Cilia: Structure and Motility (eBook)

Stephen M. King, Gregory J Pazour (Herausgeber)

eBook Download: PDF | EPUB

2009 | 1. Auflage
344 Seiten
Elsevier Science (Verlag)
978-0-08-096106-4 (ISBN)

Infos

Along with its companion volume on intraflagellar transport, ,this book provides researchers with ,a ,comprehensive and up-to-date source ,of ,methods for the analysis cilia and flagella,  ,focusing primarily on approaches that have been devised or significantly extended since the last volume of Methods in Cell Biology on this topic (volume 47, 1995). Edited by Stephen M. King and Gregory J. Pazour, the newest installment of this highly acclaimed serial will serve as an essential addition to the study of cilia and flagella.

* Covers protocols for cilia and flagella across systems and species * Both classic and state-of-the-art methods readily adaptable across model systems, and designed to last the test of time * Relevant to clinicians interested in respiratory disease, male infertility, and other syndromes, who need to learn biochemical, molecular, and genetic approaches to studying cilia, flagella, and related structures

Textauszug

Chapter 2

Electron Microscopic Imaging and Analysis of Isolated Dynein Particles

Anthony J. Roberts; Stan A. Burgess    Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom

Abstract

Despite more than 40 years of investigation since the discovery of dynein [Gibbons, I. R. and Rowe, A. J. (1965). Science149, 424–426] our understanding of how this microtubule-based motor generates force and movement remains frustratingly incomplete at the atomic level. Electron microscopy (EM) has played a major role in establishing dynein’s complex architecture and its nucleotide-dependent conformational changes. In this chapter we review recent structural studies and describe in detail negative stain EM and computational single-particle image processing techniques that have been used to investigate dynein. We describe studies of both Chlamydomonas flagellar inner arm dynein-c and recombinant cytoplasmic dynein from Dictyostelium. We also detail methods for locating green fluorescent protein (GFP) and blue fluorescent protein (BFP) tags inserted at specific locations within the dynein motor, which can be used to map subdomains and conformational changes.

I Introduction

The dynein motor is large. Its heavy chain, which contains the motor domain, is typically ∼500–540 kDa with the motor domain itself ∼380 kDa (Koonce and Samsó, 1996). This is about ten times greater than the motor domain of kinesin, the other class of microtubule motor. Despite being discovered about 20 years after dynein (Vale et al., 1985), our understanding of kinesin’s structure and mechanism far exceeds that of dynein. Nevertheless, recent progress with dynein has been made, and in this chapter we review the electron microscopy (EM) techniques that have shed new light on dynein’s mechanism of action.

Dynein’s motor domain is not only larger than kinesin’s, but is also much more extended in structure. Whereas kinesin’s motor is contained within a globular structure ∼6 nm in diameter (Vale and Milligan, 2000), dynein’s motor comprises a ringlike head domain ∼13 nm in diameter, together with an ∼15 nm-long coiled-coil stalk that protrudes from the head (Fig. 1A). The head contains dynein’s ATP hydrolysis sites, and the stalk carries at its distal end the critical ATP-sensitive microtubule-binding site within a small ∼4 nm-diameter globular domain (Fig. 1). This organization places the sites of ATP hydrolysis and microtubule binding in dynein ∼28 nm apart: about ten times the equivalent distance of their counterparts in kinesin.

Fig. 1 Overview of dynein structure. (A) Cartoon showing the domain organization of dynein, including the microtubule-binding domain (MTBD) at the distal end of the coiled-coil stalk. (B) Cytoplasmic dynein motor domain (i.e., head and stalk) imaged by negative-stain EM followed by single-particle image processing. The stalk has a kink (arrowhead) at its distal end close to the globular MTBD and corresponding closely to the kink seen in the atomic model (C). Scale bar 5 nm. (C) Atomic model of the distal stalk and MTBD (PDB accession code 3ERR; Carter et al., 2008). Small cartoon (left) is shown at approximately the same scale as in (B). Conserved prolines responsible for the kink are shown in black space fill (P3285 and P3409: mouse cytoplasmic dynein 1 sequence). The figure was made using PyMol (DeLano, W.L., 2002).

These features of dynein present significant challenges to structural studies of its intact motor domain. Dynein’s large size rules out structure determination by nuclear magnetic resonance (NMR). Crystallographic studies are problematic because the motor is extended and the stalk flexible (Burgess et al., 2003). Thus, while numerous atomic resolution structures have been obtained of kinesin (and also of the actin-based motor myosin) in various nucleotide states, no atomic resolution structures exist for the entire motor domain of dynein. Truncating the motor domain of dynein to isolate subdomains renders the motor nonfunctional (Gee et al., 1997), making any resulting structures potentially difficult to interpret (or even to assay for functionality and correct folding). Nevertheless, this approach has been applied to dynein’s microtubule-binding domain (MTBD), producing the first atomic resolution structure of dynein’s heavy chain (Carter et al., 2008). This significant breakthrough was achieved by creating a chimeric protein in which the MTBD was fused to the coiled coil of a small compact protein, seryl tRNA synthetase (Gibbons et al., 2005). This required a considerable amount of work to characterize the behavior of the resulting fusion proteins (assayed for MT-binding affinity). This approach seems less suitable for the rest of the motor, making EM as the most feasible technique for structural studies of the intact dynein motor.

Structures of kinesin and myosin bound to their cytoskeletal tracks have been obtained by exploiting the ability of these motors to saturate fully the lattices of their tracks. Because their tracks have helical symmetry, it is possible to obtain 3D reconstructions from electron micrographs of frozen-hydrated specimens. Indeed, this is the major technique used to obtain structural information about motors bound to their tracks, and recent advances have improved the resolution of the kinesin–microtubule complex to ∼9 Å—sufficient to resolve secondary structure elements (Sindelar and Downing, 2007). Unfortunately, this approach is unfeasible for intact dynein as its motor domain is too large to fully saturate all available binding sites on the microtubule lattice. Conversely, it has been used successfully to obtain the structures of various MTBD fragments of dynein bound to the microtubule (Carter et al., 2008; Mizuno et al., 2004). To date, the resolution achieved in these studies (∼20–30 Å) has not yet approached the best obtained for kinesin-microtubule reconstructions, but the MTBD-microtubule density maps do suggest the geometry of attachment of the distal stalk and the site on tubulin where binding occurs.

The earliest and arguably most striking dynein EM images are those from freeze-etch replicas of cilia and flagella, showing the outer dynein arms in situ in unprecedented detail for their time (Goodenough and Heuser, 1982). Indeed, these remain some of the best images of the motor bound to its microtubule track—not only because they show the motors in situ, but also because the coiled-coil stalks are visible directly in micrographs without any image processing. The advantage of this technique is that the shadowing material (in this case platinum/carbon) favorably deposits on the fine structure of the stalk, thereby revealing its presence. The disadvantage is that only the outermost features of the shadowed surface are visible, so heavy chains closer to the interior of the axoneme are obscured.

Recent improvements in cryo-electron tomography (see Chapter 1 by Nicastro, this volume) have produced remarkable structural insights into the entire intact axoneme, including the multiple dynein heavy chains of the inner and outer dynein arms (Bui et al., 2008; Ishikawa et al., 2007; Nicastro et al., 2005, 2006). The chief advantage of this approach is that the entire 3D structure is visualized rather than just the surfaces of favorable fracture planes (as in the freeze-etch technique). However, to date, the resolution of this technique (30–40 Å) does not reveal the stalks, leaving the motor-track interaction elusive. There is optimism, however, that technical improvements may overcome this limitation.

Other cryo-EM techniques have also been applied to in vitro dynein-microtubule systems. Microtubules sparsely decorated with monomeric cytoplasmic dynein and processed by single-particle techniques have shown in 3D the geometry of attachment of the head domain (Mizuno et al., 2007). In an alternative approach, microtubules polymerized in the presence of flagellar outer arm dyneins created pairs of helically arranged microtubules cross-linked by two rows of ordered dynein complexes (Oda et al., 2007). Processed by helical methods this study showed new details of the 3D arrangement of the heads and tails of the three heavy chains in this species of outer arm. However, neither of these studies showed the stalks. This was most likely caused by their limited resolutions (∼26 and ∼27 Å, respectively). With larger data sets and perhaps with improvements in processing, these approaches offer greater hope of higher resolution and visualization of the entire dynein motor in contact with its microtubule track. A recent development in revealing the stalk of an intact dynein motor bound to the microtubule has been obtained by a technique termed cryo-positive staining (Ueno et al., 2008). In this in vitro technique, cytoplasmic microtubules and purified dimeric outer arm flagellar dyneins were mixed in...

Produkt-Details

• Erscheint lt. Verlag: 27.11.2009
• Sprache: englisch
• Themenwelt: Geisteswissenschaften ► Psychologie ► Entwicklungspsychologie
• Themenwelt: Studium ► 1. Studienabschnitt (Vorklinik) ► Physiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zoologie
• Themenwelt: Technik
• ISBN-10: 0-08-096106-1 / 0080961061
• ISBN-13: 978-0-08-096106-4 / 9780080961064