Background Information

1) The dynein family

Dyneins are protein complexes consisting of one or more heavy chains and a variety of different light, intermediate and light-intermediate chains. The heavy chain is around 500kDa (4000-5000 amino acids) in size and contains a motor domain that is related to the AAA+ family of proteins. Higher eukaryotes typically contain 10-15 dynein heavy chain genes, which are related by their highly conserved, C-terminal motor domain. In contrast, the N-terminal third of the heavy chains, known as the tail region, is very divergent.

Phylogenetic tree of dynein heavy chain genes

Members of the dynein heavy chain gene family include one cytoplasmic dynein gene, which is responsible for most of the transport toward the minus ends of microtubules in the cell (although some is also carried out by minus-end-directed kinesins). There is also typically one intraflagella (IFT) dynein gene that plays a role in the assembly of the bundles of microtubules known as axonemes that make up the core of cilia and flagella. All the remaining dynein genes are known as axonemal dyneins and are responsible for powering the beating motion of axonemes. The axonemal dyneins are themselves divided in outer arm dyneins and inner arm dyneins depending on their location within the axoneme.

There is a large variation in number of dynein heavy chain genes found in eukaryotes [1, 2]. Cytoplasmic dynein is an essential gene in many eukaryotes [3], but has been lost from others such as higher plants and some fungi. Axonemal dyneins are missing in a number of species, including plants, fungi and the nematode worm C. elegans. In other species, such as the ciliate Tetrahymena thermophila, the number of axonemal innerarm dynein heavy chain genes has undergone a large expansion to 25 copies [4].

2.1) Roles of cytoplasmic dynein in the cell

In interphase cells cytoplasmic dynein is responsible for carrying a diverse range of cargos back towards the nucleus. These include membrane-bound organelles such as components of the endosome pathway [5], golgi vesicles [3] and peroxisomes [6]); as well as transcription factors [7]; aggregated proteins [8]; and mRNA containing particles [9]. In neurons, dynein drives retrograde transport back along axons towards the cell body [10, 11].

Cytoplasmic dynein also plays a fundamental role in mitosis [12-14]. It has been found at the cortex, where it pulls on microtubules attached to the spindle poles [15-17], and at the spindle pole, where it accumulates after transporting factors required for focusing of the poles [18, 19]. It also localises to the kinetochore, where it has a number of possible functions [20], including playing a role in the checkpoint that monitors correct attachment of the spindle to the chromosome [21, 22].

Roles of cytoplasmic dynein in interphase microtubule transport and in mitosis.

2.2) Roles of dynein in disease

Given the multiple roles of cytoplasmic dynein in many higher eukaryotes, it is perhaps unsurprising that it should be associated with a number of disease processes. The process of viral infection, for example, involves dynein. Viruses require active transport to the nucleus as they are too big to reach it purely by diffusion. This is especially apparent in the case of herpes viruses, which enter sensory neurons at the nerve termini and then travel back to the cell body in order to set up a latent infection [23].

In addition to viral transport in sensory neurons, dynein has been implicated in many other neuronal diseases. Perhaps the best studied is Lissencephaly. Mutations in the dynein regulatory protein Lis1 result in defects in dynein mediated movement of nuclei within certain neurons. This results in failure of these neurons to migrate during brain development, leading to brains with an unusual smooth appearance as well as severe retardation and early death [24].

Dynein may also have a part to play in a number of neurodegenerative diseases. In mice, mutations in the tail region of dynein lead to phenotypes that resemble patients with motor neuron disease [25]. Furthermore a mutation (G59S) in one of the dynactin subunits, called p150Glued, has been linked to motor neuron disease in both humans and mice. One of the established functions of dynein is to transport aggregated proteins [26], possibly collecting them up so they can be disposed of via phagocytosis. The link between protein misfolding and aggregation in many neurodegenerative diseases, such as Parkinsons and Lewy Body dementias, implies that dynein may have a role to play in these disesase as well. Lewy Bodies themselve resemble aggresomes and may be formed by a dynein dependent process [26].

Finally the role of dynein at the spindle assembly checkpoint means it is involved in a process that is crucial for cancer cell division. This is highlighted by the fact that a number of anticancer compounds, such as vinblastine, are thought to act by blocking this checkpoint for long enough to drive dividing cells into apoptosis.

3) Components of cytoplasmic dynein

The cytoplasmic dynein complex and some of the cargos that interact with it.

There are 45 different kinesin motors in humans each with different functions. There is only a single cytoplasmic dynein heavy chain, however, which raises the question of how one protein can carry out so many functions. At least part of the answer may lie with the many accessory factors associated with the core dimer of dynein heavy chains as shown in the figure.

Different cargos appear to interact with the cytoplasmic dynein complex via different accessory chains. This may be important to allow their attachment to be regulated in order to ensure that only the correct cargo is transported. Specificity may also come from the use of different splice isoforms of the light (LC), light-intermediate (LIC) and intermediate chains (IC). In effect there would be multiple species of dynein within the cell that each carry different cargos and can be individually regulated. For example LIC1 but not LIC2 has been shown to be responsible for transport of pericentrin, a centrosomal component [27]. In contrast only the LIC2 isoform is present in the pool of dynein that mediates fast retrograde transport in axons [28].

The cytoplasmic dynein complex is more than just a set of scaffolding proteins linked to a motor domain. A number of the factors, such as Lis1 and dynactin, have been reported to have a direct effect on dynein’s ATPase activity. It is also likely that the dynein will be regulated both in terms of its activity and in when it interacts with its cargos. Understanding this complex web of interactions will require a much better knowledge of the structures of the whole complex. Currently crystal structures of some of the components, including Lis1 [29] and the light chains [30] are known, but much needs to be done to work out how all the components fit together.

4) Structure of the motor domain

Structure of the dynein protein and model of how the elements are arranged in the motor. The core of the motor is a ring of six AAA domains (blue). The linker and C terminal region probably lie on top of the ring. The almost one complete turn of antiparallel coiled coil that makes up the stalk emerges from between AAA4 and AAA5 and has the microtubule binding domain (MTBD) at its tip.

The minimal motor domain of dynein contains the C terminal ~3000 amino acids of the heavy chain gene [31 ]. It contains six putative AAA+ domains [32], which form an ~15 nm diameter ring structure. The microtubule binding domain is at the tip of a 15nm long stalk, made of an antiparallel coiled coil that emerges from between AAA4 and AAA5 [33]. The N terminal ~400 amino acids of the motor domain form a structure called the “linker” (or sometimes the “stem”) which stretches across the face of the ring. This linker changes position in the different nucleotide states [34], so that in the absence of nucleotide it exits the ring close to AAA4, whereas in the presence of ATP it appears to move nearer to AAA2 [35, 36]. This rearrangement of the position of the linker domain corresponds to the powerstroke that drives motility [37]. Finally other elements, such as the very C terminus of the motor are also likely to sit on top of the AAA ring.

The motor domain of dynein is much larger than that of other motor proteins (kinesin and myosin), is evolutionarily unrelated and clearly moves by a very different mechanism [1]. Some of the interesting mechanistic questions that remain to be answered include: whether one [38] or two [36, 39] AAA+ domains hydrolyze ATP per dynein step; exactly how the AAA+ ring communicates with the distant MT binding domain [40]; how flexible the orientation of the stalk is as cytoplasmic dynein moves and how rearrangements in the AAA+ domains are amplified to produce the swing of the linker domain.

Next section: Research Highlights

References

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