007). However, the exact mechanism(s) involved and roles of the two additional mammalian members of the end binding (EB) protein family, EB2 and EB3, in ciliogenesis are unclear. Ebs contain a highly conserved N‐terminal calponin homology (CH) domain, which is involved in MT binding. The C‐terminal region is characterized by a coiled coil domain, which mediates EB homo‐ or heterodimerization. All three EB species exist as homodimers in vivo and in vitro, and EB1 and EB3 also form a heterodimeric complex that is likely to be functionally distinct from the homodimeric complexes (Komarova et al., 2009; De Groot et al., 2010).
This thesis is based on experiments using small interfering (si) RNA and dominant‐negative constructs to show that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured human cells. The EB3 ‐ siRNA ciliary phenotype could be rescued by GFP‐EB1 expression, and GFP‐EB3 over expression resulted in elongated cilia. Transmission electron microscopy (TEM) revealed that EB3‐depleted cells possess stumpy cilia, a disorganized centrosomal MT array and abnormally long centriole‐associated rootlet filaments. Cells lacking EB1 also had stumpy cilia and a disorganized centrosomal MT array, but rootlet filaments appeared normal. Further, live imaging revealed increased release frequency of MTs from the centrosome upon EB1 or EB3 depletion, indicating a role for both EB1 and EB3 in MT minus end anchoring at the centrosome. Finally, partial depletion of p150 Glued inhibited cilia formation indicating possible cooperation between Ebs and p150 Glued during ciliogenesis.
Collectively, these studies suggest that Ebs affect ciliogenesis by two different mechanisms: by promoting MT minus end anchoring at the basal body and by stabilizing axonemal MTs. In addition, this thesis proposes that EB3 plays a specific role in regulating the formation of centriole‐associated rootlet filaments.