ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level

Abstract

The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies.

Load Response of the Flagellar Beat

“…In our theory, we assume an active flagellar driving force that is independent of external load. This assumption is consistent with recent experiments, which show that flagellar ATP consumption is rather insensitive to beat frequency and mechanical load [39]. Accordingly, any increase in load is compensated by a reduction in speed, not an increase in fuel consumption.…”

“…Previous estimates for η reported values in the range η = 0.1 − 0.4 [36][37][38][39], see also SM. In our theory, we assume an active flagellar driving force that is independent of external load.…”

Post‐ejaculatory modifications to sperm (PEMS)

“…The consequent determination of gains, losses and modifications of sperm (or sperm‐associated seminal or female) proteins will provide molecular precision to our understanding of the sequence of changes in sperm as they reside in the FRT. Combining this approach with cryo‐electron‐tomography (Nicastro, McIntosh, & Baumeister, ) to reveal structural changes in the sperm and in the positions or shapes of complexes on or in them, and methods for single‐cell measurement of energy consumption (Chen et al ., ) in sperm will further extend the molecular precision of what is involved in PEMS. This in turn will provide a foundation for systematic efforts to examine the in vivo functional importance of PEMS, in organisms where genetic manipulation is possible, and establish molecular networks governing post‐copulatory sperm–female interactions critical to sperm viability and fertility (McDonough et al ., ).…”

Diurnal variations in the motility of algal populations

“…Further, using a hydrodynamic power balance, we were able to infer from the speed the corresponding diurnal variation in mean beat frequency. A study of demembraned Lytechinus pictus sea urchin sperm flagella has demonstrated ATP concentration correlates positively with flagellar beat frequency (34). We can reasonably hypothesise that a similar correlation will apply qualitatively for C. reinhardtii.…”

“…In particular, our data would suggest that during the dark period the amount of ATP available to flagella drops in value to a lower level that is maintained during the day; flagellar ATP then surges at the onset of division. To test if the flagellar frequency does indeed chart the variation of flagellar ATP, it would be very interesting to carry out experiments similar to (34) assaying the diurnal variation of ATP in the flagella of C. reinhardtii.…”