Abstract
BackgroundPrimary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood.ResultsEndogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium.ConclusionThe regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium.
Highlights
Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling
Localization of I-2 in the primary cilium of human retinal pigment epithelial cells We found using laser confocal fluorescent microscopy that endogenous phosphatase inhibitor 2 (I-2) concentrated in the primary cilium of confluent human diploid retinal epithelial ARPE-19 cells (Fig. 1)
Immunoblotting identified acetylated tubulin, PP1C and I-2 in the cilia fraction from post-confluent cells that were not in the cilia fraction of sub-confluent cells (Fig. 4C). These results showed that acetylated tubulin, I-2 and PP1C were recovered in the primary cilium by biochemical fractionation, which reinforces the immunostaining of I-2 in the primary cilium of individual cells
Summary
Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. Cilia are projections from the surface of cells that are similar to flagella. The axoneme of a primary cilium is made up of microtubules. Almost every cell in vertebrates has a single primary cilium with a 9+0 arrangement [2], which lacks the central pair of microtubules seen in the 9+2 arrangement of flagella and motile cilia. Recent studies suggest that a variety of human syndromes are related to defects in the assembly, maintenance and function of the primary cilium, including renal dysfunction, diabetes, and retinal degeneration [3,4]
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