] i ) in sperm significantly alters swimming direction during chemotaxis (3, 4). Recent studies of human sperm suggest that progesteronemediated Ca 2+ influx through a Ca 2+ channel, CatSper, plays essential role in motility changes, such as hyperactivation and chemotaxis (5, 6), although both phenomena are argued to be separated (7). However, Ca 2+ is a primary factor regulating symmetry of flagellar waveform (8, 9). Chemotactic movements are achieved by continuous changes in waveform symmetry of sperm flagella and subsequent changes in swimming direction to access the egg (10). Ca 2+ -dependent regulation of flagellar beating is reportedly governed by Ca 2+ -binding proteins that likely regulate axonemal dynein (8, 9). These proteins have not been identified, however, and the molecular mechanism of Ca 2+ -dependent control of flagellar asymmetry in sperm chemotaxis remains uncharacterized.Marine invertebrates are excellent models to study sperm motility because their sperm show clear motility changes and are produced in quantities sufficient for biochemical analysis. In the ascidian, Ciona intestinalis, a transient flagellar [Ca 2+ ] i burst is induced by a chemoattractant called sperm activating and attracting factor (SAAF), which triggers rapid sperm-turning followed by straight swimming toward eggs (4, 11). In a search for candidates that regulate Ca 2+ -dependent flagellar movement, we recently identified an axonemal Ca 2+ -binding protein, calaxin, which binds to outer-arm dynein in sperm flagella of C. intestinalis (12). Calaxin is highly conserved in metazoa, including mouse and human. In the present study, using Ciona sperm we show that calaxin is essential for Ca 2+ -dependent modulation of sperm movement necessary for chemotaxis toward the egg. We use in vitro motility assays to demonstrate that calaxin directly suppresses microtubule sliding driven by outer-arm dynein. Results and DiscussionTo test the function of calaxin in regulation of sperm motility in chemotaxis, we used an inhibitor of neuronal calcium sensor family proteins, repaglinide, which specifically binds to calaxin in sperm flagella (Fig. S1) (13). We first asked whether calaxin plays a critical role in sperm chemotaxis. During chemotactic movements, sperm show a unique turning movement associated with a flagellar change to an asymmetric waveform, followed by a straight-ahead movement (11). We observed sperm chemotactic movement toward a glass capillary filled with SAAF in the absence and presence of repaglinide (Fig. 1A). Sperm in control artificial sea water (ASW) with 0.5% (vol/vol) solvent (DMSO) showed very strong chemotaxis toward the glass capillary. However, sperm in the ASW containing 150 μM repaglinide did not exhibit the unique turn movement and showed less-effective chemotaxis (Fig. 1A). Linear equation chemotaxis index (LECI) (11) analysis quantitatively showed significantly decreased chemotactic property promoted by repaglinide at >100 μM (Fig. 1B). Sperm-swimming velocity showed no dramatic change following repagli...

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca 2+ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca 2+ -binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized.Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca 2+ -binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF-hand Ca 2+ -binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross-linking experiment showed that calaxin binds to β-tubulin in situ. Overlay experiments further indicated that calaxin binds the β-dynein heavy chain of outer arm dynein in the presence of Ca 2+ .Conclusions. These results suggest that calaxin is a potential Ca 2+ -dependent modulator of outer arm dynein in metazoan cilia and flagella.

Immotile cilia at the ventral node of mouse embryos are required for sensing leftward fluid flow that breaks left-right symmetry of the body. However, the flow-sensing mechanism has long remained elusive. In this work, we show that immotile cilia at the node undergo asymmetric deformation along the dorsoventral axis in response to the flow. Application of mechanical stimuli to immotile cilia by optical tweezers induced calcium ion transients and degradation of Dand5 messenger RNA (mRNA) in the targeted cells. The Pkd2 channel protein was preferentially localized to the dorsal side of immotile cilia, and calcium ion transients were preferentially induced by mechanical stimuli directed toward the ventral side. Our results uncover the biophysical mechanism by which immotile cilia at the node sense the direction of fluid flow.

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