3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography

Abstract

We have used cryo-electron tomography to investigate the 3D structure and macromolecular organization of intact, frozenhydrated sea urchin sperm flagella in a quiescent state. The tomographic reconstructions provide information at a resolution better than 6 nm about the in situ arrangements of macromolecules that are key for flagellar motility. We have visualized the heptameric rings of the motor domains in the outer dynein arm complex and determined that they lie parallel to the plane that contains the axes of neighboring flagellar microtubules. Both the material associated with the central pair of microtubules and the radial spokes display a plane of symmetry that helps to explain the planar beat pattern of these flagella. Cryo-electron tomography has proven to be a powerful technique for helping us understand the relationships between flagellar structure and function and the design of macromolecular machines in situ.axoneme ͉ dynein ͉ microtubule ͉ motility ͉ sperm E ukaryotic cilia and flagella are highly ordered organelles that are used by a great variety of species and cell types to generate motion. Despite the diversity of their motile functions, the basic architecture of these machines is remarkably conserved (for review, see ref. 1). All such structures are built on an ordered assembly of microtubules known as the axoneme. The most widely distributed form of the axoneme consists of nine outer microtubule doublets (MTDs) and two singlet microtubules (2, 3). The combination of many structural and enzymatic studies with in vitro motility assays and computer-based simulations has laid a framework for our understanding about how flagellar motion is generated. Sliding between pairs of outer MTDs is powered by the dynein ATPases and converted into flagellar bending as a result of constraints imposed on interdoublet sliding by protein cross-links, such as nexin, which lie between adjacent MTDs (for reviews, see refs. 4 and 5). For an axoneme to generate the complex motions typical of beating cilia and flagella, there must be precise control of dynein's action, both around the circumference and along the length of the axoneme.Because of their biological and medical importance and their utility as a model for other forms of microtubule-based motility (6), flagella and cilia have been studied extensively (for reviews, see refs. 3, 5, and 7-9). However, despite the increase in knowledge about isolated components of the axoneme, much remains to be learned about the mechanical and regulatory mechanisms underlying flagellar motion (3, 10). To elucidate the role of all of the players in axoneme motility, one will need a better understanding of the organization of axonemal elements in situ at a resolution that will characterize the structural changes that they undergo during functional cycles.Sea urchin sperm tails are an admirable material for this sort of study because they are built with a classic ''9 ϩ 2'' axoneme, and their beat pattern is mainly planar, greatly simplifying the geometry of their function in compari...

Thinking about flagellar oscillation

“…Myosin in skeletal muscle is presumed to operate in this manner, and this type of operation has been assumed in modeling studies such as Brokaw [1999Brokaw [ , 2002. However, the structure and spacing of outer arm dyneins are such that mechanical interactions appear to be inevitable [Goodenough and Heuser, 1982;Burgess, 1995;Nicastro et al, 2005], just as hydrodynamic and/or mechanical interactions are inevitable in closely spaced arrays of cilia. The mechanical interactions might cause synchronous cycling of groups of dyneins, or possibly self-organized metachronal coordination along a row of dyneins, with regular phase differences between the mechanochemical cycles of individual dyneins.…”

Keeping an eye on I1: I1 dynein as a model for flagellar dynein assembly and regulation

“…1). This pattern has been elucidated in exquisite detail by rapid freeze-deep etch microscopy [Goodenough and Heuser, 1985b], analysis of inner arm mutants [Piperno et al, 1990], image analysis of thin sections [Mastronarde et al, 1992;Porter et al, 1992;Perrone et al, 1998], and most recently by cryoEM tomography of axonemes [Nicastro et al, 2005[Nicastro et al, , 2006. The I1 dynein is located in the proximal position of the 96-nm repeat, in close proximity to radial spoke S1, and is found along the entire length of the axoneme (Fig.…”

“…Although it is presumed that I1 dynein is found on all nine outer doublets, this has not yet been determined by direct localization at the ultrastructural level. Based on analysis in a number of organisms, the organization of the 96-nm repeat is a highly conserved feature of the axoneme [Goodenough and Heuser, 1985b;Woolley, 1997;Nicastro et al, 2005Nicastro et al, , 2006. Additional features of the 96-nm repeat module are the single headed inner arm dyneins, two radial spokes (S1 and S2), and the dynein regulatory complex (DRC)-structures that are critical for regulation of dynein function [Gardner et al, 1994;Piperno, 1995;Smith and Yang, 2004], as well as novel structures connecting I1 dynein and the DRC to specific outer dynein arms within the 96-nm module [Nicastro et al, 2006].…”

Single particle cryoelectron tomography characterization of the structure and structural variability of poliovirus–receptor–membrane complex at 30 Å resolution

“…Initially, this approach was applied to determine the structures of macromolecular complexes in vitro (Beck et al, 2004;Cardone et al, 2007;Chang et al, 2007;Cheng et al, 2007;Deng et al, 2007;Grunewald et al, 2003;Harris et al, 2006;Nicastro et al, 2005;Nicastro et al, 2006;Nickell et al, 2007;Zhu et al, 2006). Perhaps more excitingly, the approach has begun to be used to study macromolecular complexes within the context of intact cells.…”