The Role of Central Microtubules in the Beating of Eukaryotic Flagella, Revealed by High-Speed Holographic Microscopy

Abstract

Understanding the mechanics of the eukaryotic flagellum is a key challenge in biophysics. As well as being of scientific interest, there are clear therapeutic applications, not least in reproductive medicine. The axoneme lies at the heart of the flagellum, and its structure is known: nine microtubule doublets surround a central pair of microtubule singlets. The role of the central pair in this canonical ‘9+2’ arrangement has been the subject of speculation for some time, though they are known to assist in regulating the flagellar beat. Our group has developed high-speed holographic microscopy that allows us to numerically refocus a digital image off-line. By generating a stack of refocused images from each frame in a video, we obtain scans of the sample volume at the frame rate of our video camera. Effectively, this allows us to image 500-1000 volumes per second. By analyzing this volumetric data, we can measure the waveform of a eukaryotic flagellum to within 200 nm in three dimensions, and with millisecond time resolution. We have previously used this method to measure the waveform of a naturally occurring, isolated flagellum: the microgamete of the rodent malaria parasite Plasmodium berghei. In order to assess the role of the central pair, we take advantage of a newly-available mutant strain that is lacking one or both central microtubules. By comparing the three-dimensional shape and movement observed in mutant flagella to that in the wild-type, we can get some insight into how the central pair helps to regulate the beating action.