Proteomic analysis of mammalian primary cilia - PubMed (original) (raw)
Proteomic analysis of mammalian primary cilia
Hiroaki Ishikawa et al. Curr Biol. 2012.
Abstract
The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna. Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium-shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia-specific proteins. These possible primary cilia-specific proteins include EVC2, INPP5E, and inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Figures
Figure 1
The calcium shock method efficiently isolates primary cilia from IMCD3 cells. (A, B) Immunofluorescence images of IMCD3 cells stained with acetylated-tubulin (green; primary cilia), ZO-1 (red; cell-cell junctions) and DAPI (blue; nuclei). Untreated cells (A) and cells treated with deciliation solution (B). (C) Isolated fraction includes primary cilia, which stained with acetylated-tubulin. Scale bars, 10 μm.
Figure 2
Fractionation of isolated ciliary proteins and protein correlation profiling. (A) Western blotting images of isolated primary cilia fractions, fractionated on a 30–45% continuous sucrose gradient. Acetylated-tubulin was used as a marker for ciliary proteins, while actin represents a typical non-ciliary contaminant. (B) Quantification of band intensities from Western blotting in A showing displaced but overlapping peaks of acetylated-tubulin and actin. (C, D) Graphs display the normalized abundance profiles of a set of known ciliary proteins (IFT proteins: C) and known non-ciliary proteins (focal adhesion proteins: D) based on MudPIT mass spectrometry analysis of the fractions.
Figure 3
Nocodazole and cold treatment generate non-ciliated cells for subtractive proteomic analysis. (A, B) Immunofluorescence images of IMCD3 cells stained with acetylated-tubulin (green; primary cilia), ZO-1 (red; cell-cell junctions) and pericentrin (magenta; centrosomes). Untreated cells (A) and cells treated with cold and nocodazole to retract cilia (B). Scale bars, 10 μm. (C) Western blots of isolated fractions from normal IMCD3 cells and non-ciliated (cold and nocodazole treated) cells, probed with antibodies to acetylated-tubulin (Ac-tubulin) and actin. These non-ciliated cells were used to prepare a reference fraction for subtractive proteomics.
Figure 4
Localization of candidate primary cilia proteins. (A–E) GFP-tagged candidate primary cilia protein constructs (GFP-fusion protein, green) stably expressed in transfected IMCD3 cells. Acetylated-tubulin (Ac-tubulin, red) antibody stains primary cilia. Pericentrin antibody stains centrosomes (magenta). Far left column shows merged image and DAPI (blue; nuclei). Right four columns are enlarged images. Scale bars, 5 μm.
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