A dynein light chain of sea urchin sperm flagella is a homolog of mouse Tctex 1, which is encoded by a gene of the t complex sterility locus.

The outer-arm dynein of sea urchin sperm flagella contains six light chains with molecular masses of 23.2, 20.8, 12.3, 11.5, 10.4 and 9. 3kDa. We have cloned a cDNA for the 12.3kDa polypeptide (light chain 3) and found that this protein is highly homologous to mouse Tctex1, a protein encoded by a member of the multigene family in the t complex region that is involved in male sterility and the development of the germ cells. Tctex1 has recently been shown to be homologous to a light chain of cytoplasmic dynein. Therefore, the cytoplasmic dynein light chain has been implicated in the mechanism for the transmission ratio distortion (meiotic drive) that is characteristic of t haplotypes in mice. Our present finding, however, indicates that axonemal light chain 3 must be considered equally important.

Microtubule translocation caused by three subspecies of inner-arm dynein from Chlamydomonas flagella.

To help understand the function of inner-arm dynein in flagellar motility, dynein samples from an outer arm-missing mutant of Chlamydomonas (oda1) were examined for the ability to translocate microtubules in vitro. High-salt extract of axonemes containing inner-arm dynein was separated by ion-exchange chromatography into 7 peak fractions with ATPase activities. Of these, three fractions containing different sets of dynein heavy chains translocated microtubules. The maximal velocities were all between 3 and 5 microns/s, which were comparable to the microtubule sliding rate in disintegrating oda axonemes.

The binding of nonmuscle caldesmon from brain to microtubules. Regulations by Ca(2+)-calmodulin and cdc2 kinase.

Nonmuscle caldesmon from bovine brain bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with values of Ka 4.5 x 10(5) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. The phosphorylation of caldesmon by cdc2 kinase also eliminated the microtubule-binding activity. These results suggest that caldesmon may play a physiological role in the functions of microtubules.

The fastest actin-based motor protein from the green algae, Chara, and its distinct mode of interaction with actin.

The endoplasmic streaming in Characean cells is an actin-dependent movement. The motor protein responsible for the streaming was partially purified and characterized. It was soluble at low ionic strength, an ATPase of a molecular mass of 225 kDa and activated more than 100 times by muscle F-actin. Surprisingly, in an in vitro motility assay, the motor protein moved muscle F-actin at 60 microns/s, which is similar to the velocity of streaming in a living cell and 10 times faster than muscle myosin. Proteolytic cleavage of actin impaired movement crucially on muscle myosin, but did not affect movement at all on the Chara motor protein, suggesting that the Chara motor protein would interact with actin via a set of sites different from those of muscle myosin.

Amino acid sequence of horseshoe crab, Tachypleus tridentatus, striated muscle troponin C.

The amino acid sequence of troponin C obtained from horseshoe crab, Tachypleus tridentatus, striated muscle was determined by sequence analysis and alignments of chemically and enzymatically cleaved peptides. Troponin C is composed of 153 amino acid residues with a blocked N-terminus and contains no tryptophan or cysteine residue. The site I, one of the four Ca2+-binding sites, is considered to have lost its ability to bind Ca2+ owing to the replacements of certain amino acid residues.

Strikingly low ATPase activities in flagellar axonemes of a Chlamydomonas mutant missing outer dynein arms.

The ATPase activities in Chlamydomonas axonemes were compared between wild type and a mutant (oda) that lacks entire outer dynein arms, at various ionic strengths and pH values, and in the presence of different concentrations of high-molecular-mass dextran. Over a 0-0.2 M KCl concentration range, the ATPase activity of oda axonemes was found to be 5-12 times lower than that of the wild-type axonemes. The low activity in oda is surprising since outer arm-depleted axonemes of sea urchin sperm have been reported to retain about 50% of the normal activity. In both wild type and oda, the ATPase activity of dynein was higher when contained within the axoneme than when released from it with 0.6 M KCl. The ATPase activation within the wild-type axoneme was inhibited by high ionic strengths or by the presence of dextran. The activation in oda axonemes, on the other hand, was not inhibited by these factors. These significantly different ATPase properties suggest that the inner and outer dynein arms perform somewhat different functions in this organism.

Tctex2-related outer arm dynein light chain is phosphorylated at activation of sperm motility.

When the motility of sperm is activated, only one light chain of flagellar outer arm dynein is phosphorylated in many organisms. We show here that the light chain to be phosphorylated was shown to be light chain 2 (LC2) in rainbow trout and chum salmon sperm and LC1 in sea urchin sperm. Molecular analyses of the phosphorylated light chains from sperm flagella of the salmonid fishes and sea urchin revealed that the light chains are homologs of the mouse t complex-encoded protein Tctex2, which is one of the putative t complex distorters. These results suggest that mouse Tctex2 might also be a light chain of flagellar outer arm dynein and that the abortive phosphorylation of Tctex2/outer arm dynein light chain might be related to the less progressive movement of sperm.

Characterization of smooth muscle caldesmon as a microtubule-associated protein.

We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54-56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best-characterized in all caldesmon species. Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 x 10(6) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with alpha-chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34-kDa C-terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross-linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules.

Isolation of two species of Chlamydomonas reinhardtii flagellar mutants, ida5 and ida6, that lack a newly identified heavy chain of the inner dynein arm.

Two novel Chlamydomonas mutants, ida5 and ida6, that lack subsets of inner-arm dynein have been isolated and mapped to discrete loci on the right arm of linkage group XIV. Of the seven different inner-arm dynein subspecies (a, b, c, d, e, f and g) identified by ion-exchange chromatography, ida5 lacks a, c, d and e, while ida6 lacks e alone; these are the only mutants that have been shown to lack subspecies e. Both strains can swim, albeit more slowly than the wild type. Hence, subspecies e must contribute to flagellar movement although it is unnecessary for the generation of undulating movement.

Locomotion of neutrophil fragments occurs by graded radial extension.

The only kinematic description of cell locomotion that relates the dynamics of actin filaments to whole cell movement is the graded radial extension (GRE) model for fish keratocytes, which glide without changing their shapes or sizes. To test whether the GRE model is applicable to other cell types, we analyzed the detailed shape changes during locomotion of heat-induced motile fragments of human polymorphonuclear leukocytes (PMNs). These fragments, called cytokineplasts, were loaded uniformly with a fluorescent cytoplasm-staining dye and their motility and shape changes were analyzed by fluorescence-video microscopy and digital image processing. Two-dimensional (2-D) analysis showed that cytokineplasts only changed their shapes and sizes slightly and apparently maintained their roughly circular shapes, whereas fluorescence-intensity analysis revealed distinct changes in their cytoplasmic thickness profiles. Furthermore, small structures on the cytoplasmic margins behaved as predicted by the GRE model, which therefore is probably also applicable to the parental PMNs, which show complex shape changes. This is the first indication that the GRE model operates in non-fish-keratocyte cells and may, therefore, be a universal model of cell locomotion.

Significantly enhanced stability of glucose dehydrogenase by directed evolution.

An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution. The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes. Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the p Trc99A expression vector and expressed in the host, Escherichia coli. In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B. megaterium IWG3, was also subjected to directed evolution by the family shuffling method. A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries. This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes. The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl. In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66 degrees C), while the half-life of isolated mutant DN-46 was 540 min at 66 degrees C, i.e., 415-fold more thermostable than mutant F20. The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.