The tissues and organs of most animal bodies are lined by ceaselessly beating microscopic hairs – from the structures that waft cerebrospinal fluid around the brain to lung-cleansing cilia, and many single-cell organisms, such as Paramecium, use similar structures for propulsion in the pursuit of food and to avoid difficult conditions. Junji Yano and Judith Van Houten from the University of Vermont, USA, explain that the relentlessly beating motion of Paramecium cilia is driven by calcium currents passing through protein channels within the microscopic structures that switch the direction in which the cilia beat to send the cell into reverse. The true identity of the proteins that comprise these channels had remained elusive for almost half a century until Van Houten's team identified components of the calcium channel in the cilia of Paramecium tetraurelia in 2013. But there was still no direct evidence that these channels were the origin of the essential calcium current, so Yano, Sukanya Lodh and Van Houten set about directly testing the role of the channel in Paramecium swimming behaviour.
RNA interference (RNAi) – where segments of RNA that match the sequence of a target gene are fed to organisms to prevent production of the protein that is encoded by the gene – is a powerful technique that allows scientists to assess the physiological role of specific genes. As Van Houten's team had previously sequenced three calcium channel alpha 1 subunits (CaV1a, 1b, 1c), Lodh and Yano produced RNA fragments containing lengthy sections of the three calcium channel subunits, fed them to P. tetraurelia for up to 72 h and then transferred the cells to a solution that triggers swimming in reverse to assess how losing channel components affected swimming performance. Comparing Paramecium cells that had received the RNAi diet with those that had not, the team found that cells that had lost one channel subunit could only reverse for ∼3–5 s and cells that had lost all three barely reversed at all (∼2 s); however, cells that not been fed RNAi swam backward for ∼23 s. The CaV channel proteins found in the cilia were responsible for Paramecium’s about-turn.
However, the team needed further convincing that the CaV1a–c channel subunits provided the essential calcium current, so they turned their attention to a mutant form of P. tetraurelia – known as ‘Pawn’ – which, just like its chess piece namesake, is unable to reverse. As there were two possible explanations for the Pawn mutants’ inability to reverse – either they lacked CaV channels in their cilia or they had the channels, but the proteins were inactive for some reason – Lodh and Yano added a tag to the end of the Pawn CaV1c gene and allowed the cells to grow before collecting the cilia. Then they tested for evidence of the tagged channel protein in the cilia, but there was none, suggesting Pawn mutants lack CaV channels in their cilia.
Lodh and Yano then inserted normal copies of the genes that are known to be defective in Pawn cells (known as PWA and PWB) and tested the modified cells; not only had they regained the ability to reverse but also the essential CaV channel proteins were present in the cilia. In addition, the normal PwB protein appears to be associated with the CaV1c protein in the endoplasmic reticulum, where proteins are synthesised, suggesting that PwB may be involved in transporting CaV1c to the cilia, where the protein contributes to production of the calcium current that is essential for the change in direction of P. tetraurelia’s beating cilia.
