New Tetrahymena basal body protein components identify basal body domain structure

Abstract

Basal bodies organize the nine doublet microtubules found in cilia. Cilia are required for a variety of cellular functions, including motility and sensing stimuli. Understanding this biochemically complex organelle requires an inventory of the molecular components and the contribution each makes to the overall structure. We define a basal body proteome and determine the specific localization of basal body components in the ciliated protozoan Tetrahymena thermophila. Using a biochemical, bioinformatic, and genetic approach, we identify 97 known and candidate basal body proteins. 24 novel T. thermophila basal body proteins were identified, 19 of which were localized to the ultrastructural level, as seen by immunoelectron microscopy. Importantly, we find proteins from several structural domains within the basal body, allowing us to reveal how each component contributes to the overall organization. Thus, we present a high resolution localization map of basal body structure highlighting important new components for future functional studies.

Figure 1.

Basal body protein preparations. (A) Pellicles were made from cells expressing γ-tubulin (with basal bodies) and cells depleted of γ-tubulin (without basal bodies) for 24 h. An enrichment of basal bodies in the γ-tubulin–expressing cells was found by several methods. The loss of basal bodies was observed by decreased and mislocalized centrin staining in pellicles prepared from cells depleted of γ-tubulin compared with wild-type cells (top). At least a ninefold decrease in the number of basal bodies in γ-tubulin–depleted pellicles compared with pellicles expressing γ-tubulin was observed by electron microscopy (not depicted). Both samples were analyzed by mass spectrometry, and the proteins were tabulated. The Venn diagram describes the total number of proteins found by MudPIT analysis. All proteins identified in the sample lacking γ-tubulin were removed from the study. (B) Western blots of pellicles from cells with (+GTU) or without γ-tubulin (−GTU) show a decrease in three basal body proteins: γ-tubulin, α-tubulin, and centrin. Equal numbers of pellicles were loaded for each lane. (C) Basal body proteins were also prepared using isolated oral apparatuses. Oral apparatuses in whole cells and after biochemical isolation were visualized using GFP–α-tubulin. Basal bodies were prepared using modified methods (see Basal body lysate preparation section in Materials and methods; Wolfe, 1970). Isolated oral apparatuses are shown by fluorescence (left; inset of a single isolated oral apparatus) and electron microscopy (several rows of basal bodies are shown in cross section). Basal body proteins were extracted from the oral apparatus network producing the oral apparatus lysate. The electron micrograph of the extracted oral apparatuses (pellet) shows that basal bodies are no longer present; however, the underlying substructure of the oral apparatus remains. The final oral apparatus lysate was analyzed by MudPIT.

Figure 2.

24 new basal body proteins were identified by mass spectrometry. (A) Based on BLAST analysis and gene ontology codes, proteins were classified into eight broad categories: (1) basal body, proteins known to associate with basal bodies/centrioles; (2) candidate, conserved proteins chosen as likely basal body candidates (BLAST e value <10⁻⁶); (3) cilia, proteins known to associate with cilia; (4) other, a broad category of proteins involved in a variety of biological processes other than centriole function; (5) metabolism, proteins involved in metabolic processes; (6) mitochondria, proteins involved in mitochondrial function; (7) transcription/translation (TX/TN), proteins involved in RNA and protein production; and (8) not conserved, proteins with no vertebrate homologues (BLAST e value >10⁻⁶). Detailed information for each protein is provided in Tables S1 and S2 (available at http://www.jcb.org/cgi/content/full/jcb.200703109/DC1). (B) Basal body components found and verified by live cell fluorescence imaging and immunoelectron microscopy. Protein name, domains, fluorescence localization, and ultrastructural localization are described. The percentages refer to the percentages of total gold particles localized to a specific domain by immunoelectron microscopy. The ultrastructural domains are illustrated in Fig. 3 D. BB, basal body; NA, no immunoelectron microscopy was performed; MT, microtubule.

Figure 3.

Localization of selected basal body components. (A) Live cell images of GFP-tagged Cen1, Bbc23, Bbc78, Bbc82, Sas6a, Bbc31, Bbc14, and Bbc20. Cen1, a known basal body component and a proteomics hit, is shown as a control. (B) Schematic of the organization of basal bodies in a T. thermophila cell. Basal bodies are found in cortical rows and at the oral apparatus. (C) Immunoelectron microscopy of longitudinal basal body sections from cells expressing GFP fusion proteins: Cen1, Bbc23, Bbc78, Bbc82, Sas-6a, Bbc31, Bbc14, and Bbc20. Cells were indirectly labeled with anti-GFP antibodies and gold-conjugated secondary antibodies. Locations of the gold particles are highlighted with arrowheads. (D) Schematic of the organization of a basal body. The structural domains shown are the (1) site of nascent basal body assembly, (2) cartwheel, (3) microtubule scaffold, (4) collar, (5) midpoint, (6) transition zone, (7) lumen, and (8) postciliary microtubules. The posterior and the anterior sides of the basal body in reference to the cell geometry are indicated. Bars (A), 10 μm; (C) 200 nm.

Figure 4.

New T. thermophila basal body components were shown to localize to discrete structural domains. Protein localizations were assigned to specific domains if at least 20% of all gold particles in the immunoelectron microscopy compilation images are associated with the region. New protein components are listed in Fig. 2 B and in Figs. S1–3 (available at http://www.jcb.org/cgi/content/full/jcb.200703109/DC1). Color reference and domain are listed for each new basal body component described.