The mechanics of cilia and flagella: What we know and what we need to know.

In this review, we provide a condensed overview of what is currently known about the mechanical functioning of the flagellar/ciliary axoneme. We also present a list of 10 specific areas where our current knowledge is incomplete and explain the benefits of further experimental investigation. Many of the physical parameters of the axoneme and its component parts have not been determined.

The flagellar germ-line hypothesis: How flagellate and ciliate gametes significantly shaped the evolution of organismal complexity.

This essay presents a hypothesis which contends that the development of organismic complexity in the eukaryotes depended extensively on propagation via flagellated and ciliated gametes. Organisms utilizing flagellate and ciliate gametes to propagate their germ line have contributed most of the organismic complexity found in the higher animals. The genes of the flagellum and the flagellar assembly system (intraflagellar transport) have played a disproportionately important role in the construction of complex tissues and organs.

The many modes of flagellar and ciliary beating: Insights from a physical analysis.

The mechanism that allows the axoneme of eukaryotic cilia and flagella to produce both helical and planar beating is an enduring puzzle. The nine outer doublets of eukaryotic cilia and flagella are arranged in a circle. Therefore, each doublet pair with its associated dynein motors, should produce torque to bend the flagellum in a different direction.

Functional anatomy of the mammalian sperm flagellum.

The eukaryotic flagellum is the organelle responsible for the propulsion of the male gamete in most animals. Without exception, sperm of all mammalian species use a flagellum for swimming. The mammalian sperm has a centrally located 9 + 2 arrangement of microtubule doublets and hundreds of accessory proteins that together constitute an axoneme.

Mechanics of the eukaryotic flagellar axoneme: Evidence for structural distortion during bending.

The sliding doublet mechanism is the established explanation that allows us to understand the process of ciliary and flagellar bending. In this study, we apply the principles of the sliding doublet mechanism to analyze the mechanics of the counterbend phenomenon in sea urchin sperm flagella. When a passive, vanadate-treated, flagellum is forced into a bend with a glass microprobe, the portion of the flagellum distal to the probe exhibits a bend of opposite curvature (counterbend) to the imposed bend.

The geometric clutch at 20: stripping gears or gaining traction?

It has been 20 years since the geometric clutch (GC) hypothesis was first proposed. The core principle of the GC mechanism is fairly simple. When the axoneme of a eukaryotic flagellum is bent, mechanical stress generates forces transverse to the outer doublets (t-forces).

The physiological role of ADP and Mg2+ in maintaining a stable beat cycle in bull sperm.

Sperm flagella derive their motive power from the motor protein dynein. In this study, we show that maintenance of the flagellar beat cycle in detergent-extracted bull sperm models is highly dependent on the ratio of Mg(2+) to adenosine triphosphate (ATP). An excess of either ATP un-complexed with Mg(2+) , or an excess of Mg(2+) without an equivalent concentration of ATP, results in the loss of beat amplitude and a reduced curvature development in the beat cycle.

Ultrastructural evidence that motility changes caused by variations in ATP, Mg2+ , and ADP correlate to conformational changes in reactivated bull sperm axonemes.

We report the results of an ultrastructural study of Triton X-100-extracted, Mg-adenosine triphosphate (ATP)-reactivated bull sperm. We utilized a rapid fixation method to look for differences in the flagellar apparatus that correlate to the state of motility of reactivated sperm models. In a companion article, we examined the motility characteristics induced in bull sperm models by varying the concentration ratio of ATP and Mg(2+) and the stabilizing effect of adenosine diphosphate (ADP) on coordinated beating.

The effects of Ca2+ and ADP on dynein switching during the beat cycle of reactivated bull sperm models.

Calcium regulation of flagellar motility is the basis for chemotaxis, phototaxis, and hyperactivation responses in eukaryotic flagellates and spermatozoa. Ca2+ is the internal messenger for these responses, but the coupling between Ca2+ and the motor mechanism that generates the flagellar beat is incompletely understood. We examined the effects of Ca2+ on the flagellar curvature at the switch-points of the beat cycle in bull sperm.

The calcium response of mouse sperm flagella: role of calcium ions in the regulation of dynein activity.

Triton X-100-extracted mouse sperm treated with 0.1 mM ATP and 1.0 mM Ca(2+) exhibit an extremely coiled configuration that has been previously described as a curlicue.

Experimental evidence for the geometric clutch hypothesis.

The cilia and flagella of eukaryotic cells are complex filamentous organelles that undulate rapidly and produce propulsive force against the fluids that surround the living cell. They provide a number of important functions in the life cycle of higher organisms including humans. A flagellum propels the spermatozoa to the site of fertilization and cilia move the egg through the oviduct to the uterus and have a role in left-right asymmetry in the developing embryo and contribute to normal brain morphology.

Flagellar and ciliary beating: the proven and the possible.

The working mechanism of the eukaryotic flagellar axoneme remains one of nature's most enduring puzzles. The basic mechanical operation of the axoneme is now a story that is fairly complete; however, the mechanism for coordinating the action of the dynein motor proteins to produce beating is still controversial. Although a full grasp of the dynein switching mechanism remains elusive, recent experimental reports provide new insights that might finally disclose the secrets of the beating mechanism: the special role of the inner dynein arms, especially dynein I1 and the dynein regulatory complex, the importance of the dynein microtubule-binding affinity at the stalk, and the role of bending in the selection of the active dynein group have all been implicated by major new evidence.

Functional deficiencies and a reduced response to calcium in the flagellum of mouse sperm lacking SPAG16L.

The Spag16L gene codes for a protein that is localized to the central apparatus which is essential for normal sperm motility and male fertility. Sperm from mice homozygous for a targeted deletion of the Spag16L gene were examined to assess their flagellar motor functions compared with age- and strain-matched control sperm. Sperm were also demembranated with Triton X-100 and examined for their ability to respond to free calcium, as well as for their ability to undergo microtubule sliding driven by dynein action.

Detergent-extracted models for the study of cilia or flagella.

Methods for using non-ionic detergents to produce demembranated and reactivated cilia and flagella are described in detail. Demembranated and reactivated cell models are useful as a research tool for studying motility function in flagella and cilia. When the plasma membrane is removed, the factors regulating motility can be studied under standardized experimental conditions that otherwise would be impossible.

Mechanical properties of the passive sea urchin sperm flagellum.

In this study we used Triton X-100 extracted sea urchin spermatozoa to investigate the mechanical behavior of the basic 9+2 axoneme. The dynein motors were disabled by vanadate so that the flagellum is rendered a passive structure. We find that when a proximal portion of the flagellum is bent with a glass microprobe, the remainder of the flagellum distal to the probe exhibits a bend in the opposite direction (a counterbend).

The motor activity of mammalian axonemal dynein studied in situ on doublet microtubules.

Flagellar dynein generates forces that produce relative shearing between doublet microtubules in the axoneme; this drives propagated bending of flagella and cilia. To better understand dynein's role in coordinated flagellar and ciliary motion, we have developed an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8.

Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm.

Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.

Evidence for axonemal distortion during the flagellar beat of Chlamydomonas.

In order to understand the working mechanism that governs the flagellar beat it is essential to know if the axoneme undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell was able to preserve the waveform of Chlamydomonas flagella much as it appears during normal flagellar beating [Mitchell, Cell Motil Cytoskeleton 2003;56:120-129]. This conservation of the waveform suggests that the stress responsible for the production of bending is also trapped by the fixation procedure.

The geometric clutch as a working hypothesis for future research on cilia and flagella.

The Geometric Clutch hypothesis contends that the forces transverse to the flagellar axis (t-forces) act on the axonemal scaffold to regulate flagellar beating. T-forces develop as the product of the curvature and the accumulated tension or compression on the doublet microtubules. In this respect, t-force is a mediator of self-organizing behavior.

The counterbend phenomenon in dynein-disabled rat sperm flagella and what it reveals about the interdoublet elasticity.

Rat sperm that have been rendered passive by disabling the dynein motors with 50 muM sodium metavanadate and 0.1 mM ATP exhibit an interesting response to imposed bending. When the proximal flagellum is bent with a microprobe, the portion of the flagellum distal to the probe contact point develops a bend in the direction opposite the imposed bend.

Testing the geometric clutch hypothesis.

The Geometric Clutch hypothesis is based on the premise that transverse forces (t-forces) acting on the outer doublets of the eukaryotic axoneme coordinate the action of the dynein motors to produce flagellar and ciliary beating. T-forces result from tension and compression on the outer doublets when a bend is present on the flagellum or cilium. The t-force acts to pry the doublets apart in an active bend, and push the doublets together when the flagellum is passively bent and thus could engage and disengage the dynein motors.

Direct measurement of the passive stiffness of rat sperm and implications to the mechanism of the calcium response.

Glass microprobes were used to measure the stiffness of the flagella of Triton X-100-extracted rat sperm models. The sperm models were treated with 50 microM sodium vanadate and 0.1 mM Mg-ATP to evaluate the stiffness of the passive flagellar structure without the influence of the dynein motor proteins.