[email protected] focus of this chapter is on the highly conserved axonemal structures involved in the generation and regulation of ciliary and flagellar motility and the current models for bend propagation and waveform control are outlined. The activation of dynein motors by second messengers and phosphorylation are described in Chlamydomonas and other protists. 10.1 INTRODUCTION

The dynein motors were originally defined by Ian R. Gibbons in his studies of the ciliary axoneme using biochemical fractionation, in vitro reconstitution and electron microscopy, revealing that the arm-like structures-dynein arms-contain the ATPase and motor activity responsible for motility [44]. Working with Barbara Gibbons, he then went on to develop the sea urchin sperm as a very powerful system for study of flagellar motility. Early advances included development of experimental conditions for successful in vitro ATP-induced reactivation of axonemal bending and recognition that both the outer dynein arms and inner dynein arms (Fig. 10.1) contribute to axonemal bending [42, 43]. Moreover, taking advantage of dark-field microscopy, Summers and Gibbons were the first to observe dynein-driven microtubule sliding of axonemal outer doublet microtubules [159, 160]. This was a key Handbook of Dynein Edited by Keiko Hirose and Linda A. Amos Copyright © 2012 Pan Stanford Publishing Pte. Ltd. www.panstanford.com

advance, demonstrating that the dynein motors convert the energy of ATP into translocation and sliding between adjacent microtubules.Here, we review the structure of the 9 + 2 axoneme, emphasizing the axis of the axoneme relative to the bending plane. We also emphasize and review the numerous axonemal dynein isoforms, each unique in composition and function and anchored in a regular, highly conserved 96 nm repeating module defined by flagellar mutants from Chlamydomonas and high resolution analysis of structure, including recent advances in cryoEM tomography ([20, 21, 61, 108]; reviewed in Chapter 11 and [83, 112]). We review evidence for a sliding microtubule model for axonemal bending and discuss a “switching” model for alternating forward and reverse bends; models supported, in part, by studies from Peter Satir, Charles Brokaw, Ian Gibbons, Keichi Takahashi, and colleagues. We briefly review evidence that the axoneme and dynein motors have the inherent capacity to generate oscillatory bending, likely responding to mechanical feedback for switching bend direction, results featured in the experimental work from Chikako Shingyoji, Ritsu Kamiya, and colleagues. Among the unsolved problems is how axonemal dynein activity is turned on or off and locally regulated to produce oscillatory bending or bend propagation (discussed in an outstanding review by Brokaw [16]). Theoretical discussions and mathematical models for regulation of dynein and axonemal bending are also considered in important papers by Charles Brokaw and Charles Lindemann [16, 93, 94] and are only briefly discussed in this chapter. We then focus on other axonemal structures-including the central pair apparatus, radial spokes, and the dynein regulatory complex (DRC)—and conserved, axonemal proteins that respond to second messengers-calcium and cyclic nucleotides-to specifically alter dynein activity to control the beat frequency or the size and shape of the flagellar bend. The mechanism of regulation often involves changes in phosphorylation of dynein subunits, particularly the IC138 intermediate chain subunit of I1 dynein (also called dynein f). However, the mechanism for how phosphorylation changes dynein activity is not known. The chapter ends with an overview of challenges and opportunities for understanding the role of the ciliary dyneins. 10.2 AXONEME STRUCTURAL ORGANIZATION AND

DYNEIN SUBFORMSThe axoneme is a highly conserved, cytoskeletal structure that forms the core of cilia and flagella organelles. It harbors structural elements that generate and regulate ciliary movement. The axonemal dyneins, the inner and outer dynein arms, drive outer doublet microtubule sliding, the

mechanism underlying ciliary bending. This chapter primarily focuses on the regulatory structures, mechanisms, and signaling molecules, revealed in studies using Chlamydomonas and other experimental systems, that control axonemal bending. This section will describe the complexity of the axonemal dynein motors and the specific organization of these dynein motors on the microtubule lattice of the axoneme. Furthermore, we will introduce axonemal structures that are critical for regulation of axonemal dynein function. 10.2.1 Basic Axonemal Structure, Axis of Bending and the

96 nm RepeatThe axoneme is a highly ordered, highly conserved structure. When viewed in cross-section (Fig. 10.1A), a typical 9 + 2 arrangement of axonemal microtubules is observed in motile cilia; nine outer doublets (each composed of an A-tubule and an incomplete B-tubule that is fused to the A-tubule) and two central microtubules. Attached to the A-tubule are the dynein arms and T-shaped structures called the radial spokes-extending inwards toward the central pair apparatus. The central pair is composed of two singlet microtubules, C1 and C2, along with a series of asymmetrically shaped appendages. As detailed below, the inherent asymmetry of the central pair complex is an important feature for generating signals that regulate dynein activity. In order to effectively generate and propagate a ciliary bend, the dynein motors must be tightly regulated in a temporal and spatial manner.