Table of Contents

I History and Evolution

Chapter 1 Discovery of Dynein and its Properties: A Personal Account
I. R. Gibbons

Abstract: This memoir describes my life as a researcher, beginning as a physics PhD student in the Electron Microscopy Unit of the Cavendish Laboratory in Cambridge in 1954, shortly after the development of a thin sectioning procedure for biological tissue samples. After moving to the Biology Department of Harvard University in 1958, my enthusiasm for studying cilia and flagella was sparked when I happened to take a set of electron micrographs showing the complex fibrous substructure of these organelles with exceptional clarity for that period. Shortly thereafter, my familiarity with previous research on the motile mechanism of striated muscle encouraged me to begin a biochemical study of cilia from Tetrahymena. With the benefit of advice from my wife, Barbara, who was then a PhD student in protein chemistry, I and my coworkers were able to purify and characterize the two principal axonemal proteins, dynein ATPase and tubulin. After deciding to focus our future research on the role of dynein in axonemal motility, Barbara and I moved to the Kewalo Marine Laboratory at the University of Hawaii in 1967 and spent the next 30 years collaborating on varied approaches that used the sea urchin sperm flagellum as an accessible model system of dynein-based motility. When we retired and returned to the mainland in 1997, I continued research on a part-time basis in the Molecular and Cell Biology Department of the University of California, Berkeley, where I worked in collaboration with Joan Garbarino and Andrew Carter until 2010, principally on defining the mechanism by which the ATP-sensitive microtubule-binding domain (MTBD) of dynein communicates changes in its binding status through the extended coiled-coil stalk that connects it to the site of ATP hydrolysis in the core of the dynein motor, and also on obtaining an X-ray crystal structure for this MTBD and a portion of its stalk.

Chapter 2 Evolutionary Biology of Dyneins
Bill Wickstead and Keith Gull

Abstract: The advent of whole-genome sequencing for multiple eukaryotes has allowed unprecedented analysis of the evolution of dyneins by resolving the full repertoires of dynein heavy chains (HCs), intermediate chains (ICs), and light-intermediate chains (LICs) encoded by different organisms. Phylogenetic analyses have established a consensus of nine major dynein HC families, eight of which are associated with ciliary/flagellar functions. All of the dynein HC families were established prior to the radiation of eukaryotes from their common ancestor. However, since that time all families have experienced multiple losses, with the result that no family is ubiquitous and at least three eukaryotic lineages now entirely lack dynein motors. Rooting of dynein HC phylogenies has significant consequences for the likely evolutionary path by which the cilium formed. Reconstructing the evolution of dynein function from prokaryotic origins is still very challenging, but the identification of the closest extant relatives of dynein HCs tentatively suggests descent from a complex with chaperone-like activity.

II Structure and Mechanics of Dynein Motors

Chapter 3 The AAA+ Powerhouse-Trying to Understand How it Works
Paul A. Tucker

Abstract: A brief introduction to the common features of AAA+ (ATPases associated with various cellular activities) domains is given, with specific reference to what is known from structural and mutational studies. There follows a sequence-based analysis of what information might be gained about the dynein motor "ring," which contains six AAA+ domains. Experimental data defining which of the six domains bind nucleotide and which domains are active ATPases are summarized. Finally, experimental data on the structural features and the changes in structure of a single motor "ring" during the duty cycle are described.

Chapter 4 Dynein Motor Mechanisms
Michael P. Koonce and Irina Tikhonenko

Abstract: Dynein is a microtubule-based, minus-end-directed molecular motor that drives organelle and axonemal movements in eukaryotic cells. This chapter describes the organization of the key motor components and summarizes our understanding of how they work. Analyses using combinations of fluorescence resonance energy transfer tags, mutations, inhibitors, and ultra-structural methods have provided a new level of detail in our understanding of how the motor operates. Unlike the other cytoskeleton-based motors, single dyneins contain multiple ATP-binding modules that are arranged in a ring-shaped core, an extended microtubule-binding domain (MTBD), and an arcing linker arm that undergoes a unique winch-like powerstroke. Dyneins, kinesins, and myosins form the three major families of cytoskeleton-based motor proteins that together are responsible for most of the macro-scale movements in eukaryotic cells. All three families utilize an ATP catalytic cycle to make nm-sized steps along polarized filaments/tubules, and largely do so in a predetermined direction. The catalytic cycles of the motors generate pN-scale forces and couple force production with modulating high and low substrate-binding affinities. Most of these motors are actively engaged in moving cargo, although some isoforms also provide important anchoring or polymer length control functions. Despite engaging different cytoskeleton substrates, the core structures of kinesins and myosins are strikingly similar, indicating that these two families share a common ancestral origin. Dynein, on the other hand, is considerably different. As covered in Chapter 2, dyneins likely emerged from an ancestral enzyme distinct from the primordial kinesin or myosin [1]. Not only are dynein motor domains much larger in mass than kinesins or myosins, but they are also arranged in a significantly different, ring-shaped architecture. How, then, does the dynein motor produce similar movements? This chapter reviews recent highlights on the mechanics of the dynein engine, including ring dynamics, linker arm movement, and microtubule affinity. We emphasize mutagenesis and structural work that has been performed with cytoplasmic dynein. Although there are differences in motor activities among the axonemal and cytoplasmic isoforms, the basic motor design of all dyneins is similar.

Chapter 5 Structural Analysis of Dynein Intermediate and Light Chains
John C. Williams, Amanda E. Siglin, Christine M. Lightcap and Amrita Dawn

Abstract: The dynein accessory proteins, the intermediate chains (ICs), the light-intermediate chains (LICs), and the light chains (LCs) play an essential role in coupling the force produced by the dynein heavy chain (HC) with the positioning of organelles, the separation of chromosomes, or the beating of cilia. While these subunits, especially the dynein LCs, have been ascribed to multiple functions, structural and biochemical studies have led to a comprehensive picture of how the bivalent nature of the dynein motor complex permits the selection and regulation of cargo binding. A review of these investigations is presented herein.

Chapter 6 Biophysics of Dynein In Vivo
Jing Xu and Stephen P. Gross

Abstract: This chapter will summarize what we know about how single-molecule properties relate to ensemble function, and how such properties can be regulated. We describe the functions determined for single dynein motors in vitro, in a purified and controlled environment, and then turn to how the motors function in groups in vitro. From these studies, we will discover what properties emerge from groups of motors but also which aspects of the single motors are important for their function as a group.

III Dyneins in Ciliary Biology

Chapter 7 Composition and Assembly of Axonemal Dyneins
Stephen M. King

Abstract: Axonemal dyneins form the inner and outer arms associated with doublet microtubules within cilia and flagella. These enzymes provide the motive force necessary for ciliary/flagellar beating by driving the sliding of micro-tubules with respect to each other. In order to generate complex waveforms and to allow for waveform conversion, axonemal dyneins must be subject to stringent regulation. Consequently, these enzymes contain not only components necessary for motor function and assembly at the correct location within the axoneme but also factors that allow their activity to be controlled in response to a broad array of regulatory signals. Many of these axonemal dynein components are highly conserved over a wide phylogenetic range and defects in some have now been directly linked to primary ciliary dyskinesia (PCD) phenotypes in humans. This chapter discusses the proteins that comprise the axonemal dyneins and also details our current understanding of how these complex enzymes are assembled in the cytoplasm prior to their transport into the cilium.

Chapter 8 Organization of Dyneins in the Axoneme
Takashi Ishikawa

Abstract: Axonemal dyneins are force-generating motors essential for flagellar/ciliary bending motion. On the microtubule doublets of the axoneme not less than 11 isoforms of dynein heavy chains (HCs) build inner and outer arms, together with light chains (LCs) and intermediate chains (ICs). Dynein arms interact with neighboring microtubules and other molecules. To elucidate the bending mechanism, we must characterize dyneins in the axoneme as well as their molecular networks with regulatory and cytoskeletal proteins as a whole system. In this chapter we will review the recent progress in our understanding of axonemal dynein in the context of flagellar/ciliary bending motion, focusing mainly on structural studies using electron microscopy/tomography but also correlating these studies with functional motility assays and theoretical studies. Significant questions to be addressed in the near future will also be presented.

Chapter 9 Genetic Approaches to Axonemal Dynein Function in Chlamydomonas and Other Organisms
Toshiki Yagi and Ritsu Kamiya

Abstract: For studying the function of complex axonemal dyneins, use of mutants that lack specific dynein species is essential. Forward genetic screens of Chlamydomonas reinhardtii motility mutants have provided by far the largest number of such mutants. Studies using these mutants have identified various subunits of inner-arm and outer-arm dyneins as well as proteins responsible for their assembly and transport. The variety of axonemal dyneins and the subunit organization of each type of dynein are largely conserved among all ciliated eukaryotes, including humans. In organisms other than Chlamydomonas, reverse genetic approaches, such as targeted gene knockout, have been used to infer the function of particular dynein species in ciliary movements, and in cilia-dependent higher-order biological functions; for example, body development and organogenesis. This chapter introduces what kinds of dynein-deficient mutants and strains have been obtained in Chlamydomonas and other organisms, and what these mutants have shown.

Chapter 10 Regulation of Axonemal Outer-Arm Dyneins in Cilia
Ken-ichi Wakabayashi

Abstract: Axonemal outer-arm dynein (OAD) is an ~2 MDa protein complex bound to the A-tubules of outer doublet microtubules. It is composed of two or three heavy chains (HCs) that exhibit ATPase/motor activities, several intermediate chains (ICs), and light chains (LCs). Many biochemical studies have shown that OAD activity is regulated by various kinds of signals such as phosphorylation, Ca2+, and redox via subunits with various motifs to receive those signals. Furthermore, in vitro microtubule-sliding experiments using OADs from Chlamydomonas mutants lacking individual HC motors suggest there are intra-and inter-HC regulatory systems. In this chapter, these regulation mechanisms for OAD are discussed.

Chapter 11 Control of Axonemal Inner Dynein Arms
Lea M. Alford, Maureen Wirschell, Ryosuke Yamamoto and Winfield S. Sale

Abstract: In this chapter, we focus on the composition, organization, and regulation of the axonemal inner dynein arms as well as their role in ciliary/flagellar motility. In contrast to the outer dynein arms, there are seven major and three minor species of inner dynein arms. Each inner dynein arm is distinct in composition and targeted to a precise and unique location within the 96 nm axonemal repeat. These conclusions are based on biochemical and ultrastructural analysis of Chlamydomonas mutants lacking subsets of dyneins. These Chlamydomonas mutants also revealed the importance of the inner dynein arms for control of the size and shape of the axonemal bend, features of motility referred to as "waveform. " We review data revealing that second messengers, including calcium and cyclic nucleotides, can control ciliary motility by phosphorylation and modulation of dynein activity. In particular, we focus on I1 dynein ("dynein f") and its regulation by a conserved phosphorylation pathway that includes signals from the central pair, the radial spoke, and a network of axonemal kinases and phosphatases that are physically located in the axoneme. We discuss how I1 dynein and the other inner dynein arms contribute to control of axonemal bending.

Chapter 12 Flagellar Motility and the Dynein Regulatory Complex
Mary E. Porter

Abstract: This chapter focuses on the relationship between the nexin link and a complex of axonemal polypeptides known as the dynein regulatory complex (DRC). Nexin was first identified in electron microscope images of isolated axonemes as an extensible linker that repeats every 96 nm between adjacent outer-doublet microtubules. The nexin links are thought to be the protease-sensitive structures that limit dynein-driven microtubule sliding during the ciliary beat cycle. The DRC was first described in genetic screens for extragenic suppressor mutations that restore motility to paralyzed radial-spoke or central-pair mutants. The DRC is thought to identify intermediates in the signaling pathway between the central pair, radial spokes, and dynein arms. Cryo-electron tomography (cryo-ET) of drc mutant axonemes has recently revealed that the DRC is located within the nexin link. The implications of these findings for future studies of ciliary and flagellar motility are also discussed.

Chapter 13 Regulation of Dynein in Ciliary and Flagellar Movement
Chikako Shingyoji

Abstract: Cyclical beating is a prominent feature of cilia and flagella. The regular arrays of dynein molecules on the doublet microtubules are responsible for the movement, in which the function of dynein is to move the adjacent doublet microtubule by using the energy of ATP hydrolysis. How the motile activity of dynein is controlled to produce the characteristic oscillatory movement is poorly understood. Notwithstanding the physiologically high and constant ATP concentration in the beating flagella, at any moment in the oscillatory cycle some dynein molecules are active while others are not, so that the motile activity of dynein oscillates temporally and spatially in the axoneme. The "switching" of dynein activity, which is thought to occur in intact axonemes according to the phase of beating and the position in the 9+2 structure, has been demonstrated at high ATP concentrations in axonemes of sea urchin sperm flagella chemically dissected with elastase. It is likely that the basic mechanism underlying the highly dynamic control of dynein activity involves ATP-dependent inhibition and ADP-dependent activation (or release of inhibition) of dynein, which respectively induce crossbridging and microtubule sliding between the doublet microtubules in the flagellum. How the inhibition and activation are induced in beating flagella is still unknown. It has, however, been demonstrated that the mechanical signal of bending is involved in switching dynein activity. This chapter provides an overview of the mechanism regulating dynein motility to produce oscillatory flagellar movement, focusing on the study of sea urchin sperm flagella.

Chapter 14 Dynein and Intraflagella Transport
George B. Witman

Abstract: Intraflagellar transport (IFT) is the active movement of particles and proteins from the base of a cilium or flagellum to the organelle’s tip (anterograde IFT) and then back to the base (retrograde IFT). The motor for retrograde IFT is cytoplasmic dynein 2, which contains two copies each of identical dynein heavy chains (HCs), intermediate chains (ICs), and light-intermediate chains (LICs) that are specific to this dynein, as well as two copies of the light chain LC8, which is shared with other two-headed dyneins. Cytoplasmic dynein 2 serves to recycle the IFT machinery for reuse in anterograde IFT, removes products of axonemal turnover and disassembly from the cilium, and transports membrane proteins such as polycystin-2 and signaling proteins such as those that mediate hedgehog signaling out of the cilium and into the cell body. Cytoplasmic dynein 2 also may have roles within the cell body, although much less is known about these functions.

IV Cytoplasmic Dynein Biology

Chapter 15 Cytoplasmic Dynein Function Define by Subunit Composition
K. Kevin Pfister and Kevin W. H. Lo

Abstract: Cells use microtubule-based motor proteins to move and properly position organelles and protein complexes. Members of the kinesin family are primarily used for microtubule plus-end transport and cells have approximately 45 different genes for this family of motors. However, for transport to microtubule minus ends they have only one gene for the cytoplasmic dynein motor. Cytoplasmic dynein complexes contain six subunits, and there are two genes for each of the five other subunits. Building on our knowledge of the functional roles of the subunits, this chapter discusses evidence supporting the hypothesis that cytoplasmic dynein complexes composed of different subunit isoforms have different roles both for cellular housekeeping functions and differentiated cell-specific functions.

Chapter 16 Studies of Lissencephaly and Neurodegenerative Disease Reveal Novel Aspects of Cytoplasmic Dynein Regulation
Kassandra M. Ori-McKenney, Richard J. McKenney and Richard B. Vallee

Abstract: Cytoplasmic dynein is a microtubule motor complex responsible for myriad intracellular functions. To accomplish its wide-ranging tasks, dynein is subject to complex inter-and intra-molecular regulation. Defects in dynein regulation have recently been found to contribute to human pathologies including lissencephaly and neurodegeneration. Conversely, analysis of the molecular basis for altered dynein behavior has yielded new insights into the mechanisms of dynein regulation. This chapter focuses in particular on the role of the lissencephaly gene, LIS1, in regulating dynein force production, and the molecular defects identified in the Loa mutant mouse dynein molecule. Studies in these two systems have revealed a novel mechanism for dynein force production by the LIS1 gene product, and a new form of long-range, intramolecular communication between the dynein tail and motor domains.

Chapter 17 Insights into Cytoplasmic Dynein Function and Regulation from Fungal Genetics
Xin Xiang

Abstract: Studies in fungal model organisms have contributed significantly to our current knowledge of dynein function and regulation. For example, fungal molecular genetics studies first uncovered the function of cytoplasmic dynein in the positioning of nuclei/spindles. Genetic approaches have led to the identification of several important regulators in the dynein pathway, including LIS1, the product of a causal gene for lissencephaly, a human brain disease. Fungal studies have also provided insights into the mechanisms of microtubule plus-end accumulation of cytoplasmic dynein and how plus-end dynein is functionally involved in spindle positioning and the movement of membranous cargoes. Finally, the feasibility of introducing specific mutations into a genome by homologous recombination has facilitated the characterization of proteins and their specific domains involved in dynein function, and these studies have significantly refined our understanding of the dynein motor and its regulatory mechanisms in vivo.

Chapter 18 Genetic Insights in to Mammalian Cytoplasmic Dynein Function Provided by Novel Mutations in Mouse
Anna Kuta, Majid Hafezparast, Giampietro Schiavo and Elizabeth M. C. Fisher

Abstract: In this chapter we look briefly at how we can use mammalian genetic approaches to understand gene function in health and disease. We then we move on to look at the specific effects of mutations in cytoplasmic dynein subunit genes. We discuss what these have told us about cytoplasmic dynein biology. We discuss the impact of mutation in dynein subunits on the whole mammalian organism -- mice in all cases so far -- and how this illustrates the disparate roles of dynein in terms of its housekeeping functions in all cell types and its specialized roles in the nervous system. We also discuss how findings in mammals relate to those of other organisms. Finally, we look at future directions for the study of the dynein complex and its individual components in mammals.

Chapter 19 Role of Dynactin in Dynein-mediated Motility
Trina A. Schroer and Frances K. Y. Cheong

Abstract: Ever since its initial discovery as a factor required for long-range, cytoplasmic dynein-dependent translocation of membrane vesicles in vitro, dynactin has intrigued the motor community. Yet how exactly dynactin "activates" dynein remains a mystery. The two functions first ascribed to dynactin, (1) serving as an adaptor molecule that helps dynein bind cellular structures and (2) providing an auxiliary microtubule-binding activity, still hold. But cytoplasmic dynein can interact with and move some cargoes independently of dynactin, and dynactin variants with attenuated microtubule-binding activity appear to operate normally in some contexts. These incongruities raise the possibility that dynactin contributes to cytoplasmic dynein activity in ways that are not yet discovered. In this review, we will detail what is known about dynactin binding to microtubules, dynein, and subcellular "cargoes"; discuss existing evidence for the two classic roles for dynactin; and speculate on novel ways in which dynactin might have an impact on dynein activity.

Chapter 20 Roles of Cytoplasmic Dynein during Mitosis
Kevin T. Vaughan

Abstract: Cytoplasmic dynein is a ubiquitous microtubule motor protein implicated in the transport of membranous organelles, RNA particles, viruses, and axonal proteins. During mitosis, dynein has been shown to play critical roles in spindle assembly and integrity, spindle rotation and positioning, and kinetochore activities including microtubule capture, chromosome movement, and checkpoint silencing. This chapter summarizes the major advances that support these models for mitotic dynein function. For each locus, the binding partners implicated in targeting dynein are discussed. Model systems responsible for important findings are also presented with a consideration of how universal some mechanisms of action might be. Because cell-cycle-stage-specific isoforms of dynein have not been identified, molecular signatures for each form of dynein are highlighted. Finally, the potential role of mitotic phosphorylation of dynein is analyzed in the context of new models for mitotic dynein targeting.

V Dynein Dysfunction and Disease

Chapter 21 Does Dynein Influence the Non–Mendelian Inheritance of Chromosome 17 Homologs in Male Mice?
Stephen H. Pilder

Abstract: Natural populations of the various subspecies of Mus musculus possess two homologous forms of the proximal one-third of chromosome 17, a region known as the t-complex. About 80-90% of mice carry a wild-type (+) form, while a substantial minority contain t-haplotypes (t), members of a family of region-spanning inversion polymorphisms recently descended from a common ancestor. Interestingly, male +/t-heterozygotes transmit t en bloc to 95-99% of their progeny, while males carrying two t-haplotypes (t/t) are invariably sterile. Approximately one quarter of a century ago, Lyon proposed that two to three cis/trans-acting t-distorter factors (Tcd-1, Tcd-2, Tcd-3) operating on a cis-acting t-responder gene product (Tcr) were responsible for the observed non-Mendelian transmission of t-chromosomes (transmission ratio distortion (TRD)), and that homozygosity for these Tcds was the basis of the observed sterility. Subsequent gene mapping and cloning studies linked two mutant dynein light chains (LCs) to the Tcd-1 and Tcd-3 loci, and an altered dynein heavy chain (HC) to the Tcd-2 locus. Evidence that TRD is caused by defective sperm flagellar function in roughly one-half of the caudal sperm population from +/t-males raised the possibility that the mutant axonemal dynein components were the Tcds. Here, I discuss whether, in light of new evidence, the dynein subunits linked to the Tcd loci retain any or all of their original mystique as genes that affect the non-Mendelian inheritance of mouse chromosome 17.

Chapter 22 Role of Dynein in Viral Pathogenesis
Andrew J. Mouland and Miroslav P. Milev

Abstract: Viruses make use of distinct ligandecell receptor interactions to bind to and infect specific cell types. Bound virus particles are then internalized at the plasma membrane by various cellular mechanisms. This is just the beginning as viruses are obligate parasites and must utilize the host cell machineries for all subsequent steps of their replication cycles. Intracellular transport of viral components requires the co-opting of cellular trafficking machineries during ingress (trafficking steps toward the nucleus) and egress (outbound trafficking steps toward the plasma membrane). Dynein is made use of by several viruses for intracellular trafficking of genetic material: capsids as well as viral structural proteins by direct virus dynein interactions or by co-opting intracellular membranes derived from endosomal compartments or exchanges with other organelles in the cell. This chapter will emphasize those facets of virus dynein interactions that not only show how viruses have adapted to interact with and co-opt dynein function and activity during ingress and egress but also how these interactions may enhance the virus’ ability to counteract innate anti-viral host cell responses to infection.

Chapter 23 Cytoplasmic Dynein Dysfunction and Neurodegenerative Disease'
Armen J. Moughamian and Erika L. F. Holzbaur

Abstract: Cytoplasmic dynein is an essential intracellular motor in higher eukaryotes. While dynein has multiple functions in cell division and intracellular trafficking that are critical in most cell types, dynein motor activity is particularly important in neurons. Evidence for this comes from the analysis of mutations in dynein or dynein-associated proteins such as dynactin, Lis1, and huntingtin, which result in pronounced neurodevelopmental and neurodegenerative phenotypes. Here we review the multiple functions of dynein in the neuron and discuss the effects of dynein pathway dysfunction in the pathogenesis of neurodegenerative disease.

Chapter 24 Dynein Dysfunction as a Cause of Primary Ciliary Dyskinesia and Other Ciliopathies
Anita Becker-Heck, Niki T. Loges and Heymut Omran

Abstract: Cilia are hair-like structures extending from almost every cell surface and can be grouped as having motile or non-motile axonemes. Motile cilia display specific structures that contribute to specific axonemal bending patterns. Among many distinct cilia-related disorders, primary ciliary dyskinesia (PCD) refers to a group of disorders characterized by inborn defects of cilia beating. Since the early 1900s, when the first cases of the situs abnormalities bronchiectasis and chronic sinusitis (Kartagener syndrome) were reported, many molecular defects responsible for PCD have been identified. This work has been greatly aided by molecular findings obtained in the unicellular flagellate Chlamydomonas reinhardtii, which serves as a model organism for studies of flagellar motility.