Handbook of Dynein (Second Edition)

Keiko Hirose

• Publisher: Jenny Stanford Publishing
• Year: 2019
• Print ISBN: 9789814800013
• eBook ISBN: 9780429664717
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Dyneins are molecular motors that are involved in various cellular processes, such as cilia and flagella motility, vesicular transport, and mitosis. Since the first edition of this book was published in 2012, there has been a significant breakthrough: the crystal structures of the motor domains of cytoplasmic dynein have been solved and the previously unknown details of this huge and complex molecule have been unveiled. This new edition contains 14 chapters written by researchers in the US, Europe, and Asia, including 3 new chapters that incorporate new fields. The other chapters have also been substantially updated. Compared with the earlier edition, this book focuses more on the motile mechanisms of dynein, especially by biophysical methods such as cryo-EM, X-ray crystallography, and single-molecule nanometry. It is a major handbook for frontline researchers as well as for advanced students studying cell biology, molecular biology, biochemistry, biophysics, and structural biology.

Chapter 1


Dyneins: Ancient Protein Complexes Gradually Reveal Their Secrets

Linda A. Amos^a and Keiko Hirose^b

^aMRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK

^bBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 350-8565, Japan

[email protected]

The history of research on dynein began with EM images showing dynein ‘arms’ forming crossbridges between the doublet microtubules in a ciliary axoneme. In the half-century since then, our view of these amazing macromolecules has gradually reached a point where some of its protein domains including the dynein motor domain have been seen at atomic resolution. Also, the origin and evolution of the various dynein components are now becoming clearer.

1.1 Introduction

Dynein was first identified and named by Ian Gibbons in the 1960s as an ATPase that could be extracted from cilia and flagella. The number of papers written per year has increased steadily ever since (Fig. 1.1). The complex structural and functional secrets of this microtubule (MT) motor were very gradually unlocked. In Fig. 1.2 we summarize some of the important advances that were made in the past half-decade. Recently, however, progress has accelerated greatly, thanks to a variety of tools that were not available originally, such as sequencing of whole genomes, success in producing recombinant dynein, X-ray crystallography, EM cryo-microscopy and tomography, and single-molecule measurements. This makes it difficult to summarize all important new contributions. Current understanding of dynein’s structure and motile mechanism, and its wide range of roles in vivo, are described in more detail in subsequent chapters.

Figure 1.1 Increasing number of published papers on dynein.

1.2 Dynein Molecular Structure Coming into View

1.2.1 Axonemal Arms

Dynein was first seen in an electron microscope (EM) as two rows of ‘arms’ on each doublet MT in thin sections of flagella (see Chapter 11, Chapter 12 and Chapter 13) whose fine structure had been preserved with a new chemical fixative, glutaraldehyde [1]. The image in Fig. 1.3i is an example of similar results obtained by Gibbons and Grimstone [39], who improved the contrast in their sections by introducing a novel staining method. A few years later, a protein having ATPase activity was extracted from Tetrahymena cilia and named ‘dynein’ by Gibbons and Rowe [41]. A fraction that was characterized by ultracentrifugation as 14S molecules was seen by EM (Fig. 1.3ii) as individual globular particles; another fraction, consisting of larger complexes appeared to be a longish linear polymer (30S dynein), whose identity is still a little mysterious. A decade later, outer arm of Tetrahymena cilia, before and after extraction from axonemes, appeared in negative stain as a linear complex of three subunits [160] (Fig. 1.3iii). It became also clear that outer arms are arranged with a periodicity of ∼24 nm in axonemes.

Figure 1.2 Dynein discovery timeline. Abbreviations: EM, electron microscopy; MT, microtubule; HC, heavy chain; IC, intermediate chain; LC, light chain; ODA, outer dynein arm; IDA, inner dynein arm; OAD, outer-arm dynein (proteins); CD, cytoplasmic dynein; IFT, intraflagellar transport; MTBD, microtubule-binding domain; EB1, end-binding protein. ‘arms’ are the projections seen extending from doublet MTs (see Fig. 1.3i).

Conformational changes were observed between the dynein arms crossbridging two doublet MTs and those unbound to the B-tubule [159] (Fig. 1.3iv), and between the arms in the presence and absence of ATP [143]. It was found that the binding of arms to the B-tubule is nucleotide-dependent, but there was controversy as to whether the arms dissociate from the B-tubule with ATP. In 1982, Goodenough and Heuser [46] first saw a thin stalk extending from the globular dynein head, in axonemes that had been rapidly frozen to preserve their structure (Fig. 1.3v–viii). Conformational changes in the presence/absence of ATP were also clearly demonstrated [46, 126] (Fig. 1.3v,vii). At this point, therefore, the main structural features of dynein molecules (Chapter 2, Chapter 3 and 4, and 13) had already been observed but they could not properly be understood until the HC sequence had been determined in 1991 [38, 101], which paved the way both for identification of the MT-binding region in 1997 [32, 75] and recognition of dynein as a member of the superfamily of AAA+ proteins in 1999 [98].

Figure 1.3 Electron micrographs of axonemal dynein arms, showing the gradual increase in resolution and understanding of the structure, (i) TEM image of a stained thin section cut through glutaraldehyde-fixed plastic-embedded flagella [39]. (ii) Top-left: ‘30S dynein’ polymers extracted from Tetrahymena axonemes and shadowed with platinum; bottom-left: 30S dynein image after translational averaging with 14 nm steps; right: separate ‘14S dynein’ subunits viewed in negative stain [41]. (iii) Negatively stained Tetrahymena dynein arms arranged on a doublet MT (bottom) and extracted ‘14S dynein’ subunits (top), both showing three subunits [160]. (iv) Tetrahymena doublet MTs crossbridged with outer arms, in two conformational states [159]. (v–vii) Chlamydomonas (v–vi) and sea urchin (vii) axonemes, demembranated and quick-frozen before being shadowed with platinum [46, 126]. In v and vii, the side views of axonemes fixed in the ATP-free solution to trap the dynein arms in the rigor state (top) and those relaxed by incubation in vanadate plus ATP (bottom) show markedly different conformations of dynein arms. The stalks connecting the dynein arms to the B-tubule of the next doublet-MT are clearly observed in both states. The basal end is on the right. In vi, an axoneme is viewed from tip to base and the arrow indicates a relaxed dynein outer arm. (viii) View of a doublet MT from inside an axoneme. The radial spokes (S), in groups of three, are seen end-on and, next to them, the dynein inner arms form two pairs (D) of distinct individual heads, then a triad (T) [47], which correspond to 7 different dynein species [63]. (ix–xiii) 3D tomographic images of quick-frozen axonemes. ix is a view similar to viii, showing inner dynein arms [13] (see Chapter 13). (x) A doublet MT with outer and inner arms, viewed from tip to base [52]. (xi) Native Chlamydomonas outer arms [58, 99]; the two images show very similar details, each arm being a stack of three dy...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• 1 Dyneins: Ancient Protein Complexes Gradually Reveal Their Secrets
• 2 Structural and Functional Analysis of the Dynein Motor Domain
• 3 Electron Microscopy Studies of Dynein: From Subdomains to Microtubule-Bound Assemblies
• 4 Subunit Architecture of the Cytoplasmic Dynein Tail
• 5 Measuring the Motile Properties of Single Dynein Molecules
• 6 Mechanics of Dynein Motility
• 7 Interactions of Multiple Dynein Motors Studied Using DNA Scaffolding
• 8 Cytoplasmic Dynein Force Regulation in vitro and in vivo
• 9 Dynein in Endosome and Phagosome Maturation
• 10 Dynein in Intraflagellar Transport
• 11 Diversity of Chlamydomonas Axonemal Dyneins
• 12 Motility of Axonemal Dyneins
• 13 Axonemal Dyneins in Cilia and Flagella
• 14 Regulatory Mechanism of Axonemal Dynein
• Index