Asymmetry of the central apparatus defines the location of active microtubule sliding in Chlamydomonas flagella

Abstract

Regulation of ciliary and flagellar motility requires spatial control of dynein-driven microtubule sliding. However, the mechanism for regulating the location and symmetry of dynein activity is not understood. One hypothesis is that the asymmetrically organized central apparatus, through interactions with the radial spokes, transmits a signal to regulate dynein-driven microtubule sliding between subsets of doublet microtubules. Based on this model, we hypothesized that the orientation of the central apparatus defines positions of active microtubule sliding required to control bending in the axoneme. To test this, we induced microtubule sliding in axonemes isolated from wild-type and mutant Chlamydomonas cells, and then used electron microscopy to determine the orientation of the central apparatus. Transverse sections of wild-type axonemes revealed that the C1 microtubule is predominantly oriented toward the position of active microtubule sliding. In contrast, the central apparatus is randomly oriented in axonemes isolated from radial spoke deficient mutants. For outer arm dynein mutants, the C1 microtubule is oriented toward the position of active microtubule sliding in low calcium buffer, but is randomly oriented in high calcium buffer. These results provide evidence that the central apparatus defines the position of active microtubule sliding, and may regulate the size and shape of axonemal bends through interactions with the radial spokes. In addition, our results indicate that in high calcium conditions required to generate symmetric waveforms, the outer dynein arms are potential targets of the central pair-radial spoke control system.

The radial spokes and central apparatus: Mechano‐chemical transducers that regulate flagellar motility

“…In each case, output involves changes in the physical and chemical properties of the radial spokes on subsets of doublet microtubules for local control of dynein activity. Localized interactions of the radial spokes and central apparatus are predicted to differentially alter dynein activity on one side of the axoneme relative to the propagating bend [Mitchell, 2003a;Wargo and Smith, 2003]; this axonemal axis is defined by the inherent asymmetry of the central apparatus.…”

“…Wargo and Smith [2003] have analyzed the orientation of the central apparatus in flagellar axonemes isolated from Chlamydomonas cells using an in vitro microtubule sliding assay. Their results indicate that the orientation of the C1 central microtubule correlates with the position of active sliding for subsets of doublet microtubules.…”

“…However, in this case the specific orientation of C1 does not always correlate with dynein activity as noted above for isolated axonemes induced to slide in vitro. Based on the combined results of Wargo and Smith [2003] and Mitchell [2003a], the sliding assay appears to capture the equivalent of one phase of flagellar beating, the principal bend of the effective stroke.…”

Three types of cilia including a novel 9+4 axoneme on the notochordal plate of the rabbit embryo

“…Each of the central pair microtubules is equipped with a unique set of accessory proteins distinguishing the two tubules from each other and creating intrinsic asymmetry in the CP: the C1 tubule shows two long and two short projections, the C2 tubule is equipped with 3 short projections (Adams et al, 1981;Dutcher et al, 1984;Mitchell, 2003). In some species, the CP rotates during bending of the cilium, thereby modulating outer doublet dynein activity via the radial spokes, which may account for the variety of waveforms and beating frequencies observed in different cilia and flagella (Omoto et al, 1999;Wargo and Smith, 2003). In cells with planar ciliary beat, the CP is fixed in its position and the plane of beating is perpendicular to the CP (Gibbons, 1981;Marshall and Nonaka, 2006).…”

Analysis of microtubule sliding patterns inChlamydomonasflagellar axonemes reveals dynein activity on specific doublet microtubules

“…Genetic and functional analyses indicate that simple oscillatory bending is a feature of the dynein arms, outer doublets, and a feedback system that controls microtubule sliding (Kamiya, 2002). Several studies have contributed to a model in which the asymmetry of the central apparatus plays a role in modulating the switch-point to activate sliding on specific microtubules (reviewed by Satir, 1985) (see also Lindemann, 2003;Wargo and Smith, 2003).…”