Dynamic microtubules captured in new study
A group of researchers led by Professor Ilan Davis in the department has identified how certain types of cells move molecular cargoes to specific regions of the cell.
The study, published in the Journal of Cell Biology1, reveals for the first time details of how the underlying cytoskeleton of the cell is organised to achieve this intracellular transport.
The researchers carried out their study in the immature egg cell (oocyte) of the fruit fly Drosophila melanogaster, where the movement of mRNA cargo is crucial for the development of the future embryo.
Drosophila is very tractable for this type of study as it is easy to grow and can be genetically manipulated. As well as this, there is a wide availability of fluorescent markers which can be used to label and track different proteins inside cells.
During its development towards maturity, the oocyte becomes highly polarised. It acquires an anterior and posterior axis, and dorso-ventral polarity which will define the polarity of the future embryo.
The establishment of this polarity is dependent upon the directional movement of specific mRNAs which are transported to specific cellular locations on a network of cytoskeletal filaments known as microtubules. Microtubules are polymers which grow and shrink dynamically and which have polarity themselves.
Similar mechanisms for transporting mRNAs exist in a wide range of polarised cell types including neurons and fibroblasts. But it is not clear whether the underlying orientation of the microtubules in oocytes and other cells matches the directionality of cargo transport.
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Scheme of biased random cargo movement
'People have generally said that when cells are polarised, they have microtubules that are really polarised', explains Professor Davis. The most extreme scenario is that all the microtubules are lined up in a single direction. Alternatively, there may be a subset of specifically orientated, biochemically distinct microtubules that the cargo travels on.
The posterior cargo Staufen (red) is transported on microtubules (green) at the oocyte posterior. (Click to enlarge)
But recent work from Professor Davis' lab together with Professor Daniel St Johnston and colleagues at the Gurdon Institute in Cambridge has suggested that, at least in the Drosophila oocyte, the explanation is much more subtle.
What the researchers observed was a biased random movement of posterior localising cargo. The cargo was moving in different directions on what appeared to be a complicated microtubule network, but with a bias so that the net movements were towards the posterior.
The tools were not good enough at the time to follow the microtubules directly and explore the underlying cause of the biased random movement. This was what Professor Davis and colleagues set out to address in the new study.
Using state-of-the-art live cell imaging, and tracking more than one marker at a time, they followed the dynamics of the microtubules whilst imaging the transport of the cargo. Alongside this, they worked on developing more sophisticated tools for handling the data generated.
'We tracked the organisation of microtubules manually at first,' explains Dr Richard Parton, the postdoctoral researcher who carried out the imaging. The painstakingly slow manual analysis was replaced by an automated tracking tool, devised together with Dr Graeme Ball in the lab. This was essential to get sufficient numbers for the data to be meaningful.
At the same time, Dr Russell Hamilton in the lab developed a programme that could take the data, analyse them, and plot diagrams showing the overall directionality of the microtubules.
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Dynamic microtubules support posterior localising cargo transport. Cargo is labelled in red and the microtubules in green
The results from this detailed analysis explain the previously reported subtle biased random transport of cargoes. The researchers saw that the cargo is transported on the entire dynamic microtubule network, but that the overall bias in cargo movement directionality matches precisely the bias in microtubule orientation.
The group also looked at distribution of the initiation of microtubule formation in the oocyte. Mutations in a protein called PAR-1 disrupt microtubule organisation and lead to mislocalisation of specific mRNAs. Using oocytes which lacked PAR-1, the researchers showed that the protein acts by preventing microtubule initiation at specific regions of the oocyte, consistent with the effect the mutant has on disruption of localisation of mRNAs.
Dr Parton comments that the study is a big step up. 'It's the combination of both improved imaging and analysis. They've kind of met in the middle – the point at which we can get sensible information.'
'The key thing that we've shown is that you can have microtubule distribution that's only biased – it's not completely polarised - and that's sufficient to generate polarity,' adds Professor Davis.
How widespread is a subtly biased microtubule network such as this likely to be? The group believes that it could be a general mechanism for initiating and maintaining strong cell polarity whilst ensuring that cargoes are distributed as required throughout the cytoplasm. The techniques developed in this study can be transferred to other cell types so that researchers can begin to answer this question.
1. Parton RM, Hamilton RS, Ball G, Yang L, Cullen CF, Lu W, Ohkura H and Davis I (2011). A PAR-1-dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte. J Cell Biol. Jul 11;194 (1):121-35