Computer-assisted image analysis of human cilia and Chlamydomonas flagella reveals both similarities and differences in axoneme structure

Abstract

In the past decade, investigations from several different fields have revealed the critical role of cilia in human health and disease. Because of the highly conserved nature of the basic axonemal structure, many different model systems have proven useful for the study of ciliopathies, especially the unicellular, biflagellate green alga Chlamydomonas reinhardtii. Although the basic axonemal structure of cilia and flagella is highly conserved, these organelles often perform specialized functions unique to the cell or tissue in which they are found. These differences in function are likely reflected in differences in structural organization. In this work, we directly compare the structure of isolated axonemes from human cilia and Chlamydomonas flagella to identify similarities and differences that potentially play key roles in determining their functionality. Using transmission electron microscopy and 2D image averaging techniques, our analysis has confirmed the overall structural similarity between these two species, but also revealed clear differences in the structure of the outer dynein arms, the central pair projections, and the radial spokes. We also show how the application of 2D image averaging can clarify the underlying structural defects associated with primary ciliary dyskinesia (PCD). Overall, our results document the remarkable similarity between these two structures separated evolutionarily by over a billion years, while highlighting several significant differences, and demonstrate the potential of 2D image averaging to improve the diagnosis and understanding of PCD.

Figure 1. Axoneme cross sections from Chlamydomonas flagella and human cilia reveal similarities and differences.

(A) Typical cross section of a Chlamydomonas axoneme shows the typical 9 +2 arrangement of outer microtubule doublets surrounding a central pair complex. Doublet microtubules contain attached inner and outer dynein arms with doublet number one missing the outer arm (*). (B) Axonemes isolated from human respiratory cilia also contain the classic 9+2 arrangement of microtubules with all outer doublets containing an outer dynein arm. Central pair projections and radial spokes surround the central pair microtubules. (C) Chlamydomonas average containing 140 outer doublet cross sections. The outer dynein arm contains distinct lobes including an outer density (arrow). (D) Human ciliary average containing 110 doublets shows outer dynein arm density is reduced compared to Chlamydomonas (arrow). Bar = 50 nm A,B and 25 nm C,D.

Figure 2. Central pair (CP) complex averages from Chlamydomonas and human cilia.

(A) Chlamydomonas average from 45 CP complexes. (B) Schematic of CP complex organization in Chlamydomonas based on the densities present in the Chlamydomonas average (modified from Lechtreck et al. 2008; Mitchell and Smith 2009). The CP microtubules (C1 and C2) are connected by a bipartite bridge (B) and diagonal linker (DL). CP projections extend out from the CP microtubules and the density of C1a is enhanced relative to human (*). (C) Human cilia average from 46 central pair complexes. Radial spokes are enhanced in the average. (D) Schematic of CP complex organization in human cilia based on the densities present in the human average. CP microtubules are connected by a bipartite bridge (B) and a diagonal linker (DL) is present. C1 and C2 projections are present. The density of the C2 projections is enhanced relative to Chlamydomonas (*). An additional density adjacent to C1c projection is present (*). Bar = 25 nm.

Figure 3. The complexity of inner arm organization is revealed in longitudinal averages from human cilia.

(A) Axonemes in longitudinal view are selected that contain two doublets separated by the central pair microtubules. The top doublet contains outer and inner dynein arms and radial spokes. Model points (black dots) show the center of the 96nm inner dynein arm repeat used for averaging. (B) Individual average containing the 12 repeats shown above. (C) Grand average from 13 axonemes and a total of 120 repeats. (D) Combined grand average from 2 people containing a total of 25 axonemes and 223 repeats. Bar = 100 nm (A); 25 nm (B-D)

Figure 4. Analysis of axoneme longitudinal averages from Chlamydomonas flagella and human cilia.

(A) Averages from Chlamydomonas flagella based on a 96 nm repeating unit centered around 2 radial spokes. 2D averages resolve at least 10 densities in the inner dynein arm region. (B) Averages from human cilia show remarkable similarity in inner arm organization. Three radial spokes are present. (C) Diagram of densities within the Chlamydomonas 96 nm repeat and difference plots showing statistically significant differences between Chlamydomonas and human. Inner dynein arm organization is similar but human cilia have a reduced density corresponding to the dynein regulatory complex and a reduced density corresponding to a lobe distal the DRC. (D) Composite diagram showing inner arm organization along the 96 nm repeat with structures unique to Chlamydomas shaded in light gray and structures unique to human in dark gray (model modified from Porter and Sale 2000). Bar = 25 nm.

Figure 5. Image averaging is useful for analyzing defects in human ciliary axonemes.

(A) Average of 110 microtubule doublets imaged in cross section from axonemes of a normal individual. (B) An average of 132 doublet microtubules observed in cross-section from axonemes of a PCD patient. The density of the inner dynein arm is clearly reduced. (C) Average from a patient with cystic fibrosis (disease control, containing 109 doublets) shows dynein organization similar to the average from the normal individual. (D) Average of longitudinal sections from normal axonemes (25 axonemes/ 223 repeats) and (E) average of longitudinal sections obtained from the PCD patient (8 axonemes/ 65 repeats. (F) Difference image between normal and PCD axonemes shows the reduced inner dynein arm density in the axonemes from the PCD patient.