Tuesday, 29 July 2014

ἀνάφάσις: it's all Greek to me? Molecule of the month: the anaphase promoting complex

Paul Andrews/University of Dundee
The remarkable picture on the left was taken by a good friend of mine, who, not long after his PhD in my lab at Sheffield became the microscopy imaging maestro at the University of Dundee, one of the UK's leading Life Science Departments. Paul Andrews now heads up his own Stem Cell consulting company, Stem Cell Solutions. His beautiful image shows migration of sister chromatids, which have just split from their parent chromosomes, to the opposite "poles" of the cell prior to completion of either mitosis or meiosis. You can read about anaphase at the wiki site and the whole sequence of events during the cell cycle here. The pioneering work of scientists that include the 2001 Nobel Prize winning trio of Sir Paul Nurse, Tim Hunt and Lee Hartwell, opened the door to our understanding of a molecular process that hold one of the major the keys to understanding and ultimately managing cancer. The controlled division of cells is at the heart of this disease and considerable efforts have been, and continue to be made, in elucidating the core mechanism as well as the network of associated molecular events in cell division. 

The cell cycle (RHS) is described in more detail at the above links, but briefly comprises 4 major stages. G1 (or the first Gap phase): this is a period of preparation for S phase, in which synthesis of DNA occurs. This is followed by the second Gap phase, which ensures the genome is ready for Mitosis, the phase that leads to cell division. During these phases, there are mechanisms of surveillance or quality control that make sure everything is present and correct for cell division. These processes are called checkpoints and are a major focus of cell cycle research. You can read more here at Boundless. It is the process of Mitosis that we are concerned with today (I will reserve meiosis until another time). Mitosis itself comprises Prophase, Metaphase, Anaphase and Telophase which result in Cytokinesis. Another set of intimidating terms, I'm afraid: Pro comes from the Greek, before (think pro-logue, pro-active). Meta in this context means after, or the following phase (metamororphosis, following a change in shape: you will have heard me use this word in the context of the life cycle of the mealworm, or I can thoroughly recommend the short story of the same name by Franz Kafka). Ana comes from the Greek for up, specifying in this case the polar movement of the chromatids, and finally Telo relates to completion or the end of a process (again from the Greek, remember telomere, the end or tip of a chromosome). All of these words are prefixes that allow us to "organise" the steps of Mitosis, which is itself derived from the Greek for a threading (Mito) process (sis) and for completeness Meiosis means a reducing process since the chromosome number is halved. Finally, Cytokinesis is the result of all of this classical activity and this is derived from Cyto (meaning a container) and kinesis which means movement (think of kinetics from your Physics and Chemistry).

This month, following years of effort and a number of intermediate publications the group of Professor David Barford at the Institute of Cancer Research in London (and now at the Laboratory of Molecular Biology in Cambridge) have determined a molecular structure for the Anaphase Promoting Complex, or APC. The significance of these results have been nicely summarised by Ian Foe and David Toczyski in a Nature News and Views article, and their schematic diagram for this complex is shown left. The protein complex comprises 13 different polypeptide chains and represents not only a major advance in our understanding of this giant enzyme, but also advances our technology in the analysis of large multiprotein complexes in general. The team used insect cells in culture combined with the multibac cloning system that allows the complete set of genes (or more accurately the open reading frames) to be expressed simultaneously and purified for analysis. 

The determination of the structural details of a complex of this size requires the use of many techniques, but one of the most powerful methods is called cryo-electron microscopy, in which single particles are analysed at low temperatures and their resultant contours mapped against data from other X-ray studies and the structures of similar proteins (inferred from sequence and functional similarities). The importance of mass spectrometry, which is becoming a key method in projects of this kind, should also be mentioned in dispatches. The structure on the RHS is too small for you to observe the detail, but if you focus on the representation of structural elements (the coloured cylinders are alpha helices) they are mapped onto an envelope of electron density that is derived from electron microscopy. This not only defines the "molecular envelope", but begins to give us insight into the internal structure of APC. This image is taken from the Nature paper recently published by the Barford lab.

Cancer cell division, Paul Andrews
(University of Dundee)
How does this structural work help? I have always been impressed by the beauty of structural biology and the structure of APC is undoubtedly a biochemical tour de force. Similar landmarks in recent years have included the ribosome and the RNA Polymerase (PolII) machinery from yeast. However, there must be a value in these structures beyond scientific aesthetics. This is now the challenge that must be addressed. The understanding of the sequence of events and the specific role played by each of the APC polypeptides in anaphase will undoubtedly follow. We will then be faced with understanding the catalytic events, which are much more of a challenge, and then we will try and establish whether we can interfere with this process in a predictable manner. Ideally we would like to be able to modulate the cell cycle in a controlled and tissue specific way, in order to inhibit unregulated cell division in cancer (as shown in another or Paul Andrews' lovely images, top left). So, the determination of the structure is indeed a momentous moment, but many would say that this is now where the work begins. As students of molecular biology, we must assimilate and understand these elegant experimental structures, but we must design experiments to test their significance: we must pick up the baton now and run with it. I look forward to watching the impact of this work over the coming years and hope it stimulates an interest in the cell cycle.

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