Rootletin forms centriole-associated filaments and functions in centrosome cohesion

Abstract

After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA–mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.

Rootletin Interacts with C-Nap1 and May Function as a Physical Linker between the Pair of Centrioles/Basal Bodies in Cells

“…While this manuscript was in preparation, an article was published by another group of investigators that had reached a similar conclusion (Bahe et al, 2005). The authors of that study identified the interaction between rootletin and C-Nap1 of human origin by yeast two-hybrid analysis.…”

The Centrosome and Its Duplication Cycle

“…5A) (Fry et al 1998;Mayor et al 2000;Bahe et al 2005;Yang et al 2006). Depletion of C-Nap1 or rootletin causes centrosome splitting regardless of the cell-cycle stage (Mayor et al 2000;Bahe et al 2005).…”

“…5A) (Fry et al 1998;Mayor et al 2000;Bahe et al 2005;Yang et al 2006). Depletion of C-Nap1 or rootletin causes centrosome splitting regardless of the cell-cycle stage (Mayor et al 2000;Bahe et al 2005). Immuno-EM revealed C-Nap1's localization to the proximal ends of the connected centrioles but not between them, whereas rootletin is present both at ends and between the centriole pairs in nonciliated cells and, in addition, connected to basal bodies in ciliated cells (Fry et al 1998;Mayor et al 2000;Bahe et al 2005;Yang et al 2006).…”

“…Cep68 also decorates fibers emanating from the proximal ends of centrioles, dissociates from centrosomes during mitosis, and requires rootletin and C-Nap1 for centrosome localization. Depletion of rootletin does not affect the association of C-Nap1 with centrosomes, whereas either depletion of C-Nap1 or overexpression of its fragments affects the centrosomal localization of rootletin (Bahe et al 2005;Yang et al 2006). Phosphorylation of both proteins by Nek2A (Fry et al 1998;Helps et al 2000; Bahe al.…”

The Set1/COMPASS Histone H3 Methyltransferase Helps Regulate Mitosis With the CDK1 and NIMA Mitotic Kinases in Aspergillus nidulans

“…The mitotic functions of NIMA are conserved in eukaryotic cells, with NIMA being the founding member of the family of NIMA-related kinases called the Nek kinases (Fry et al 2012). Human Nek kinases have been shown to share NIMA's function in regulating the bipolar spindle formation as well as the permeability of the nuclear envelope at mitotic entry (Fry et al 1998;Bahe et al 2005;Bahmanyar et al 2008;Bertran et al 2011;Laurell et al 2011). Consistent with these conserved functions, ectopic expression of NIMA in organisms such as fission yeast, Xenopus, and human cells results in premature mitotic characteristics, including chromatin condensation (O'Connell et al 1994;Lu and Hunter 1995).…”