Cryo-electron tomography reveals conserved features of doublet microtubules in flagella
Daniela Nicastro a, 1, Xiaofeng Fu a, b, Thomas Heuser a, Alan Tso a, Mary E. Porter c, and Richard W. Linck c
Author Affiliations
Edited by J. Richard McIntosh, University of Colorado, Boulder, CO, and approved August 24, 2011 (received for review May 3, 2011)
Fig. 1. Cryo -ET provides an overview of the 3D structure of DMTs. Tomographic slices (A and B) and isosurface renderings (D–F) of averaged axonemal repeats from Chlamydomonas pseudo-WT (pWT ; Table 1) show cross-sectional (A and D), longitudinal (B), and oblique (E and F) views of the DMT. The red lines in A indicate the cutting plane of the slice shown in B. In the surface renderings, only the DMT core is shown, whereas all peripheral structures [e.g., inner or outer dynein arm (IDA or ODA, respectively)] were removed but their positions are indicated in A (surface rendering overview with associated structures is shown in Fig. S1). PF numbers [according to Linck and Stephens (16)] are colored pink in the A-tubule (At) and dark blue in the B-tubule (Bt). In B, prominent left-handed helical lines with an 8-nm axial periodicity are apparent, probably corresponding to the helical lattice of tubulin subunits (28, 49). The IJ and trimeric outer junction (OJ) have distinct structures. Colored arrowheads point to MIP1 (light blue), MIP2 (red), MIP3 (yellow), and MIP4 (orange). DMT cross-sections are viewed from a proximal orientation (flagellar base) toward a distal (flagellar tip) orientation, and in the longitudinal view, the left side is proximal. The DMT orientations, labels, and colors shown here are used consistently in all subsequent figures unless otherwise noted and are valid for all panels. (C) Resolution of the DMT averages used in this study ranged from 3.3 to 3.9 nm (0.5 criterion of the Fourier shell correlation method). More details are provided in Table 1. (Scale bar: 10 nm .)
Abstract
The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin . The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the “beak” structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.
microtubule stability cilia axoneme ciliopathies cytoskeleton
Footnotes
1To whom correspondence should be addressed. E-mail: nicastro@brandeis.edu.
Author contributions: D.N. designed research; D.N., X.F., T.H., and A.T. performed research; M.E.P. contributed new reagents/analytic tools; D.N., X.F., T.H., and R.W.L. analyzed data; and D.N. and R.W.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Author Summary on page 17249.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106178108/-/DCSupplemental.
FREE PDF GRATIS: PNAS
