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Elegant cryoelectron tomography reveals that the nexin link between microtubule doublets in 9 + 2 axonemal structures, critical for their ability to bend, is the dynein regulatory complex., Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending.

Cilia and flagella are found on a variety of cell types, ranging from single cell protozoa and sperm to the ciliated epithelia of the respiratory and reproductive tracts. Despite this diversity, most motile cilia and flagella contain a highly ordered structure, the 9 + 2 axoneme ([Fig. 1][1] A)

We have used computer averaging of electron micrographs from longitudinal and cross-sections of wild-type and mutant axonemes to determine the arrangement of the inner dynein arms in Chlamydomonas reinhardtii. Based on biochemical and morphological data, the inner arms have previously been described as consisting of three distinct subspecies, I1, I2, and I3. Our longitudinal averages revealed 10 distinguishable lobes of density per 96-nm repeating unit in the inner row of dynein arms. These lobes occurred predominantly but not exclusively in two parallel rows. We have analyzed mutant strains that are missing I1 and I2 subspecies. Cross-sectional averages of pf9 axonemes, which are missing the I1 subspecies, showed a loss of density in both the inner and outer portions of the inner arm. Averages from longitudinal images showed that three distinct lobes were missing from a single region; two of the lobes were near the outer arms but one was more inward. Serial 24-nm cross-sections of pf9 axonemes showed a complete gap at the proximal end of the repeating unit, confirming that the I1 subunit spans both inner and outer portions of the inner arm region. Examination of pf23 axonemes, which are missing both I1 and I2 subspecies, showed an additional loss almost exclusively in the inner portion of the inner arm. In longitudinal view, this additional loss occurred in three separate locations and consisted of three inwardly placed lobes, one adjacent to each of the two radial spokes and the third at the distal end of the repeating unit. These same lobes were absent ida4 axonemes, which lack only the I2 subspecies. The I2 subspecies thus does not consist of a single dynein arm subunit in the middle of the repeating unit. The radial spoke suppressor mutation, pf2, is missing four polypeptides of previously unknown location. Averages of these axonemes were missing a portion of the structures remaining in pf23 axonemes. This result suggests that polypeptides of the radial spoke control system are close to the inner dynein arms.

A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella less than 6 microns long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions causing complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner arm row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.

Termination of peripheral filaments of the axoneme of gill cilia of fresh-water mussels (Elliptio or Anodonta) occurs in characteristic fashion: (a) subfiber b of certain doublets ends leaving a single simplified tubular unit; (b) the wall of the unit becomes thick and may even obliterate the interior; and (c) the filament drops out of the 9 + 2 pattern. The order in which doublets begin simplifying is also characteristic. This may be determined by numbering the filaments, those with the bridge being 5–6, with the direction of numbering determined by the apparent enantiomorphic configuration (I to IV) of the cross-section. Shorter filaments can be identified in simplifying tips with mixed double and single peripheral units. In this material, laterofrontal cirri show a morphological specialization in the region where individual cilia simplify. The cilia studied run frontally from the body of the cirrus and point in the direction of effective stroke. The longest filaments (Nos. 3, 4, 5, 6, 7) appear as the doublets at the bottom of the cross-section, nearest the surface of the cell of origin. Above them, and above the central pair, a dark band (a section of a dense rod) runs through the matrix. The remaining filaments are the single units. Effective-pointing frontal and lateral ciliary tips end in a fashion similar to laterofrontal tips, although no dense band is present. For all effective-pointing tips studied, the order in which the peripheral filaments end appears to be Nos. (9, 1), 8, 2, 7, 6, 3, 4, 5. However, recovery-pointing lateral tips show a different order: Nos. 7, 6, 8, 5, 9, 4, 1 (3, 2), although the longer filaments are still at the bottom of the cross-section. In simple models of ciliary movement involving contraction of the peripheral filaments, filaments at the top of the cross-section should be longer, if any are. Such models are not supported by the evidence here. These results can be interpreted as supporting sliding-filament models of movement where no length change of peripheral filaments occurs.