The Molecular Architecture of Axonemes Revealed by Cryoelectron Tomography

Abstract

Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for the way dynein generates force to slide microtubules. They also reveal two dynein linkers that may provide "hard-wiring" to coordinate motor enzyme action, both circumferentially and along the axoneme. Periodic densities were also observed inside doublet microtubules; these may contribute to doublet stability.

Functional protofilament numbering of ciliary, flagellar, and centriolar microtubules

“…Cryo-EM tomography studies on sea urchin flagella by Nicastro et al [2006] resolved the 10 B-PFs and the B-11th component between PFs A 1 and B 10 , but not its possible axial periodicity or discontinuity. The cryo-EM tomography of Sui and Downing [2006] on sea urchin flagella also resolved 10 B-PFs, but differs from other reports in that it suggests a continuous gap between the A-and B-tubules with no component in the gap; instead, a linker was described running from PFs A 12-13 across the B-lumen to PFs B 9-10 (converting their PF numbering to the convention presented here- Fig.…”

“…All views are from the proximal base, looking toward the distal tip. The binding positions (brackets) of the radial spokes (RS) and inner dynein arms (IDA) are approximated from Nicastro et al [2006]; these positions are not bracketed in panel (a), due to the uncertainty of the PF identities in that diagram.…”

“…(iii) The ''seam'' (the mismatch between a-and b-tubulin subunits along the 3-start monomer helix of the B-lattice) is reported to lie between PFs A 1 and A 2 [Song and Mandelkow, 1995]. (iv) Most of the ''action'' seems to be focused on the first four PFs, A 1 -A 4 , along which the major forceproducing and regulatory components are anchored, including inner dynein arms, the dynein regulatory complex, radial spokes, and interdoublet nexin link components Heuser, 1982, 1985;Stephens et al, 1989;Porter and Sale, 2000;Lindemann, 2003;Rupp and Porter, 2003;Nicastro et al, 2006;Ralston and Hill, 2006;Setter et al, 2006;Wirschell et al, in press].…”

“…1, has a number of compelling advantages. Aside from the historic precedent of the base-totip CCW numbering [Tilney et al, 1973;Linck, 1976;Nojima et al, 1995;Song and Mandelkow, 1995;Nicastro et al, 2006;Setter et al, 2006], a CCW convention permits PF B 10 to lie adjacent to the ephemeral B-11th component, rather than the more confusing CW juxtaposition of PF B 1 and the B-11th component. Even though the B-11th component will eventually be identified and appropriately named, there are a sufficient number of reports of its existence that its adjacent B-tubulin PF be numbered B 10 .…”

“…The wide-ranging and essential functions of these organelles help to explain the prevalence of the many varied diseases and developmental disorders that arise from mutations in ciliary, flagellar and centriolar genes [Afzelius, 2004;Eley et al, 2005;Keller et al, 2005;Pazour et al, 2005]. Several recent studies have now highlighted the importance of the underlying arrangement and composition of the microtubule protofilaments (PFs) and their associated proteins in ciliary/flagellar assembly and their motile/sensory functions [Marshall and Nonaka, 2006 -for review;Rupp and Porter, 2003;Nicastro et al, 2006;Ralston and Hill, 2006;Setter et al, 2006;Sui and Downing, 2006;Wirschell et al, in press].…”