摘要:

AbstractThe development and locomotion of the amoeboid sperm of the nematode,Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly‐disassembly‐reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHiwas 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHito 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re‐establishment of the pH gradient, and re‐initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHioccurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH‐sensitive MSP‐membrane interaction. © 1994 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970270302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractEjaculated ram sperm were demembranated with Triton X‐100, separated from the detergent‐soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264–273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was ∼100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP‐dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5–24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5–24) prior to reactivation. The cytosol‐free models retained>90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X‐100 alone, but only by cAMP and Triton X‐100 in combination. We conclude that cAMP‐dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg‐lys‐arg‐ala‐arg‐lys‐glu, which has been reported to be a selective inhibitor of cGMP‐dependent pro

ISSN:0886-1544    

DOI:10.1002/cm.970270303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractWe analyzed the distribution and orientation of transitory microtubule structures, microtubule converging centers, during interphase and mitosis in endosperm of the higher plantHaemanthus.In interphase the pointed tips of microtubule converging centers are associated with the nuclear envelope. Their orientation gradually reverses during prophase, and the tips tend to point away from the nucleus. From prometaphase through early telophase, microtubule converging centers are present predominantly in the cytoplasm at the polar region. They are either “free” or associated with chromosomes or microtubule bundles. In late telophase, pointed tips of microtubule converging centers are again associated with the reconstructed nuclear envelope and, additionally, they often appear in the phragmoplast area. The orientation of microtubule converging centers seems to be directly correlated to the previously determined microtubule polarity, with the converging tip being minus and the diverging one, plus.Elevated temperature (35°–37°) enhances the number of microtubule converging centers in the cytoplasm and at the nuclear envelope. This is especially pronounced during the telophase‐interphase transition and in some interphase cells, indicating temperature and stage dependence.Our data imply that microtubule converging centers bind together MT minus ends and, thus, control the predominant direction of elongation and shortening of microtubule arrays. We argue that these configurations are instrumental during the reorganization of interphase cytoskeleton and mitotic spindle inHaemanthusendosperm. © 1994 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970270304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractMicrotubule‐associated protein 2 (MAP‐2) is an abundant component of the cytoskeleton present in dendrites and cell bodies of neurons of the CNS. To examine the biological function of MAP‐2, two MAP‐2 antisense (AS) oligonucleotides complementary to the 5′ region of the rat MAP‐2 cDNA were added to rat primary embryonic day 17–18 (E17‐18) cultured cortical neurons 24 h after plating and neurite outgrowth and morphology studied. The treatment of primary cortical cultures with either of the two MAP‐2 AS oligonucleotides resulted in decreased MAP‐2 and reduction in the number of neuritic processes relative to the control or MAP‐2 sense‐treated cultures. By immunostaining and light microscopy the AS‐treated neurons appeared smaller, more rounded, and less intensely stained for MAP‐2 than the untreated or the MAP‐2 sense‐treated cultures. By electron microscopy disorganized microtubules and a reduction in the number of microtubules within neurites of the AS‐treated cultures were observed. We conclude that MAP‐2 continues to be required for microtubule spacing and stability within neurites once they

ISSN:0886-1544    

DOI:10.1002/cm.970270305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe morphogenesis of functional myofibrils in chick skeletal and cardiac muscle occurs in greatly different time spans, in about 7 and 2 days, respectively. In chick skeletal myogenic cells, one isoform of the 250 kD actin‐binding protein (ABP) filamin is associated with stress fiber‐like structures of myoblasts and early myotubes, then disappears for approximately 4 days, whereupon a second filamin isoform reappears at the Z‐disc periphery. We sought to determine if cardiac myogenesis involves this sequence of appearance, disappearance, and reappearance of a new filamin isoform in a compressed time scale. It was known that in mature heart, filamin is localized at the Z‐disc periphery as in mature (fast) skeletal muscle, and is also associated with intercalated discs. We find that myocardial filamin has an apparent molecular weight similar to that of adult skeletal muscle filamin and lower than that of smooth muscle filamin, and that both skeletal and cardiac muscle contain roughly 200 filamin monomers per sarcomere. Two‐dimensional peptide mapping shows that myocardial filamin is very similar to skeletal muscle filamin. Myocardial, slow skeletal, and fast skeletal muscle filamins are all phosphorylated, as previously shown for filamin of non‐striated muscle. Using immunofluorescence, we found that filamin could not be detected in the developing heart until the 14‐somite stage, when functional myofibrils exist and the heart has been beating for 3 to 4 hours. We conclude that in cardiac and skeletal myogenesis, different sequences of filamin gene expression result in myofibrils with similar filamin distributions and isoforms. © 1994 W

ISSN:0886-1544    

DOI:10.1002/cm.970270306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractPrevious experiments have suggested that 3T3 cells were able to extend pseudopodia toward latex particles up to 60 μm away from the cell body if the particles were irradiated by an infrared beam in the range of 700–900 nm [Albrecht‐Buehler, 1991:J. Cell Biol.114:493–502]. The present article reports that this response of cells to infrared light can be inhibited if the cell center is simultaneously irradiated with a beam of the same light. In marked contrast, the cells responded normally to the presence of infrared light scattering particles if the second beam irradiated other parts of the cell body. The results imply that the cellular mechanism of infrared detection is located at the cell center. The infrared sensing mechanism remains intact in enucleated cells and in cells which were incubated in monensin to vesiculate their Golgi apparatus and inhibit their Golgi functions. Accordingly, it is proposed that the centrosome which contains the centrioles is the only remaining candidate in the cell center for a cellular detection device for the direction of infrared signal sources. The results support an earlier suggestion that centrioles may be such detection devices [Albrecht‐Buehler, 1981:Cell Motil. Cytoskeleton1:237–245]. © 1994 Wile

ISSN:0886-1544    

DOI:10.1002/cm.970270307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe single alpha‐tubulin gene ofTetrahymena thermophilawas isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha‐tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha‐tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha‐tubulins. The single alpha‐tubulin ofTetrahymenamust be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha‐tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha‐tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system ofTetrahymena, alpha‐tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha‐tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms,Tetrahymenaadjusts alpha‐tubulin message amounts via message decay. However the complex control of alpha‐tubulin mRNA during theTetrahymenalife cycle involves regulation of both decay and transcription rates.

ISSN:0886-1544    

DOI:10.1002/cm.970270308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970270309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970270301

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY