Trypanosomes yield their secrets in new study on chromosome segregation
Trypanosomes appear to use a completely novel set of proteins to ensure the correct separation of their chromosomes during cell division.
Diagram showing the kinetochore complex (green) providing a bridge between the spindle microtubules (red) and the chromosomes (blue)
This is the finding of research from Dr Bungo Akiyoshi, a Sir Henry Dale Fellow in the department, together with Professor Keith Gull in the Sir William Dunn School of Pathology.
Their work is described in a recent paper in Cell (1). It opens up new opportunities for the development of drugs against these organisms which cause tropical diseases, and provides insight into the enigmatic history of the earliest eukaryotes.
As a postdoc in Professor Gull’s lab, Dr Akiyoshi took the opportunity to continue his work on the kinetochore, a protein complex which links the chromosomes to the mitotic spindle ensuring that the chromosomes segregate correctly. But he decided to shift his research away from yeast to trypanosomes.
‘Kinetochore components had been identified in various eukaryotes and it was assumed that all eukaryotes utilise common components for building kinetochores,’ he explains. ‘But no kinetochore protein was identifiable in kinetoplastids [to which trypanosomes belong]. Although people wanted to find out more, no-one could work on the kinetochores in these organisms because they couldn't identify components based on bioinformatics analysis.’
Dr Akiyoshi decided to tackle the problem using a different approach. He screened for genes that are up-regulated during mitosis and was lucky enough to identify a protein that he suspected was a kinetochore protein. This key finding led to a flurry of further discoveries.
‘I was thinking of investing up to two years in identifying one protein but I was very lucky – I found one within a year,’ he says. ‘Once I’d done this, I could identify others by affinity purification and mass spectrometry of the co-purifying proteins. Within 6 months or so, I found a further 18 proteins in total.’
Using inducible RNAi knockdown on a number of these proteins, he showed that they are essential for chromosome segregation and cell growth.
Sequence analysis of the proteins, named KKT proteins, revealed why they had not been picked up before. ‘They look completely different from conventional kinetochore proteins found in other eukaryotes,’ explains Dr Akiyoshi.
Although the trypanosome kinetochore serves the same function as all other kinetochores, bridging between DNA and the microtubules of the mitotic spindle, the components appear to be completely different.
The finding is intriguing from both an evolutionary perspective and the potential for drug development.
Trypanosome cell with a YFP-tagged kinetochore protein (yellow) and DNA stained with DAPI (purple) (Click to enlarge)
‘We still don’t know much about the early histories of the eukaryotes,’ explains Dr Akiyoshi. ‘People draw the eukaryotic tree without a root because they don’t know which organisms are the earliest branching eukaryotes. It’s a big unanswered question in biology.’
‘One idea is that trypanosomes might be the earliest branching eukaryote, based on some unique biological features that have been found in them. The finding from my work - that kinetochores in trypanosomes are very different from those in other eukaryotes - supports this hypothesis.’
‘I think this work will encourage other people to look closer at trypanosomes and related species and see if there are other unique features.’
The uniqueness of the kinetoplastid kinetochore proteins also makes them an attractive drug target for diseases caused by these organisms - sleeping sickness and Chagas disease from Trypanosoma species and leishmaniasis from Leishmania species. Of immediate interest are the 4 kinases identified within the group of 19 proteins. These appear to be functionally important and are potentially druggable.
Dr Akiyoshi’s current work is focussed on trying to understand how the KKT proteins perform their function - binding to DNA and to microtubules.
He admits that it is quite a challenge in the absence of any clues about which proteins may be carrying out which functions. At the primary sequence level, he has not been able to identify any possible microtubule-binding regions although he adds that this sort of analysis is often difficult until the structure of the protein has been elucidated.
‘I’ve been using localisation patterns of the proteins as a guide,’ he says. ‘Often the proteins that bind to the DNA are the ones that are constitutively localised to the base of the kinetochore. The microtubule-binding proteins tend to come to the kinetochore only during mitosis and do their job then.’
He has some idea about candidates for DNA binding and is planning to express those proteins and carry out in vitro biochemical assays to test their function. He will then go back to cell culture to test how the proteins behave in cells. Alongside this approach, he will also be determining the structure of the proteins using X-ray crystallography.
Gradually, he hopes to build up a detailed picture of how the trypanosome’s novel set of kinetochore proteins fulfils the same role as the conventional proteins found in all other eukaryotes.
‘By understanding unique kinetochores in trypanosomes and comparing with insights obtained from other eukaryotes, I am trying to reveal fundamental requirements for building a kinetochore - a very fascinating molecular machine,’ he adds.