ABSTRACT

[email protected] dynein is the major minus-end-directed microtubule-based motor in nearly all eukaryotic cells. Due to its large size and subunit complexity, dissecting the motile properties of dynein has been challenging. However, recent advances in recombinant approaches to purify dynein, as well as studies with the native motors, have begun to reveal the details of how dynein steps along microtubules and responds to externally applied loads. Compared to studies on the other cytoskeletal motors, myosin and kinesin, studies of dynein are still in their infancy, leading to a number of controversies regarding the dynein motile mechanism. However, a consensus is beginning to emerge from single-molecule studies that dynein is a highly processive motor, which can take forward, backward and diagonal steps related in size to the minimum repeat unit of the microtubule (8 nm). Here we discuss some of the more controversial aspects of the dynein stepping mechanism and response to load. We also review what is known about dynein regulation by its multiple ATP-binding sites and associated cofactors, the dynactin complex, LIS1, and NudE. 8.1 IntroDuCtIonCytoplasmic dynein (referred to as dynein in this chapter) performs nearly all minus-end-directed microtubule (MT)-based transport in eukaryotic cells. All Handbook of Dynein Edited by Keiko Hirose and Linda A. Amos Copyright © 2012 Pan Stanford Publishing Pte. Ltd. www.panstanford.com

eukaryotic genomes that have been sequenced contain a single cytoplasmic dynein heavy chain gene that is expressed in both ciliated and nonciliated cells, with the exception of higher plants, which have no dynein genes, but an expanded family of minus-end-directed kinesins [76]. As the major minus-end-directed transporter, dynein has a diverse set of cargo, ranging from organelles, to RNAs, to signaling proteins. Dynein also has multiple functions in cell division and migration and can be hijacked by nonphysiological cargo such as viruses [23]. In this chapter we will focus on studies of dynein motility, primarily the motile properties of the purified enzyme in vitro. The cytoplasmic dynein holoenzyme is composed of dimeric subunits of a motor (or head)-containing heavy chain (HC), intermediate chain (IC), light intermediate chain (LIC), and light chains (LC) [53] (Fig. 8.1). There are three LC families: TCTEX, LC7/Roadblock, and LC8, all of which are also present in two copies per holoenzyme and bind directly to the dynein IC [53]. Only the LC8 light chain is present in all organisms that also have a dynein HC gene. In addition to the holoenzyme subunits, a number of other proteins and protein complexes are required for dynein’s function in cells (reviewed in [21]). In Section 8.5 we will discuss the role of those that have been shown to regulate dynein’s motile properties, the dynactin complex, LIS1, and NudE. One of the challenges for studying the motile properties of dynein has been its enormous size and complexity. While the holoenzyme alone is approximately 1.2 MDa, adding the dynactin complex, LIS1, and NudE brings the total complex size to ~2.5 MDa. The HC itself is quite complicated and distinctly different evolutionarily from the other cytoskeletal motor proteins, kinesin, and myosin. The domain structure of the HC is shown in Fig. 8.1A. Briefly, dynein’s amino-terminal “tail” domain represents ~30% of the entire mass of the dynein HC and is required for dimerization and the association of most dynein subunits and associated proteins. Situated between the tail and motor domains, is a recently discovered element, the “linker” domain, which shifts position relative to the dynein motor ring during the ATPase cycle and is required for motility [2, 27, 55, 57] (Fig. 8.1B, see Chapters 3 and 4). Following the linker domain, and comprising ~60% of the mass of dynein, is the motor domain, which is made up of a hexameric ring of concatenated AAA+ ATPase domains. The first four of these AAA+ domains are expected to bind ATP or ADP based on the phenotypes of mutants [4, 28, 54, 67]. Between AAA+ domains 4 and 5 is a 10-15 nm antiparallel coiled coil “stalk” capped by the dynein microtubule-binding domain (MTBD), whose atomic structure was recently solved ([3], Chapter 6). Thus, some of the striking features of the dynein molecule in comparison to kinesin and myosin include the high number of ATP molecules that can bind per dimer (up to 8 for dynein vs. 2 for kinesin and myosin) and the large distance between the primary site of ATP hydrolysis (AAA1 in dynein) and the site of filament binding (20-25 nm for dynein vs. a few nm for myosin and kinesin).