Centrin, an EF hand Ca 2ϩ 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 Ͼ 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 2ϩ control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca 2ϩ or the Ca 2ϩ -mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca 2ϩ , 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 2ϩ responses, including ciliary reversal or chemotaxis.

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.

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed ϳ70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.Cilia are ubiquitous cellular nanomachines, found in protists and multicellular eukaryotes, including man, whose repetitive beat depends on a microtubule-based cytoskeleton, powered by molecular motors, the outer and inner rows of dynein arms (outer arm dynein, 22 S dynein; and inner arm dyneins, 14 S dynein; respectively). The arrangement of the dynein arms along the axonemes is complex (1). Dynein arm mechanochemistry is thought to regulate beat frequency and beat form by signal transduction mechanisms that change the parameters of microtubule sliding within the axoneme, such that the outer arm dyneins principally regulate beat frequency whereas the inner arm dyneins control beat form (cf. Refs. 2 and 3).cAMP specifically increases ciliary beat frequency (4), normally measured by an increase of swimming speed, in the protozoan ciliate, Paramecium tetraurelia. The increase occurs in living cells and in cells that have been permeabilized with Triton X-100 and reactivated with Mg 2ϩ -ATP; it persists in the permeabilized cells even when cAMP is subsequently removed and it is quenched by simultaneous addition of Ca 2ϩ to the medium (5-8). We previously reported on a molecule, p29, whose phosphorylation both in vivo and in vitro correlated with the cAMP-dependent Ca 2ϩ -sensitive increase in swimming speed. Further studies revealed that p29 is a component of outer arm dynein (6,8,9), which specifically binds to one heavy chain isoform of the three-headed 22 S outer arm dynein. Phosphorylation of p29 increases in vitro microtubule translocation velocity by outer ...

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.

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.