Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".     

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

Acrosomal actin bundles of Asian and American horseshoe crab sperm differ in protein composition

Acrosomal actin bundles were isolated from the sperm of horseshoe crabs from four different sources, three from Asia and one from North America, and their protein constituents and structures were compared. The bundle from the American Limulus polyphemus sperm was composed of actin and two associated proteins of MW 95,000 and MW 52,000, as reported previously (Tilney, L. G. (1975) J. Cell Biol. 64, 289-310). However, those from the three Asian species (Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda) were composed only of actin and the protein of MW 95,000. Electron microscopic and optical diffraction studies indicated that both the helical structures and the interfilament spacing of the actin filaments composing the acrosomal bundle were indistinguishable among the four species. These results suggest that the MW 95,000 protein crosslinks actin filaments in the bundle. Moreover, they support the idea that Limulus and the three Asian species have evolved independently from a common ancestor.     

Secretory carcinoma of the endometrium

A case of secretory carcinoma of the endometrium in a 21-year-old woman is reported. Endometrial smears were interpreted as showing a differentiated adenocarcinoma. Smears of ascitic fluid obtained during subsequent surgery showed similar findings; periodic acid-Schiff staining of the smears revealed abundant positive material in the cytoplasm. These findings were interpreted as evidence of secretory carcinoma, which was confirmed by histopathologic study of the biopsy and surgical specimens.     

Separation of RRBC-RFC and non-rosette forming cells (non-RFC) of guinea pig T-cell populations and their functional difference. 1. Response of RRBC-RFC and non-RFC to either Con-A or LPS.

Guinea pig lymph node cells suspension (LNC-O) was filtered through a glass wool column and the effluent (LNC-G) was further filtered through a nylon column. In this effluent (LNC-NE) about 30 per cent of the lymphocytes was identified as non-rosette forming cells (non-RFC). The non-RFC fraction was separated from LNC-NE fraction by Ficol-Conray specific gravity centrifugation or effluent cells reacted previously to rabbit red blood cells (RRBC). The upper layer after centrifugation, designated non-RFC fraction, was separated. In this fraction 96% of the cells were lymphocytes and about 95% of them were non-RFC, which lacked receptors for rabbit red blood cells (RRBC) or EAC and detectable surface immunoglobulin by conventional techniques. Though the response of the lymphocytes in the non-RFC fraction to mitogenic (Con-A, LPS) or antigenic stimulation was lower in comparison with that in RFC-rich fraction, the response of non-RFC to ConA exceeded the response to LPS. These facts suggest that at least a portion of the non-RFC may be cells from the T-cell line.