Sperm lacking Bindin are infertile but are otherwise indistinguishable from wildtype sperm.

Cell-cell fusion is limited to only a few cell types in the body of most organisms and sperm and eggs are paradigmatic in this process. The specialized cellular mechanism of fertilization includes the timely exposure of gamete-specific interaction proteins by the sperm as it approaches the egg. Bindin in sea urchin sperm is one such gamete interaction protein and it enables species-specific interaction with a homotypic egg. We recently showed that Bindin is essential for fertilization by use of Cas9 targeted gene inactivation in the sea urchin, Hemicentrotus pulcherrimus. Here we show phenotypic details of Bindin-minus sperm. Sperm lacking Bindin do not bind to nor fertilize eggs at even high concentrations, yet they otherwise have wildtype morphology and function. These features include head shape, tail length and beating frequency, an acrosomal vesicle, a nuclear fossa, and they undergo an acrosomal reaction. The only phenotypic differences between wildtype and Bindin-minus sperm identified is that Bindin-minus sperm have a slightly shorter head, likely as a result of an acrosome lacking Bindin. These data, and the observation that Bindin-minus embryos develop normally and metamorphose into normal functioning adults, support the contention that Bindin functions are limited to species-specific sperm-egg interactions. We conclude that the evolutionary divergence of Bindin is not constrained by any other biological roles.

Bindin is essential for fertilization in the sea urchin.

Species-specific sperm-egg interactions are essential for sexual reproduction. Broadcast spawning of marine organisms is under particularly stringent conditions, since eggs released into the water column can be exposed to multiple different sperm. Bindin isolated from the sperm acrosome results in insoluble particles that cause homospecific eggs to aggregate, whereas no aggregation occurs with heterospecific eggs. Therefore, Bindin is concluded to play a critical role in fertilization, yet its function has never been tested. Here we report that Cas9-mediated inactivation of the bindin gene in a sea urchin results in perfectly normal-looking embryos, larvae, adults, and gametes in both males and females. What differed between the genotypes was that the bindin-/- sperm never fertilized an egg, functionally validating Bindin as an essential gamete interaction protein at the level of sperm-egg cell surface binding.

X-ray fiber diffraction analysis shows dynamic changes in axial tubulin repeats in native microtubules depending on paclitaxel content, temperature and GTP-hydrolysis.

Microtubules are key components of the cytoskeleton in eukaryotic cells. The dynamics between assembled microtubules and free tubulin dimers in the cytoplasm is closely related to the active shape changes of microtubule networks. One of the most fundamental questions is the association of microtubule dynamics with the molecular conformation of tubulin within microtubules. To address this issue, we applied a new technique for the rapid shear-flow alignment of biological filaments, enabling us to acquire the structural periodicity data of microtubules by X-ray fiber diffraction under various physiological conditions. We classified microtubules into three main groups on the basis of distinct axial tubulin periodicities and mean microtubule diameters that varied depending on GTP hydrolysis and the content of paclitaxel, a microtubule stabilizer. Paclitaxel induced rapid changes in tubulin axial repeats in a cooperative manner. This is the first demonstration of dynamic changes of axial tubulin repeats within native microtubules without fixation. We also found extraordinary features of negative thermal expansion of axial tubulin repeats in both paclitaxel-stabilized and GMPCPP-containing microtubules. Our results suggest that even in assembled microtubules, both GTP- and GDP-tubulin dimers can undergo dynamic conversion between at least two different states: short and long configurations.

Effects of the dynein inhibitor ciliobrevin on the flagellar motility of sea urchin spermatozoa.

Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 µM of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin.

Gravity-dependent changes in bioconvection of Tetrahymena and Chlamydomonas during parabolic flight: increases in wave number induced by pre- and post-parabola hypergravity.

Bioconvection emerges in a dense suspension of swimming protists as a consequence of their negative-gravitactic upward migration and later settling as a blob of density greater than that of water. Thus, gravity is an important parameter governing bioconvective pattern formation. However, inconsistencies are found in previous studies dealing with the response of bioconvection patterns to increased gravity acceleration (hypergravity); the wave number of the patterns has been reported to decrease during the hypergravity phases of parabolic aircraft flight, while it increases in centrifugal hypergravity. In this paper, we reassess the responses of bioconvection to altered gravity during parabolic flight on the basis of vertical and horizontal observations of the patterns formed by Tetrahymena thermophila and Chlamydomonas reinhardtii. Spatiotemporal analyses of the horizontal patterns revealed an increase in the pattern wave number in both pre- and post-parabola hypergravity. Vertical pattern analysis was generally in line with the horizontal pattern analysis, and further revealed that hypergravity-induced changes preceded at the top layer of the suspensions while microgravity-induced changes appeared to occur from the bottom part of the settling blobs. The responses to altered gravity were rather different between the two sample species: T. thermophila tended to drastically modify its bioconvection patterns in response to changes in gravity level, while the patterns of C. reinhardtii responded to a much lesser extent. This difference can be attributed to the distinct physical and physiological properties of the individual organisms, suggesting a significant contribution of the gyrotactic property to the swimming behavior of some protists.

Long-term storage of bionanodevices by freezing and lyophilization.

Successful long-term storage of a "smart dust" device integrating biomolecular motors and complex protein assemblies has been demonstrated using freezing or lyophilization, which implies that fabrication and application can be separated even for complex bionanodevices.

Cloning, localization, and axonemal function of Tetrahymena centrin.

Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.