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. 2009 Feb;20(3):904-14.
doi: 10.1091/mbc.e08-08-0835. Epub 2008 Dec 3.

Basal body components exhibit differential protein dynamics during nascent basal body assembly

Chad G Pearson  1 , Thomas H Giddings Jr, Mark Winey
Affiliations

Basal body components exhibit differential protein dynamics during nascent basal body assembly

Chad G Pearson et al. Mol Biol Cell. 2009 Feb.

Abstract

Basal bodies organize cilia that are responsible for both mechanical beating and sensation. Nascent basal body assembly follows a series of well characterized morphological events; however, the proteins and their assembly dynamics for new basal body formation and function are not well understood. High-resolution light and electron microscopy studies were performed in Tetrahymena thermophila to determine how proteins assemble into the structure. We identify unique dynamics at basal bodies for each of the four proteins analyzed (alpha-tubulin, Spag6, centrin, and Sas6a). alpha-Tubulin incorporates only during new basal body assembly, Spag6 continuously exchanges at basal bodies, and centrin and Sas6a exhibit both of these patterns. Centrin loads and exchanges at the basal body distal end and stably incorporates during new basal body assembly at the nascent site of assembly and the microtubule cylinder. Conversely, both dynamic and stable populations of Sas6a are found only at a single site, the cartwheel. The bimodal dynamics found for centrin and Sas6a reveal unique protein assembly mechanisms at basal bodies that may reflect novel functions for these important basal body and centriolar proteins.

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Figures

Figure 1.
Figure 1.
Differential centrin assembly at basal bodies. (A) Two possible outcomes for GFP-Cen1 expression: 1) stable incorporation only during new basal body assembly (top cell) or 2) dynamic incorporation throughout the cell cycle (bottom cell). Differentially labeled (arrow) and unlabeled (arrowhead) basal bodies indicate GFP-Cen1 assembly occurs conservatively during new basal body assembly (top cell). Basal body duplication occurs in the medial region of dividing cells with new, daughter basal bodies (inset, arrow) assembling anterior to old, parent basal bodies (inset, arrowhead). Alternatively, uniformly labeled basal bodies (lower cell) indicate proteins are incorporated independent of basal body assembly. (B) GFP-Cen1 expression for 2 h caused all basal bodies to be labeled. A fraction of the basal bodies within the cell median exhibit increased fluorescence compared with the majority of the basal bodies. GFP-Cen1 (green) was followed relative to basal bodies labeled with anti-Cen1 (red) antibodies showing uniform protein levels. The OAs (arrows), composed of basal bodies, are differentially labeled so that the old OA (arrowhead) at the cell anterior has decreased fluorescence relative to the new OA (arrow) at the cell median. After 8 h of GFP-Cen1 expression (∼3 to 4 cell cycles), the majority of basal bodies were equally labeled, indicating that most basal bodies have duplicated, incorporated, and retained high levels of GFP-Cen1. Bar, 10 μm.
Figure 2.
Figure 2.
GFP-Cen1 incorporates coincident with new basal body assembly. (A) The brightly labeled basal bodies were quantified relative to quadrants (I, II, III, and IV) within dividing cells. The greatest frequencies of bright basal bodies were found in quandrants II and III, consistent with the cellular locations of most new basal bodies (Kaczanowski, 1978). The frequency (percentage) of bright GFP-Cen1–labeled basal bodies in each quadrant is described for 15 cells (1204 basal bodies). (B) Antibodies to K-antigens (red) were used to define old basal bodies compared with GFP-Cen1 label (green). Each panel contains a segment of a representative ciliary row. The bright GFP-Cen1 signal is found at new basal bodies (arrow; low or no K-antigen), whereas dim GFP-Cen1 is coincident with K-antigen signal marking older basal bodies (arrowhead). (C) Cells were arrested by starvation before induction of GFP-Cen1 for 2 h. GFP-Cen1 exhibited low fluorescence signal at each basal body. On release into the cell cycle by refeeding with media for 2 h (release) differential labeling by GFP-Cen1 was observed. Panel width, 2.1 μm.
Figure 3.
Figure 3.
Distinct classes of basal body protein dynamics. (A) GFP-Sas6a and -Atu1 are incorporated into new basal bodies (arrow) that are not labeled with anti-K-antigen (arrowhead; old basal body). GFP-Spag6 exhibits equal intensity at new and old basal bodies. (B) GFP-Sas6a, -Atu1, and -Spag6 fluorescence intensity (green) relative to anti-Cen1 (red) was determined at 0, 2, and 8 h after GFP fusion protein expression. After 2 h, anti-Cen1 labeling (red) remained constant, whereas GFP-tagged proteins exhibited three distinct classes of assembly at basal bodies. As with Cen1, Sas6a associated with basal bodies to a low level (arrowhead), whereas the majority of incorporation occurred during new basal body assembly (arrow). Atu1 associated with basal bodies only during new basal body assembly (arrow). Spag6 incorporation at basal bodies is independent of new basal body assembly. Panel width, 2.1 μm.
Figure 4.
Figure 4.
Duplication dependent basal body protein incorporation requires cell cycle progression. (A) To determine whether basal body assembly and the differential labeling of basal bodies by each GFP-labeled protein is dependent upon cycle progression, cells were starved for ∼14 h before GFP-labeled proteins were expressed for 2 h. Cells were then refed media and grown for 2 h to determine whether the expressed protein was incorporated at basal bodies. (B) Low levels of homogenous signal (i.e., no differentially bright and dim labeled OAs) were detected at GFP-Sas6a– and -Atu1–labeled basal bodies. GFP-Spag6 brightly labeled basal bodies uniformly. (C) On release for 2 h, GFP-Sas6a and -Atu1 incorporated at basal bodies with dim and bright fluorescence. GFP-Spag6 levels of protein incorporation were constant at all basal bodies. Panel width, 2.1 μm.
Figure 5.
Figure 5.
Domain localization of bimodal Centrin and Sas6a basal body incorporation. (A) Centrin IEM localization in starved (no basal body duplication) or asynchronous (incorporation at duplicated basal bodies) cells expressing GFP-Cen1. The majority of Cen1 loaded at the basal body distal end, independent of new basal body assembly. GFP-Cen1 incorporated at all three major Cen1-localizing domains after stable and dynamics incorporation during new basal body assembly. (B) Both dynamic (starved) and dynamic and stable (asynchronous) populations of GFP-Sas6a predominantly localize to the cartwheel hub and spokes. On new basal body assembly, the GFP-Sas6a is more focused at the cartwheel. Each condition represents a total count of at least 150 gold particles for at least 30 basal bodies. Cartoons display the relative fraction of gold localization to each domain with a total of 25 red spots. Bar, 100 nm.
Figure 6.
Figure 6.
Rates of dynamic basal body protein exchange. Protein exchange at duplicated basal bodies was measured using FRAP to determine the GFP-fusion protein dynamics over a short time period (5 min). GFP-labeled Cen1 (A), -Sas6a (B), -Atu1 (C), and -Spag6 (D) (prebleach; t = −0.1 s) were photobleached with a short laser exposure (postbleach; t = 0.0 s) before the recovery was quantified in subsequent time points. The average fluorescence recovery is shown for each protein (squares) relative to the best fit exponential recovery (diamonds). GFP-Cen1 and -Sas6a exhibit similar percent recoveries but different recovery rates. Alternatively, GFP-Atu1 shows low levels of recovery, whereas GFP-Spag6 has a high level of rapid recovery. Arrows indicate the photobleached basal body. The average t1/2 recovery rate and percentage of recovery are indicated for each protein. Time, seconds. Panel width, 4.0 μm.
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