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Structure and Function of the Dynein Tail

Final Report Summary - DYNTAIL (Structure and Function of the Dynein Tail)

The size and complexity of eukaryotic cells make continuous movement of cargo within the cell an essential process. Cells rely on motor proteins to generate the force needed to power this transport. Examples of continuous cargo movement in the cell include the movement of endosomes, the collection of aggregated proteins and the reshaping of the mitotic spindle. An important unanswered question is the question of specificity. How do motor proteins associate with the correct cargos? In order to find an answer to this question, it will be necessary to have a detailed understanding of motor/cargo interaction. We proposed three objectives to address the question of specificity by looking at the structure of the cargo-binding domain (known as the tail) of one such motor protein: cytoplasmic dynein.

Aim 1: Recombinantly express and purify a functional yeast dynein tail complex to obtain the crystal structure.

To achieve the goals set out in this aim we expressed and purified a large number of differently truncated versions of the yeast dynein tail and used the crystallization facilities at the MRC-LMB to screen for a variant that would crystalize. This has proven to be a challenging task but very recently we have obtained the first crystals and are currently in the process of determining the structure of what will be the first crystal structure of the dynein heavy chain tail.

Aim 2: Recombinantly express and purify mammalian dynein tail complex and perform biochemical and EM analysis.

By purifying the complete recombinantly produced human dynein complex and not only the tail we have far exceeded the goals set out in this aim. Additionally we have succeeded in reconstituting dynein movement in an in vitro microscopy assay and provide the biochemical analysis that provides insight into the general mechanism that links cargo binding to dynein powered transport. Finally, we have been able to provide the first EM analysis of dynein bound to the accessory components that allow it to move cargo. The results of this work have recently been published in the EMBO Journal and are available for download without charge (open access).

Aim 3: Analysis of dynein/virus interactions using purified rat brain dynein.

The work on dynein/virus interactions has benefited from the success of aim 2. The reconstitution of dynein movement in vitro combined with fluorescently labeled virus particles obtained from our collaborators in Oxford has already produced very promising preliminary results in which movement of virus particles could be observed. However, the considerable optimization of the experimental conditions will be necessary.

Together the results obtained in this study have significantly advanced our understanding of the cargo binding properties of the dynein tail specifically and the dynein complex in general. The lines of research initiated in this study will continue to be pursued in the lab and we are confident that they will lead to many more discoveries that will help us understand the fundamental principles that underlie specific cargo binding and movement within cells and their role in human disease.