My (Our) project - Cell division in Plasmodium Berghei

What am I doing?

I am investigating the role of an Aurora-like protein kinase, PbARK2, in cell division in Plasmodium berghei, the rodent model for Plasmodium falciparum.

Why am I doing this?

Plasmodium undergoes cell division in a very atypical way – while most eukaryotes undergo open mitosis, in which the nuclear membrane breaks down before sister chromatids are pulled apart, Plasmodium cells maintain their nuclear envelope at all times. This is called closed mitosis. In addition to closed mitosis, their nuclear division is asynchronous – in a multinucleate cell, each nucleus divides independently of the ones around it. This suggests that there is no global control of mitosis within a cell, and a recent study highlighted that CDC20, a protein essential for mitotic regulation in all eukaryotes is in fact dispensable in the blood stages of parasite development.

Given that Plasmodium uses a drastically different system for cell division, many of the proteins involved may be very different to those in humans, making them good drug targets as off-target effects will be limited. By understanding the underlying biology of the system, we can better make predictions and investments into drug development.

How am I doing this?

ARK2 has already been shown to be essential for blood stage development in P. berghei. The plan was therefore as follows: place ARK2 under the control of an alternative promoter that is active in the blood stage to escape the lethal phenotype, allowing phenotypic analysis at later stages of development. This was to be achieved using a PTD construct (promoter trap using double homologous recombination), generated using molecular cloning techniques in the lab, before transfecting it into wild-type parasites.

Schematic representation of the PTD construct for placing Ark2 under the control of ama1

To provide additional insight into the function of ARK2, a previously generated parasite line expressing an ARK2-GFP tag was to be used to visualise the cellular distribution of the protein and determine expression patterns throughout the life cycle. This would be complimented by qPCR data produced for each life cycle stage.

What’s the story so far?

Last semester started pretty well – there are four of us MSci students in the lab doing very similar projects with different proteins so we felt like we weren’t all on our own. The initial molecular cloning went well, with the direct supervision of Declan, our resident lab-God, knower-of-all-things, provider-of-reagents and all-round-science-boss (the lab technician). With the 3’ fragment successfully inserted into the vector, we were left to do the 5’ section ourselves – this is where things started to go wrong. I managed to get lucky and only need to do one step twice, but Jack had to repeat his bacterial transformation four or five times. However, after a slightly shaky semester we had our constructs completed, sequenced to check accuracy, and ready for transfection. Two weeks before the end of the semester we were poised to begin our transfections and get on with the meat of the project. December 5^th came around, everything went well, nothing could have gone wrong, we felt confident and excited until a week later, when nothing survived the drug selection step, meaning our transfections hadn't worked.

Merry Christmas!

Four weeks later we were back and ready to get back into it. We knew the protocol better this time, we had read more about it, we wanted it more this time. And then it didn’t work. Neither did the third attempt, but the positive control worked just fine. Slightly disheartened, we decided to move on with getting our Western blots to show our protein was correct in the GFP-tagged lines. Carrie has a fantastic post telling you just how well that went… While a few weeks went by desperately hunting for meaning in the Rorschach tests we were creating, we started to learn how to use the fluorescence microscope to visualise our proteins. Jack has a detailed post talking about some of the trials and tribulations we experienced with the microscope, particularly the bit where more than four people need to use one microscope to do time sensitive investigations. Somewhere along the line we went from “just taking practice images” to being expected to produce a full collage of every life cycle stage – I’m still not sure exactly when that was, but I found that this was something I could confidently do. In addition to regular life cycle stages, we discovered that my protein was only transiently expressed during ookinete development, meaning we needed to image timepoints in ookinete development. This is where it gets exciting, because I got my first actual set of data usable in my write-up!

Collage of ookinete developmental stages showing punctate distribution of ARK2 dependent on stage.

Having finally got a decent set of images, I hoped that shortly after everything else would click into place – my Western would come out beautifully, my transfection would be successful and I’d have a clear picture of everything-ARK. However, the next transfection also failed and I still, 3 months and 14 Westerns later, don’t have a band for my protein. We’ve got a few plans for how to fix this but time is running out and it’s getting a bit worrying.

That being said, having spoken to my friends who are doing PhDs, and the post-docs in the lab, in research you have to get used to failure. The trick to being a good scientist is to persevere despite things not going right and work out how to fix it. Use the scientific method and think logically about what you can change, and maybe, just maybe, you’ll get the result you want.

If you’ve had a project come right down to the wire, or want to share any of your science woes please let me know in the comments so I can feel like it’s not just me that’s had this experience.